WO2012152063A1 - Novel biomarker of hcc, rarres2 - Google Patents

Abstract

The present invention discloses a novel biomarker of HCC, RARRES2, and more specifically discloses a polypeptide with anticancer activity, comprising sequence of YFPGQFAFS or its variant. The invention also provides the use of the polypeptide in preparing the medication for treating and preventing caner, in preparing the medication for inhibiting Akt signal pathway. The invention also discloses a detecting kit for cancer prognosis, assessing the survival time of cancer patients through detecting the expression level of RARRES2 in a sample from the object.

Description

 A new molecular marker of liver cancer retinoic acid receptor response protein 2

 The present invention relates to the field of cancer control, and in particular to the use of retinoic acid receptor-responsive protein 2 and its C-terminal active fragment in cancer control. Background technique

 Hepatocellular carcinoma (HCC) is one of the most common and most harmful cancers in the world, ranking the fifth most common cancer in the world and the third most common cancer-related cause of death, second only to lung cancer and stomach cancer. Liver cancer cells are extremely active, invasive, and easily invade the capsule and blood vessels. Because of the abundant sinusoids in the liver, intrahepatic metastasis is the most common, and most patients can have intrahepatic metastases in the early stage. Hepatoma cells invade the branches of the portal vein to form tumor thrombi, and liver cancer with portal vein tumor thrombus is considered to be one of the main indicators of poor prognosis. Extrahepatic metastasis is mainly caused by hematogenous metastasis. It is more common in advanced patients. The lung is the most common metastatic organ outside the liver of HCC, followed by bone, adrenal gland, kidney and brain. The high metastatic rate of liver cancer is an important reason for the poor prognosis of liver cancer. Therefore, it is an urgent task to study the mechanism of metastasis and deterioration of hepatocellular carcinoma and develop effective treatment strategies.

 Chemerin, also known as tazarotene-induced gene 2 (TIG2), or retinoic acid receptor responder 2 (RARRES2, retinoic acid receptor responder 2), was first identified as a new class present in psoriatic lesions. Vitamin A response gene. Later, it was identified as a secreted ligand of the orphan G protein-coupled receptor chemokine-like receptor 1 (CMKLR1, also known as ChemR23), which revealed its biological function for the first time. Further studies have shown that chemerin is also a ligand for two other receptors: chemokine (C-C motif) receptor-like (CCRL) 2 and G protein-coupled receptor (GPR) 1. However, the function of these two receptors in mammals is not yet clear.

Chemerin is usually secreted out of the cell in the form of a weakly active prochemerin, which is required to cleave several amino acids at the C-terminus into an active form, activating its receptor, ChemR23. Chemerin-154, 155, 156, 157, etc. can be obtained because the identification and shearing sequences of different enzymes are not the same. Active chemerin in various forms (corresponding to the 9, 8, 7, 6 amino acids at the C-terminus). According to reports in the literature, the C-terminus of chemerin-157, especially the penterin-9 at positions 149-157, is critical for the activation of chemerin receptors. chemerin-9 retains most of chemerin-157. Activate the activity of the receptor.

 Many species of cells involved in both innate and acquired immunity express chemerin receptors

CMKLR1, Currently, chemerin is known to act as a chemokine, recruiting these cells to ulcers and tissues. In addition, studies have reported that chemerin expression and secretion are significantly elevated during adipocyte differentiation. Moreover, in cell models, loss of chemerin or CMKLR1 expression almost completely prevented the production of adipocytes and altered the expression of important genes in glycolipid metabolism, including GLUT4, DGAT2, leptin and adiponectin. These studies reveal that chemerin is not only a chemokine but also a fatty cytokine. Although chemerin has been studied and reported in the fields of immunity, cardiovascular disease and metabolic diseases, its role in tumors and mechanisms have rarely been reported. This is exactly what we have to say in this invention. Summary of the invention

 A first aspect of the invention provides a coding sequence of retinoic acid receptor receptor protein 2 or a polypeptide thereof or an agonist thereof, retinoic acid receptor response protein 2 or a polypeptide thereof, or a polynucleotide construct comprising the same Use in the preparation of a medicament for treating or preventing cancer.

 A second aspect of the invention provides a coding sequence of retinoic acid receptor response protein 2 or a related polypeptide thereof or an agonist thereof, retinoic acid receptor response protein 2 or a polypeptide thereof, or a polynucleotide construct comprising the same The use in the preparation of a medicament for inhibiting the metastasis ability of a patient's cancer cells.

 A third aspect of the invention provides a coding sequence of retinoic acid receptor-reactive protein 2 or a polypeptide thereof or an agonist thereof, retinoic acid receptor-responsive protein 2 or a polypeptide thereof, or a polynucleotide construct comprising the same The use in the preparation of a medicament for anti-tumor angiogenesis.

 In a specific embodiment, the anti-tumor angiogenesis comprises inhibiting angiogenesis during cancer development, progression, and/or metastasis.

 A fourth aspect of the invention provides a diagnostic kit for cancer prognosis, the kit comprising an agent for detecting a level of retinoic acid receptor response protein 2 in a sample.

The fifth aspect of the present invention provides an agent capable of detecting retinoic acid receptor-responsive protein 2 in preparation for diagnosis of cancer Application in the broken kit.

 A sixth aspect of the invention provides a coding sequence of retinoic acid receptor-reactive protein 2 or a polypeptide thereof or an agonist thereof, retinoic acid receptor-responsive protein 2 or a polypeptide thereof, or a polynucleotide construct comprising the same The use in the preparation of a medicament for inhibiting the Akt signaling pathway.

 The present invention also includes a coding sequence of retinoic acid receptor-responsive protein 2 or a related polypeptide thereof or an agonist thereof, retinoic acid receptor-responsive protein 2 or a related polypeptide thereof, or a polynucleotide construct containing the same. Use in drugs that inhibit the mRNA and protein levels of VEGF.

 In a specific embodiment, the retinoic acid receptor response protein 2 or a related polypeptide thereof or an agonist thereof, the coding sequence of retinoic acid receptor response protein 2 or a related polypeptide thereof or a polynucleoside containing the coding sequence The acid construct down-regulates the level of its mRNA by inhibiting the transcriptional activity of the VEGF promoter.

 The present invention also includes a coding sequence of retinoic acid receptor-responsive protein 2 or a related polypeptide thereof or an agonist thereof, retinoic acid receptor-responsive protein 2 or a related polypeptide thereof, or a polynucleotide construct containing the same. Use in drugs that promote the differentiation of Ml macrophages.

 In a specific embodiment, differentiation of Ml macrophages produces a tumor suppressing microenvironment to achieve inhibition and/or treatment of cancer.

 In the present invention, the retinoic acid receptor response protein 2 is selected from the group consisting of

 (1) the amino acid sequence of SEQ ID NO: 2; or

 (2) An amino acid sequence which has been substituted, deleted or added with one or more amino acids in the amino acid sequence of SEQ ID NO: 2 and which retains the biological activity or function of SEQ ID NO: 2.

 In the present invention, an example of the coding sequence of retinoic acid receptor-responsive protein 2 is shown in SEQ ID NO: 1.

 In the present invention, retinoic acid receptor-responsive protein 2 includes a truncated retinoic acid receptor-responsive protein 2 having a C-terminally truncated 1 to 10 amino acids. In a specific embodiment, the retinoic acid receptor response protein 2 for use in the various methods or uses of the invention described above is as set forth at positions 21-157 (SEQ ID NO: 49) of SEQ ID NO: 2, or as SEQ ID NO: 2 shows the 21st to 156th, 21st to 155th or 21st to 154th amino acids. The invention also includes amino acids which have been substituted, deleted or added with one or several amino acids in the truncated retinoic acid receptor response protein 2 and which retain the biological activity or function of the truncated retinoic acid receptor response protein 2 sequence.

 In the present invention, the retinoic acid receptor response protein 2 related polypeptide is selected from the group consisting of:

(1) a C-terminal active fragment of retinoic acid receptor-responsive protein 2 or an active variant thereof; (2) a polypeptide comprising (1) the C-terminal active fragment or an active variant thereof;

 (3) an amino acid which has undergone substitution, deletion or addition of one or several amino acids in the amino acid sequence of (1) to (2) and retains the biological activity or function of the amino acid sequence of (1) - (2) sequence.

 In the present invention, the C-terminal active fragment of retinoic acid receptor response protein 2 comprises a C-terminal active fragment containing YFPGQFAFS, which may be 9 to 20 amino acid residues in length. Exemplary C-terminal active fragments include, but are not limited to, those set forth in SEQ ID NOS: 25-30 and 41.

 The active variants of the C-terminal active fragment of the present invention include variants of YFPGQFAFS and variants containing a C-terminal active fragment of YFPGQFAFS. Specifically, the variant comprises a C-terminal active fragment which has been substituted, deleted, added or chemically modified in YFPGQFAFS or in a C-terminal active fragment containing YFPGQFAFS, and which retains YFPGQFAFS or contains YFPGQFAFS. A variant of biological activity or function.

Exemplary variants include, but are not limited to, X!XsPGQFAFXg (SEQ ID NO: 48), wherein X, and X _{2 are} optionally an aromatic residue or a hydrophobic residue, and X ₉ is a serine residue or a C-terminal carboxyl group. The aspartic acid residue (D-CONH ₂ ) in which the hydroxyl group is substituted with an amino group.

 Exemplary variants include, but are not limited to, the amino acid sequences set forth in SEQ ID NOS: 24, 26, 31-40, and 42-47.

 The present invention also encompasses a polypeptide comprising the chemerin-binding receptor ChemR23 comprising the C-terminal active fragment or a variant thereof. Such polypeptides include, but are not limited to, polypeptides having a length of from 20 to 156 amino acid residues containing YFPGQFAFS in positions 1-157 of SEQ ID NO: 2.

 In the present invention, the polynucleotide construct comprising the coding sequence may be an expression vector which expresses retinoic acid receptor response protein 2 or a related polypeptide thereof.

 In the present invention, the cancer is selected from the group consisting of thyroid cancer, pancreatic cancer, breast cancer, head and neck squamous cell carcinoma, liver cancer squamous cell carcinoma, adrenocortical carcinoma or prostate cancer, and preferably liver cancer.

 In the present invention, the kit may comprise a nucleotide primer that specifically binds to a retinoic acid receptor response protein 2 coding sequence or a receptor thereof.

 Examples of the nucleotide primers include, for example, primers for retinoic acid receptor response protein 2 coding sequence SEQ ID NOS: 3 and 4, and primer sequences for receptor ChemR23 SEQ ID NOS: 5 and 6, and the like.

Alternatively, the kit may contain a reagent that specifically binds to retinoic acid receptor-responsive protein 2, such as a specific antibody to retinoic acid receptor-responsive protein 2, and the like. Kits of the invention may also include instructions for directing cancer diagnosis. DRAWINGS

Figure 1A shows the expression level of chemerin in monoclonalized MHCC97 cells with different metastatic potentials by real-time PCR, expressed as monoclonal cells (M, H, L) and original cell chemerin (P) expression, respectively. The ratio of the monoclonal cells of M, H and L in vitro decreased in vivo. Figure 1B shows that the expression of chemerin mRNA in 46 pairs of normal liver tissue and liver cancer tissue was detected by real-time PCR, expressed as Log ₂ (T/N) of liver cancer tissue (T) and corresponding normal liver tissue (N). .

 Figures 2A, 2B, 2C and 2D represent four cases of chemerin staining of tissue microarrays, respectively corresponding to scores 0, 1, 2, 3. 0 means no signal is detected, 1 means the signal is weak, 2 means the signal is obvious, strong, 3 means the signal is strong. Figure 2E shows that the results of tissue microarray were analyzed in combination with clinical pathology information, and chemerin expression was significantly positively correlated with patient survival (p** = 0.000333). Figure 2F shows that if "0, 1" is uniformly classified as "0", and 2, 3 is uniformly classified as "1", combined with clinical pathological information, the results of tissue microarray are reanalyzed, and the expression of chemerin is The positive correlation between patients' survival was more pronounced (p**=0.0000724).

 Figure 3 shows the effect of chemerin on the metastatic potential of hepatoma cells by the Boyden chamber assay. A, 7404 cells, 7404/v represents control cells, 7404/chemerin L, 7404/chemerin M and 7404/chemerin H represent three monoclonal clones with increasing expression of chemerin, respectively; B, infected with control virus and chemerin, respectively A pool of PVTT-1 and C, MHCC97H cells expressing the virus, wherein PVTT-1 con and MHCC97H con are control cells, PVTT-1 chemerin and MHCC97H chemerin are cells with high expression of chemerin; D, SK-Hep-1/v For control cells, SK-Hep-1/chemerin L and SK-Hep-1/chemerin H are two positive clones with different expression levels of chemerin; E, 7404/chemerin H icon is a cell infected with control virus, and 7404/ Chemerin H il and 7404/chemerin H i2 are infected with cells containing different RNAi target sequences; F, down-regulated expression of chemerin in HepG2 cells by RNAi, wherein HepG2 chemerin icon is a cell infected with control virus, HepG2 chemerin il HepG2 chemerin i2, HepG2 chemerin i3 represent cells infected with a virus containing three different RNAi target sequences, respectively.

Figure 4A shows the results of Western Blot, which shows at 7404 (left) and PVTT-1 (middle) The phosphorylation level of Akt in the liver cancer cells, control cells and cells expressing hyperchemin ( Ser473 ), and right shows the change in Akt phosphorylation level in HepG2 cells knockdown of chemerin expression by RNAi. Figure 4B shows that 7404 cells were treated with 1 nM of active chemerin recombinant protein and the change in Akt phosphorylation levels was detected by Western Blot.

 Figure 5A shows the results of semi-quantitative PCR showing mRN A levels of VEGF, including VEGF, in control cells and cells expressing hyperchemin in 7404 (left), PVTT-1 (middle) and MHCC97H (right) hepatoma cells. Two isomers of 165 and VEGF 121. Figure 5B shows the results of Western Blot showing protein levels of VEGF in control cells and cells expressing hyperchemin in 7404 (left), PVTT-1 (middle) and MHCC97H (right) hepatoma cells, including near 55 kDa. Homodimers, and monomers near 17kDa. Figure 5C shows the results of the Tube formation assay using conditioned medium from 7404/v, 7404/chemerin, PVTT-1 con, PVTT-1 chemerin, MHCC97H con, MHCC97H chemerin cells, respectively. CM), the effect of CM from control and high expression of chemerin-expressing hepatoma cells on the angiogenesis ability of HUVEC (human umbilical vein endothelial cells) cells was examined by capillary formation assay.

 Figure 6 shows the effect of chemerin on the metastatic potential of hepatoma cells by the Boyden chamber. Among them: A, SK-Hep-1 con is the control cell, SK-Hep-1/chemerin L and SK-Hep-1/chemerin H are two positive clones with different expression levels of chemerin (see C); B, 7404/ Chemerin H icon is a cell infected with a control virus, and 7404/chemerin H il and 7404/chemerin H i2 are cells infected with different RNAi target sequences; C, SK-Hep- overexpressing chemerin is detected by Western Blot 1 In the cell clone, and in the 7402/chemerin H cell clone in which chemerin was knockdown, the expression of the labeled molecule was changed.

 Figure 7 shows the use of conditioned medium (CM from A, SK-Hep-1 con and SK-Hep-1/chemerin and B, 7404/chemerin H icon, 7404/chemerin H il , 7404/chemerin H i2 cells, respectively. The effect of CM from control and high expression of hepatin cells inhibited by chemerin/chemerin expression on the angiogenic ability of HUVEC cells in vitro was examined by a tube formation experiment.

Figure 8A shows that PVTT-1 con and PVTT-1 chemerin cells were injected into the left ventricle of 6-week-old nude mice, and the luciferase-labeled PVTT-1 cells were monitored by IVIS-imaging system. Transfer in the body. Figure 8A shows the day 0 (test immediately after injection), day 14 and day 28; Figure 8B shows the results of liver injection showing PVTT-1 con group and PVTT-1 from the front and back, respectively. A representative mouse liver in the chemerin group, the white solid arrow indicates the primary lesion formed at the injection site, and the white dotted arrow indicates the surface visible foci _; Figure 8B also shows the corresponding PVTT- HE staining of liver sections of 1 con group mice (top) and PVTT-1 chemerin group mice (bottom); Figure 8C shows injection leaf surface of mice in the PVTT-1 con group and the PVTT-1 chemerin group Counting statistics of visible cancer lesions (left) and metastatic cancer lesions visible on the surface of non-injected leaves (defined these lesions as intrahepatic metastases).

 Figure 9 shows the analysis of THP-1 cells that have been differentiated into macrophages and treated with conditioned medium of PVTT-l con and PVTT-1 chemerin cells by real-time PCR, including Ml Expression of the macrophage marker molecules TNF-alpha (A), IL-12a (B), IL-6 (C), and expression of the M2 type macrophage marker molecule CCL24 (D). Control is a negative control and LPS stimulation is a positive control (see "Materials and Methods").

 Figure 10 shows the chemerin protein purified by SP Sepharose FF using AKTA purifier and Q Sepharose FF (conductivity curve reflects the change of ion concentration in the solution); B, using the purified protein for long-term treatment, and detecting Akt by Western Blot Changes in phosphorylation levels.

 Figure 11 shows the use of intrahepatic injection of fluorescently labeled PVTT-1 cells in situ for tumorigenesis and treatment with chemerin to explore the therapeutic potential of chemerin for liver cancer. A, survival curves of PBS control group and chemerin treatment group (n=10, data cut-off treatment week 6th, dotted line represents PBS control group, solid line represents chemerin treatment group); B, average of PBS control group and chemerin treatment group The body weight curve was measured from the first day (the first day of intraperitoneal injection of protein as the first day), and was weighed every four days until the 33rd day. The result was an average number of standard errors (n=10), solid. The frame lines represent the PBS control group and the open frame lines represent the chemerin treatment group.

Figure 12 shows the effect of extracellular active form of chemerin on the migration of hepatoma cells by Boyden chamber assay. A: Pretreatment (740 h) of 1040 M active forms of recombinant chemerin was performed on 7404 and PVTT-1 cells, respectively. During the experiment (7404: 8 h, PVTT-1: 6 h), control wells were taken every 2 h. Add lul PBS, add luL 100x chemerin to chemerin-treated wells to maintain the persistence of chemerin; B: use lug/ml and 10ug/ml chemerin antibodies for 7404/chemerin H cells (R&D, Cat.No: AF2324, formulated in PBS) Pretreatment (0.5 h) was performed followed by a Boyden chamber test in which the same concentration of normal goat IgG was used as a control (anti-chemerin antibody was of sheep origin).

 Figure 13 shows the transfer of empty vector and CA-Akt expressing Akt in a sustained activation form in 7404/chemerin H, respectively, followed by A: and 7404 con cells for Boyden chamber assay; B: Western blot for CA-Akt Transfer to identify. HA indicates the expression of exogenous CA-Akt, GSK-3beta is a downstream target of Akt, and elevated levels of p-GSK-3beta (Ser9) indicate activation of the Akt downstream pathway.

 Figure 14 shows the effect of chemerin on the transcriptional activity of the VEGF promoter by a dual luciferase reporter assay using a luciferase reporter vector containing the human VEGF promoter. A, B, and C are the results in three different liver cancer cell lines 7721, Huh7, and 7404, respectively.

 Figure 15 shows the effect of chemerin-9 and chemerin on the migration of 7404 cells by Boyden chamber analysis. Cells were pretreated with 0.5 nM chemerin-9 and recombinant chemerin in active form (0.5 h). During the assay (8 h), lul PBS was added to control wells every 2 h, and wells treated with chemerin-9 and chemerin. Add lul 100x stock solution to maintain the persistence of chemerin-9 and chemerin; B, treat 7404 cells with 1 nM chemerin-9 and active form of recombinant chemerin, collect samples after 2h, and detect Akt by Western Blot The change in phosphorylation level (left), right is the quantitative scan of the luminance scan of the Western Blot results. detailed description

 The chemerin used in the present invention includes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 (wherein the 1st and 20th positions are signal peptides) and an active fragment which retains the biological activity of chemerin.

The chemerin of the present invention also includes a variant form of the sequence of SEQ ID NO: 2 having the same function as the chemerin protein. These variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, still more preferably 1 -8, 1-5) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually within 20, preferably within 10, more preferably The ground is less than 5 amino acids. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the egg. White matter function. The table below shows representative amino acid substitutions.

 Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to chemerin DNA under high or low stringency conditions. The invention also provides other polypeptides, such as fusion proteins comprising a chemerin protein or a fragment thereof.

It is well known in the art that homologous proteins between different species often have similar functions. The identity of homologous proteins of chemerin in different species is 30-90%. For example, the identity of mouse chemerin and human chemerin is 63%. Therefore, the present invention also encompasses human chemerin (SEQ ID NO: 2) A polypeptide having greater than 60% identity and retaining chemerin biological activity. Preferred are polypeptides having greater than 70%, 80%, 90%, 95% or 98% identity.

 Usually, chemerin is secreted out of the cell in a weakly active prochemerin. It is necessary to cleave several amino acids at the C-terminus into an active form and activate its receptor ChemR23 [also known as 3⁄4 CMKLRl (chemokine-like). Receptor) (Gene ID: 1240 ) ). There are a variety of enzymes (such as serine proteases, including elastase secreted by neutrophils and cathepsin G, tryptase secreted by mast cells, etc.) involved in the C-terminal amino acid cleavage, and chemerin activation, resulting in chemerin- 154, 155, 156, 157, 158 and other forms (corresponding to the 9, 8, 7, 6, 5 amino acids at the C-terminus) were cut off because the recognition and cleavage sequences of the various enzymes were different. These forms of chemerin can be detected from body fluids under physiological conditions. Thus, the present invention also encompasses truncated chemerin fragments of the C-terminus that are truncated by 1 to 10 amino acids, i.e., the active form of chemerin. These polypeptides include, but are not limited to, positions 1-157, 21-157, 21-158, 21-156, 21-155, 21-154, etc. of SEQ ID NO:2.

 In addition, chemerin-157 is the most active in the active chemerins (such as chemerin-154, 155, 156, 157, 158, etc.) which are currently known to be cleaved by different enzymes. Therefore, in order to obtain a more stable protein of the active form of chemerin-157 in vivo, the amino acids of several other cleavage sites other than Serl57 can be mutated, so that the enzymes in the body cannot recognize these sites and perform cleavage. This results in a protein that is more stable and retains chemerin-157 activity. These mutated polypeptides are also included in the scope of the present invention.

It is known in the art (Wittamer V et al., The C-terminal Nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency, J Biol. Chem. 2004, 12; 279(11): 9956-62), chemerin C The terminal fragment or variant thereof also has the biological activity of mature chemerin. Therefore, these C-terminal fragments are also included in the scope of the chemerin active fragment of the present invention. In particular, the C-terminal fragment of the chemerin of the invention is a C-terminal fragment of the chemerin active form. Further, the C-terminal fragment of the chemerin of the present invention comprises amino acid residues 150-157 of SEQ ID NO: 2 (FPGQFAFS, SEQ ID NO: 24), preferably YFPGQFAFS (SEQ ID NO: 28). Typically, the fragment is 8-20 amino acid residues in length, for example 9-19 amino acid residues in length. In a preferred embodiment, the fragment is FPGQFAFS. In other embodiments, the C-terminal fragment is located in positions 139-157 of SEQ ID NO: 2 (which may include positions 139 and/or 157), It also contains FPGQFAFS, preferably containing YFPGQFAFS.

 In a preferred embodiment, the C-terminal fragment of the chemerin of the invention includes, but is not limited to, QRAGEDPHSFYFPGQFAFS (SEQ ID NO: 2, positions 139-157, SEQ ID NO: 25), QRAGEDPHSFYFPGQFAF (SEQ ID NO: 2, positions 139-156, SEQ ID NO:26), FPGQFAFS (SEQ ID NO:2, positions 150-157, SEQ ID NO:27), FYFPGQFAFS (SEQ ID NO:2, positions 148-157, SEQ ID NO:28) HSFYFPGQFAFS (SEQ ID NO:28) N0:2, positions 146-157, SEQ ID NO: 29), PHSFYFPGQFAFS (SEQ ID NO: 2, positions 145-157, SEQ ID NO: 30), YFPGQFAFS (SEQ ID NO: 2, positions 149-157, SEQ ID N0:41) and so on.

 The invention also encompasses variant forms of the above C-terminal fragments. These variant forms have one or several amino acid insertions, deletions or substitutions compared to the C-terminal fragment, while still retaining the biological activity of the C-terminal fragment, particularly the treatment or prevention of cancer described herein (especially Liver cancer, etc., inhibiting the ability of cancer cells to metastasize, anti-tumor angiogenesis, inhibiting Akt signaling pathway and/or inhibiting the activity or function of VEGF mRNA and protein levels, or binding to Chemerin receptor (CMKLR1 (chemokine-like receptor), Gene ID: 1240) Activity or function. Variations of exemplary C-terminal fragments include, but are not limited to, Wittamer V et al. (supra) and various variants listed in US Pat. No. 7,419,658 B2, the entire disclosure of each of which is incorporated herein by reference. Not limited to AFPGQFAFS (SEQ ID N0.31), YAPGQFAFS (SEQ ID NO: 32), YFAGQFAFS (SEQ ID NO: 33), YFPAQFAFS (SEQ ID NO: 34), YFPGAFAFS (SEQ ID NO: 35), YFPGQAAFS (SEQ ID NO: 36), YFPGQFAAS (SEQ ID NO: 37), YFPGQFAFA (SEQ ID NO: 38), YHSFSFPGQFAFS (SEQ ID NO: 39), YHSFFFPGQFAFS (SEQ ID NO: 40), and the like.

It is known in the art that the aromatic and hydrophobic residues in the N-terminus of YFPGQFAFS are essential for the receptor binding activity of YFPGQFAFS; and it has also been demonstrated in the art that the N-terminus of the YFPGQFAFS (including the N-terminal positions 1 and 2) is mutated. The resulting mutant retains receptor binding activity as an aromatic residue or a hydrophobic residue. Further, the C-terminus of YFPGQFAFS can be modified, for example, D is substituted for S, and amidation is performed. The product obtained by the modification still retains the biological activity of the C-terminal fragment. See, for example, US Pat. No. 7,666, 401 B2, incorporated herein by reference in its entirety herein in its entirety herein in its entirety herein in Accordingly, the invention also encompasses such modified C-terminal fragment variants including, but not limited to, LFPGQFAFS (SEQ ID NO: 42), IFPGQFAFS (SEQ ID NO: 43), FLPGQFAFS (SEQ ID NO: 44), YLPGQFAFA (SEQ ID NO: 45), YVPGQFAFS (SEQ ID NO: 46), and YFPGQFAFD-CONH ₂ (SEQ ID NO: 47).

 It will be understood that the invention also encompasses various well-known modifications in the art of chemerin, active fragments thereof (including active forms of chemerin, C-terminal fragments) or variants thereof. These modifications include, but are not limited to, hydrophobic modifications at the N-terminus, such as myristoylation, or stearic acid modification, or palmitic acid modification, or cholesterol modification; and/or C-terminal stabilization modifications, such as amidation or isoprene Alcoholization modification.

 The present invention also includes those polypeptides having a C-terminally active C-terminally active polypeptide of chemerin that binds to chemerin's receptor ChemR23.

 Furthermore, the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention may be naturally purified products, either chemically synthesized or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells). The polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.

 The technical solutions of the present invention can be implemented using various commercially available chemerin products.

 Herein, "retaining the biological activity or function of SEQ ID NO: 2 or chemerin" includes retention

The currently known biological activity or function of SEQ ID NO: 2 or chemerin also includes retention of the treatment or prevention of cancer (especially liver cancer, etc.) as described herein, inhibition of cancer cell metastasis, anti-tumor angiogenesis, and / or inhibit the activity or function of the Akt signaling pathway.

 The invention also provides a polynucleotide sequence encoding a chemerin of the invention or a conservative variant polypeptide thereof. SEQ ID NO: 1 is one of the polynucleotide sequences encoding SEQ ID NO: 2.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1 or may be a degenerate variant. As used herein, a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1.

A polynucleotide encoding a mature polypeptide of SEQ ID NO: 2 comprises: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and Non-coding sequence. The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.

 The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .

 The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 °C, etc.; or (3) at least 90% identity between the two sequences It is better to have a hybridization when it is more than 95%. Further, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.

 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is typically 15 to 30 nucleotides in length, typically 18 to 24 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate polynucleotides encoding chemerin proteins.

 The full length sequence of the chemerin protein nucleotide of the present invention or a fragment thereof can usually be obtained by a PCR amplification method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vector or chemerin protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques. The recombinant chemerin protein can be expressed or produced using the polynucleotide sequences of the present invention by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Methods well known to those skilled in the art can be used to construct expression vectors containing the chemerin protein encoding DNA sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

 Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.

 The present invention also encompasses these polynucleotide sequences or expression vectors in the preparation of a medicament for treating or preventing cancer, a medicament for preparing a cancer cell capable of inhibiting metastasis of a patient, a medicament for preparing an anti-tumor angiogenesis, and a kit for preparing a prognosis for cancer patients. Applications.

 In addition to the direct use of chemerin or its coding sequence or its expression vector for the above applications, an agonist of chemerin can also be used to carry out the technical solution of the present invention. As used herein, "agonist" refers to a substance that increases the expression or activity of chemerin. Examples of agonists include, but are not limited to: retinoids (Sunil Nagpal et. al. Tazarotene-induced Gene 2 (TIG2), a novel retinoid-responsive gene in skin. J Invest Dermatol, 1997, 109: 91-95); Interleukin-1 beta induces the novel adipokine chemerin in adipocytes in vitro, Requl Pept, 2009, 154(l-3): 102-6); insulin (can improve adipose tissue explants) Expression of chemerin) (Tan BK et. Al. Insulin and metformin regulate circulating and adipose tissue chemerin, Diabetes, 2009, 58(9): 1971-7); TNF-a (Song SH et. al., Cloning, expression analysis , and regulatory mechanisms of bovine chemerin and chemerin receptor, Domest Anim, Endocrinol, 2010, 39(2): 97-105).

 The present invention also encompasses chemerin agonists, including the agonists specifically listed above, for the preparation of a medicament for treating or preventing cancer, for preparing a medicament for inhibiting metastasis of a cancer cell of a patient, and for preparing a medicament for anti-tumor angiogenesis.

 The present invention also encompasses a test kit comprising an agent for detecting the expression level of chemerin in a sample. These reagents include, for example, primer sequences for chemerin (e.g., SEQ ID NOS: 3 and 4 specifically listed herein), primer sequences for ChemR23 (e.g., SEQ ID NOS: 5 and 6 specifically listed herein), and the like. Also included are specific antibodies to chemerin and the like.

Various formulations required to carry out the assay may also be included in the kit, including, for example, reagents required for PCR Wait. Instructions for directing the technician to perform the assay may also be included in the kit.

 The kit of the invention can be used to detect the expression level of chemerin in various samples. The sample specifically includes cancer tissues (including cancer cells). The expression level of chemerin obtained can be used as an independent risk factor for prognosis. Thus, the assay can also include detection of normal tissue or cell samples to assess the survival of the subject by comparing the expression level of chemerin in the cancerous tissue, cell sample, and its level of expression in normal tissue, cell samples. Therefore, the kit of the present invention may also include the content showing the chemerin expression level of normal tissues and cell samples.

 As used herein, the term "cancer" includes various cancers well known in the art, including, inter alia, various cancers that are mediated by or mediated by Akt activity, as well as various cancers that have a down-regulated chemerin level in cancerous tissues. The present inventors have found that inhibition of Akt activity is critical for chemerin to inhibit tumor cell migration. The PI3K/Akt signaling pathway is known to be involved in a variety of cellular functions, and abnormal activation of this pathway can be observed in at least 50% of cancers, including thyroid cancer, pancreatic cancer, breast cancer, head and neck squamous cell carcinoma, liver cancer. and many more. Therefore, these cancers are all within the scope of the "cancer" of the present invention. The present inventors have also found that the expression of chemerin is significantly reduced in liver cancer tissues. It is known in the art that the expression level of chemerin in cancerous tissues such as squamous cell carcinoma, adrenocortical carcinoma and prostate cancer is also significantly lower than that in normal tissues, suggesting that inhibition of chemerin activity may occur during the development of these cancers. To an important role, the administration of chemerin in these cancers to increase chemerin activity may be an effective means of treating these cancers. Therefore, these cancers are also included in the scope of the "cancer" of the present invention. Particularly preferably, the invention relates to the diagnosis (including prognosis), prevention and treatment of liver cancer.

 The development and progression of cancer involves many factors, such as genetic mutations, cell proliferation, cancer cell metastasis, tumor angiogenesis, and so on. Therefore, the "anticancer activity" herein may be an activity for inhibiting cancer for any one or several factors related to the development of cancer, including, but not limited to, inhibition of protooncogene expression and inhibition of cancer cell proliferation. It promotes apoptosis of cancer cells, inhibits metastasis of cancer cells, and inhibits tumor angiogenesis.

 Furthermore, the present invention also encompasses the use of retinoic acid receptor-responsive protein 2 or an agonist thereof for the preparation of a medicament for inhibiting the Akt signaling pathway.

The retinoic acid receptor-reactive protein 2 of the present invention or an agonist thereof, or a coding sequence thereof or an expression vector containing the coding sequence can be administered, for example, in the form of a pharmaceutical composition, in a manner well known in the art. Pharmaceutically acceptable carriers well known in the art may also be included in the pharmaceutical compositions. Active ingredient in a pharmaceutical composition The amount can also be determined by the skilled person according to conventional technical means.

 The invention also encompasses a method of inhibiting the Akt signaling pathway comprising administering to a subject, such as a mammal or a tissue thereof or a cell thereof, a retinoic acid receptor response protein 2 or an agonist thereof. Inhibition can be inhibition in vivo or in vitro. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.

 Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only. Materials and Methods:

 Experimental animal

 BALB/c-nu/nu male nude mice were provided by the Slack Laboratory Animal Center of Shanghai Chinese Academy of Sciences. Male nude mice were housed under standard conditions: SPF (Specific Pathogen Free Condition) conditions, constant temperature 25-27'C, constant humidity 45 -50%, placed in a laminar flow-free rack, and 5 nude mice were housed in each cage. The operation of the animal experiment strictly complied with the Shanghai Animal Science Research Institute's guidelines for the management and use of laboratory animals and was approved by the Animal Management and Use Committee. In the experiment, nude mice of 4 to 8 weeks old were mainly used and selected according to specific experimental requirements. Liver cancer tissue sample

 All normal liver tissue, primary and portal vein metastatic liver cancer samples were obtained from the Eastern Hepatobiliary Surgery Hospital. This work was approved by the Institute of Nutritional Sciences of the Chinese Academy of Sciences. Cell line

7404, 7721, Huh7 and THP-1 cells were purchased from the Chinese Academy of Sciences Cell Bank, SK-Hep-1 and HepG2 cells were purchased from ATCC, and HUVEC (human umbilical vein endothelial cells) cells were isolated according to a conventional method (Bruno Baudin et al, A protocol for isolation and culture of human umbilical vein endothelial cells, Nature Protocols 2, 481-485, 2007). MHCC97P, MHCC97L, MHCC97H, MHCC97M are monoclonal MHCC97 cells constructed by Professor Tang Yu and others from Zhongshan Hospital. MHCC97P is the original MHCC97 mother cell (Tian J, Tang ZY, Ye SL, Liu YK, Lin ZY, Chen J, Xue Q, New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer 81:814-821, 1999) MHCC97L, MHCC97H (Li Y, Tang ZY, Ye SL, Liu YK, Lin ZY, Chen J, Xue Q, Chen J, Gao DM, Bao WH. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol 7:630-636, 2001 MHCC97M (HCCLM3, Li Yan, Tang Yu, Ye Shenglong, Liu Yinkun, Chen Jie, Xue Qiong, Huang Xiaowu, Chen Jun, Bao Weihua, Yang Wei, Gao Dongmei, Chinese Journal of Medicine, May 10, 2002, Vol. 82, No. 9, 601- 605) Represented monoclonal MHCC97 cells with progressively elevated metastatic potential in vivo. PVTT-1 is the laboratory and laboratory of the Eastern Hepatobiliary Surgery Hospital. Professor jointly established a portal vein tumor thrombus from a patient cell lines (see patent applications received Pat invention "luciferase expression lentiviral vector system and its use", 201010555483.3). Reagents and equipment

 The instrument for in vivo imaging IVIS-Imaging System, as well as the substrate D-Luciferin are purchased from

Xenogen Biotechnology (Xenogen, Alameda, CA, USA). Matrigel was purchased from BD Bioscience chemerin and VEGF, and chemerin recombinant protein was purchased from R&D. The antibody to actin was purchased from Santa Cruz, P-Akt (Ser473) and Akt was purchased from Cell Signaling. Real-time PCR analysis

 Primers corresponding to the genes tested in the experiment were designed by PRIMER5 software.

 The primer sequence for chemerin is:

 F: 5, -GGTCCACTGCCCCATAGA-3, (SEQ ID NO: 3),

R: 5 '-CTTGG AG AAGGCG AACTGT-3 ' (SEQ ID NO: 4). The primer sequence for ChemR23 is -

F: 5 ' -GGTGGTCT AC AGC ATCG-3 ' ( SEQ ID NO: 5 ) ,

 R: 5'-TGGCGGCATAGGTGA-3' (SEQ ID NO: 6).

 Primer sequence of β-actin:

 F: 5,-GATCATTGCTCCTCCTGAGC-3, (SEQ ID NO: 14)

 R: 5,-ACTCCTGCTTGCTGATCCAC-3, (SEQ ID NO: 15)

 Labeling molecule primer sequence of Ml type macrophage:

 TNF-alpha:

 F: 5'-CCAGGCAGTCAGATCATCTTCTC-3' (SEQ ID NO: 16)

 R: 5'-AGCTGGTTATCTCTCAGCTCCAC-3' (SEQ ID NO: 17)

 IL-6:

 F: 5'-ATGTAGCCGCCCCACACAGA-3' (SEQ ID NO: 18)

 R: 5'-GCATCCATCTTTTTCAGCCATC-3' (SEQ ID NO: 19)

 IL-12a:

 F: 5,-GGCCCTGAATTTCAACAG-3, (SEQ ID NO: 20)

 R: 5'- AATAGTCACTGCCCGAAT-3' (SEQ ID NO: 21)

 Marker molecule primer sequence for M2 macrophage:

 CCL24:

 F: 5 ' -C ATC ATCCCT ACGGGCTCT-3 ' ( SEQ ID NO: 22)

 R: 5'- TGGCGTCCAGGTTCTTCAT-3' (SEQ ID NO: 23)

 The amplification reaction system is 20 LightCycler-DNA Master SYBR Green I mix (Roche Applied Science, Penzberg, Germany), containing 10 pmol of the bowel, 2 mmol / L MgC12, 200 mol / L deoxynucleotide triphosphate mixture , 0.5 units of Taq DNA polymerase, and universal buffer. All reactions were performed in triplicate in iCycler iQ system (Bio-Rad, Hercules, CA) and the thermal cycling conditions were set as follows: 95 ° C for 3 minutes; then 95 ° C for 20 seconds, 58 ° C for 20 seconds, 72 ° C for 20 seconds, cycle 40 times; finally 72 ° C extension for 10 minutes.

In each clinical sample or cultured cell sample, the relative mRNA levels of the detected genes based on β-actin were calculated as follows. Briefly, when a fixed number of cycles have passed, the amplified PCR product is detected and this cycle value is defined as Ct, which is used to describe the reaction. The target information in the unknown sample was quantified by measuring the Ct value, and the Ct value of β-actin was also used as an endogenous RNA reference. Each sample of the level of gene expression detected in an amount of β-actin based, normalized by the following equation - τ _Λ, Λ ι a (CtB-actin- Ct by "I gene sample)

Level detected gene / Level _fl _ _actin - T

The relative expression level of the detected gene in each pair of HCC samples is calculated by the detected gene tunw/detected gene ^ _mal when the detected gene is detected by the detected gene _n . „ ^, when the detected gene _tum . r<detected gene _n . TM _al , with - (detected gene _n . TM _al / detected gene _tum . Calculated. Paired t-test was used for statistical analysis. When P < 0.05, statistical results were considered significant. All data were analyzed. Use the SPSS for Windows program.

 We cloned human chemerin into pcDNA3.1/myc-His (-) A (Invitrogen) using the primer sequences:

 F: 5 ' - AtaG A ATTCC ACC Atgcgacggctgctgatc-3 ' ( SEQ ID NO: 7 ) ,

 R: 5 ' - ataCTCGAGGCTGCGGGGC AGGGC-3 ' (SEQ ID O: 8).

 When this plasmid was constructed, we subcloned the chemerin fragment carrying myc-His-Tag into the viral vector pSin4-EF2-IRES-Puro (purchased from Addgene) using the primer sequences:

F:5,- AtaGAATTCCACCAtgcgacggctgctgatc-3' (EcoRI) (SEQ ID NO: 9),

R: 5 '-ata ACT AGTTC AATGGTG ATGGTGATGATG-3 ' (Spel, His-Tag) (SEQ ID NO: 10).

 The construction of a viral vector expressing luciferase is described in the patent application "Lentiviral Vector Expression System of Luciferase and Its Use (201010555483.3)".

 The three target sequences for chemerin's small interfering RNA were designed by Ambion's online tool, respectively

Chemerin i 1: A AG AA ACCCGAGTGC AA AGTC ( SEQ ID NO: 1 1 ) chemerin i2: AAGTTCTGCGGGAGGCTGAGG ( SEQ ID NO: 12 ) chemerin i3: AAGCCAGCACTGAGATGCGTG ( SEQ ID NO: 13 ) Using Completely Disturbed Base Sequence, by PubMed Blast The sequence in which the G+C content is identical to the target sequence serves as a control sequence chemerin icon, which does not target any human gene coding sequence. FG 12 RNAi (Xin XF et al, Inhibiting HIV-1 infection in human T cells by lenti viral-mediated delivery of small interfering RNA against CCR5, Proc Natl Acad Sci USA., 2003, 100(1): 183-8) was used Small double-stranded RNA (small interfering RNA) is produced to inhibit chemerin expression in target cells. Construction of active form of chemerin recombinant protein expression plasmid

 The human chemerin gene (hchemerin, GeneBank Accession No. NM-002889.3) has a total length of 767 bases, and the coding sequence (CDS) is 492 bases. The first 60 bases encode a 20-aa length signal peptide, and the final 6-aa peptide needs to be excised from the immature protein to obtain the active form of the protein. Therefore, our purified chemerin recombinant protein corresponds to GLu21-Serl57, plus N-terminal Met, which is identical to the commercially available chemerin protein sequence of R&D. We used the pET-30a(+) vector (Novagen) and the primers were -

F: 5'- GGAATTCC GGAGCTCACGGAAGCCCAGCGCCG-3, (SEQ ID NO: 24, italicized Ndel restriction site)

 R: 5,- CCGCrCG^GTTAGGAGAAGGCGAACTGTCCAG-S'i SEQ ID NO:

25, italicized Xhol enzyme cleavage site)

 A 400-bp PCR product is obtained by heating, extension, annealing, etc., which is a gene sequence expressing active chemerin.

 The empty product of the product and the prokaryotic expression vector of pET30a was recovered by double digestion with Nde I and Xho I, respectively, and the target fragment and empty vector were recovered and ligated.

 The ligation product was transferred into DH5-alpha E. coli competent cells, and a single clone was picked up on a kanamycin-resistant plate and the plasmid was extracted, and the enzyme was identified by Nde l and Xho l, and the positive clone was sent to the company. Sequencing. Prokaryotic expression of rh-chemerin protein

The pET30a-chemerin plasmid was transformed into E. coli BL21 (DE3) competent cells, and the transformed monoclonal was picked and added to 5 mL of LB liquid medium containing kanamycin. The culture was shaken overnight at 37 ° C and 250 rpm, and the next day was 1:100. Inoculate in 400 mL of LB liquid medium containing kanamycin, shake culture at 37 ° C, 250 rpm until the OD ₆ Q _G value is 0.6~0.8, add IPTG to the final concentration of lmm/L, shake culture at 37 ° C, 250 rpm 4 After the hour, the bacterial solution was collected, centrifuged at 6000 rpm for 5 minutes at 4 ° C, and the supernatant was discarded, using l x PBS buffer (137 mM NaCl, 2.7 mM KC1, 4.3 mM Na ₂ HP0 ₄ , 1.4 mM KH ₂ P0 ₄ , pH 7.4). After resuspending, it was centrifuged at 6000 rpm for 5 minutes at 4 ° C, the supernatant was discarded, and the cells were weighed.

 Add 1 ml of EDTA in PBS buffer according to the weight of the cells, resuspend the cells, and ultrasonically precipitate the resuspension on ice (ultrasound for 3 seconds, interval 10 seconds, ultrasonic power 100 watts, continuous ultrasound for 30 minutes), centrifuge at 12000 rpm. After 15 minutes, the supernatant was discarded, and the precipitate was weighed. Add 1 mL/O. lg to denaturing buffer (6M guanidine hydrochloride, 1 mM EDTA, 0 nM NaCl, 50 mM Tris-HCl; pH 8.0), fully vortexed and dissolved in a dialysis bag. Dialysis was carried out in 3 steps at 4 ° C. The dialysate was followed by the addition of a refolding buffer (ImM GSH, O. lmM GSSG, 0.4 M sucrose, 0.1 M Tris) containing different concentrations of guanidine hydrochloride (4M, 2M, 0.5M). - HCl; pH 9.5), each dialysis time was 12 hours.

 The dialysis protein solution was diluted and dissolved in 10 volumes of dilution buffer (InM GSH, 0.1 mM GSSG, 0.1 M Tris-HCl; pH 9.5), adjusted to pH 7.5 with 1 M HCl, and centrifuged at 12,000 rpm at 4 ° C. In minutes, the supernatant was taken for subsequent purification. Purification of rh-chemerin protein

The rh-chemerin protein was purified by anion exchange column (Q Sepharose FF, GE) and a cation exchange column (SP Sepharose FF, GE). The protein purification instrument used was AKTA purifier 100 (GE Healthcare Life Science). ). The anion exchange column and the cation exchange column were first washed with Wash 1 (20 nM Tris-HCl, 1 mM EDTA, 25 mM NaCl; pH 7.5) and Wash Buffer 2 (50 nM NaAc-HAc, 1 mM EDTA, 25 mM NaCl; pH 4.5, respectively). After pre-equilibration, the centrifuged supernatant was passed through an anion exchange column at a rate of 2 mL/min to remove impurity proteins, and the collected effluent was adjusted to pH 4.5 with glacial acetic acid, and centrifuged at 12,000 rpm for 30 minutes at 4 ° C to remove the precipitate. Then, the rh-chemerin protein was adsorbed onto the cation exchange column at a flow rate of 2 mL of Anin, and finally the rh-chemerin protein was eluted with a gradient of 0.025 M to 1.0 M NaCl in an elution buffer, and the UV absorption peak was determined. A protein eluate containing rh-chemerin. The protein solution in the collection tube was neutralized with 1 M Tris-HCl (pH 8.5), the pH was adjusted to 7.4, and then the protein buffer was changed to PBS by ultrafiltration at 4 ° C, and then concentrated and quantified. The purified rh-chemerin protein was subjected to SDS-PAGE electrophoresis, stained with Coomassie blue, and confirmed by Western Blot, and its activity was identified by cell processing assay and Boyden chamber assay. Establishment of cell monoclonal and cell pool

 pcDNA3.1-chemerin and empty vector pcDNA3.1 were transfected into 7404 and SK-Hep-1 cells, respectively, using Lipofectamine 2000 transfection reagent. Transfected cells were screened by G418, and resistant clones were further identified by Western blotting to obtain monoclonal clones with different expression levels.

 The viral expression vector and packaging plasmid were co-transfected into 293T cells for packaging. The packaged virus was collected according to a conventional method, and the target cells were infected, and luciferase-labeled PVTT-l luci cells (cell pool obtained after sorting), PVTT-1 chemerin with high expression of chemerin, and control PVTT-1 were obtained, respectively. Con cell pool, high expression of chemerin MHCC97H chemerin and control MHCC97H con cell pool. In vitro cell migration assay

Cell migration ability was tested using the improved Boyden Chamber method. 8-um pore size polycarbonate film (Neuro Probe) coated with collagen (50mg/ml in 0.02N acetic acid) at 4°C overnight, the appropriate number of cells (5.0χ 10 ⁴ ~1.0χ 10 ⁵ , depending on cell type Depending on whether it is suspended in lOOul medium containing 1% FBS or serum-free medium, inoculated into the upper well of a 12-well Transwell (Neuro Probe), and a medium containing 10% FBS is added to the lower well to provide a serum gradient, 37 Incubate at °C for 6 to 10 hours (depending on the cell type), remove the cells remaining on the front side of the film with absorbent paper, fix the stain with eosin, and take a photo under an upright microscope. In vivo tumor metastasis experiment

Luciferase-labeled PVTT-1 cells were used. 5 X 10 ⁵ cancer cells were injected into the left ventricle of six-week-old nude mice (8 in each group, and empty vector control cells and cells overexpressing chemerin were injected separately). After the experiment, the success of the injection was verified by the IVIS-Imaging System. The luciferase substrate fluorescein was then injected weekly and the cell transfer was detected by the IVIS-Imaging System. Tube Formation Assay

On the day before the experiment, Matrigel was thawed overnight at 4 °C. On the next day, 45 ul of Matrigel was pipetted from the frozen tip, placed in a 96-well plate, and allowed to stand at 37 ° C for more than 30 minutes to allow it to solidify. digestion HUVEC cells were counted and resuspended in conditioned medium at a density of 1.2 x lO ⁵ cells/ml. After mixing, 100 ul of cell suspension was added to a 96-well plate that had been placed in Matrigel and placed in a cell culture incubator. Inside, 37 ° C, 5% CO _{2 2} was cultured and observed continuously. Generally, after 6 to 8 hours, obvious tube formation was observed, and photographing was performed with a microscope. Induction and differentiation of THP-1 cells

THP-1 cells according to the density of 1.2xl0 ⁶ cells / well were plated in 6-well plate. The negative control group was differentiated with ΙΟΟηΜ phorbol-12-myristate-13-acetate (PMA, purchased from cell signaling) (ΙΟΟηΜ) for three days, then serum-free RPMI 1640 medium (purchased from Gibco) was cultured for two days; the positive control group was differentiated with PMA (ΙΟΟηΜ) for three days, and then cultured with RPMI 1640 serum-free containing lipopolysaccharide (LPS, purchased from Sigma) (100 ng/ml). Base culture for one day; control conditioned medium (CM) group was differentiated with PMA (ΙΟΟηΜ) for two days, and then differentiated with PT plus PVA (ΙΟΟηΜ) of PVTT-1 con cells for one day, then PMA was removed, with PVTT-1 con The cells were incubated with CM for two days; the chemerin CM group was differentiated with PMA (ΙΟΟηΜ) for two days, and then differentiated with PM plus PMA (ΙΟΟηΜ) of PVTT-1 chemerin cells for one day, then PMA was removed, and PVTT-1 chemerin cells were used. The CM was incubated for two days. During the treatment, the medium is changed every day and the corresponding fresh medium is added. Intrahepatic injection of liver cancer cells

Nude mice were injected with 6% chloral hydrate (300 mg/kg) by intraperitoneal injection of nude mice 5-6 weeks old. Fix the supine position, cut the skin about 1 cm along the midline of the sternal at 2 cm at the lower edge of the sternum, separate and cut the peritoneum, expose the liver, gently press the abdomen, squeeze out the left lobe of the liver, and suspend the cell suspension with a syringe (5xl0 ⁵ The cells / 50 L) are slowly injected into the liver parenchyma under the liver capsule. The angle between the needle and the liver surface is as small as possible. The penetration depth is 2~3 mm. See the white tissue of the liver at the injection site, indicating that the injection is successful and the needle is static. After the haiji ij, then slowly pull out the needle and gently press the injection site with hemostatic cotton to prevent bleeding and cell overflow. The liver is returned to its natural position and the peritoneum and skin incision are sutured with absorbable surgical sutures. Mice that were intrahepatically injected with PVTT-l con and PVTT-1 chemerin cells were each in a group of 8 each. Using the liver injection model to study the therapeutic potential of chemerin As described above for the "intrahepatic injection of liver cancer cells" method, we injected luciferase-labeled PVTT-1 luci cells into the liver. Three days after surgery, the fluorescein-containing substrate fluorescein was injected and detected by a living body imager. The mice with the same fluorescent signal intensity in the liver were equally divided into two groups of 10 rats each. One group was the control group, intraperitoneal injection of lOOul PBS at a frequency of two days, and the other group was treated with chemerin. At the same frequency, the same volume (lOOul) chemerin was injected intraperitoneally according to the amount of 10 ug chemerin/mouse. During the treatment experiment, hepatic in situ tumor growth and distant metastasis were detected by fluorescent signals every week, and the body weight of the mice was weighed every 4 days, and the death time of the mice was recorded. The current data is due to be processed for the sixth week. Tissue chip

 The liver cancer tissue chipset consists of two independent tissue chips (the first chip and the second chip). The liver cancer tissue used in tissue microarray is derived from surgically resected tissue (from the Eastern Hepatobiliary Surgery Hospital). After confirming the normal and liver cancer tissue areas by pathological examination, the tissues of the corresponding parts were selected and the tissue array was constructed. The liver cancer tissue chipset used a total of 60 cases of normal tissue adjacent to the cancer, 372 cases of liver cancer tissue samples, and 33 cases of liver cancer portal vein tumor thrombus. The No. 1 chip contains 30 normal tissue samples, 186 liver cancer tissue samples, and 17 liver cancer portal vein thrombosis samples, totaling 233 sample points. Among them, the second chip contains 30 normal tissue samples, 186 liver cancer tissue samples, and liver cancer portal vein. There were 16 cases of cancer plugs, totaling 232 sample points. Western Blot

 The cultured cells were washed with PBS and lysed on ice for 30 minutes in RIPA buffer. The cell lysate was centrifuged at 10,000 g for 15 minutes, the supernatant was taken, the protein concentration was determined with Bradford reagent (Sigma), the gel was run using 10% or 12% SDS-PAGE, and the protein was transferred to the Immobilon membrane (Millipore, Bedford, MA). On, a specific antibody was used for immunoblotting. All immunoblots were developed using chemiluminescence (Pierce). Result

 1. Clinical significance of chemerinde expression

We performed a microarray on monoclonalized MHCC97 hepatoma cells with different metastatic potentials. Column analysis, in the expression of a significant correlation with its ability to metastasize, we selected chemerin with a negative correlation with the ability to metastasize to study. We first detected the expression of chemerin in monoclonalized MHCC97 cells by real-time PCR. The results were consistent with the results of microarray analysis. The expression level of chemerin decreased as the ability of cells to metastasize in vivo (Fig. 1A). Next, we examined the expression of chemerin in 46 pairs of matched clinical samples, and the results showed that chemerin expression was significantly reduced at the mRNA level in liver cancer tissues compared to matched normal liver tissues (Fig. 1B).

 In addition, we used tissue microstaining to detect changes in chemerin expression at the protein level. We scored the signal strength of chemerin in the tissue chip, which is divided into 4 levels, from low to high, 0, 1, 2, 3. As shown in Figures 2A and 2B, chemerin immunohistochemical staining signals were almost undetectable, or very weak, with scores of 0 and 1, respectively. In Figures 2C and 2D, chemerin showed strong or very significant high expression with scores of 2 and 3, respectively.

 We combined clinical pathology information and performed statistical analysis of chemerin expression. The results of statistical analysis showed that chemerin had a significant positive correlation with the survival of liver cancer patients when the score of tissue microarray was divided into 0, 1, 2, and 3 grades. The higher the expression of chemerin, the survival time of patients. The longer the chemerin is expressed, the shorter the patient's survival. When we classify 0, 1 as "0" and 2, 3 as "Γ, and then perform statistical analysis, the positive correlation between chemerin and liver cancer patients is more significant (p=0.0000724). [Two-level division] <p=0.000333 [four-level division]) Therefore, in the subsequent analysis of the correlation between chemerin expression and various pathological parameters, we use the "0, 1" two-level classification.

 The results of Univariate analysis revealed that chemerin was closely associated with pre-clinical liver cancer patients (p**=0.005). Multivariate analysis revealed that chemerin and patient age, tumor size were independent risk factors for the prognosis of patients with liver cancer (Table 1). Table 1

在我们所用的肝癌组织芯片中， 有 28对配对的原位肝癌和门静脉癌栓的 点 （dot) ， 对进行了免疫组化染色后的 chemerin信号强度的统计表明， 与相 对应的原位肝癌相比， 在大部分门静脉癌栓中， chemerin的表达都下调， 并且 具有显著的统计学差异（表 2 ) ， 提示 chemerin的下调可能是肝癌门静脉癌栓 生成和发展的必要因素。

In the liver cancer tissue chip we used, there were 28 pairs of matched orthotopic liver cancer and portal vein tumor plugs. The statistics of chemerin signal intensity after immunohistochemical staining showed that the corresponding orthotopic liver cancer In contrast, in most portal vein tumor thrombi, chemerin expression was down-regulated, and there was a statistically significant difference (Table 2), suggesting that down-regulation of chemerin may be a necessary factor for the development and progression of portal vein tumor thrombosis in liver cancer.

 Table 2: Analysis of chemerin signal intensity in 28 pairs of matched orthotopic liver cancer and portal vein tumors in hepatocellular carcinoma tissue microarrays

 P*=0.014, "0" and "1" are 0 and 1 in the aforementioned "two-level division".

2. chemerin inhibits the migration ability of liver cancer cells in vitro We examined the expression of chemerin in several hepatocellular carcinoma cell lines and found that in addition to the relatively low level of hematin-producing HepG2 expression, the expression level of chemerin was very low in many liver cancer cells, and many of them were difficult to detect. Therefore, we introduced chemerin into cells with very low background expression levels of 7404, PVTT-1 and the like. Stable expression strains with different expression levels of chemerin were obtained (7404/v, 7404/chemerin L, 7404/chemerin M, 7404/chemerin H; SK-Hep-l/v, SK-Hep-l/chemerin L, SK After -Hep-l/chemerin H), or cell pool (PVTT-1 con, PVTT-1 chemerin; MHCC97H con, HCC97H chemerin), we performed multiple tests. There was no difference in proliferation and apoptosis between hepatoma cells with high expression of chemerin compared with control cells. However, the results of Boyden chamber assay showed that overexpression of chemerin significantly inhibited the migration ability of hepatoma cells in vitro (Fig. 3A-3D, Figure 6A). When we down-regulated chemerin expression in 7404/chemerin H and HepG2 cells by RNAi, the in vitro migration ability of the cells was enhanced (Fig. 3E-3F, Fig. 6B), suggesting a close relationship between chemerin and liver cancer cell migration ability. We treated the liver cancer cells with the active form of the chemerin recombinant protein, and the results showed that the active form of the recombinant protein can significantly inhibit the migration ability of the cells in vitro. When we used chemerin antibodies to treat hepatoma cells overexpressing chemerin, they significantly enhanced the in vitro migration ability of cells (Figure 12). These results indicate that chemerin inhibits the migration of liver cancer cells by the active chemerin protein secreted outside the cell.

3. chemerin inhibits the migration of hepatoma cells by negatively regulating the activity of Akt

In order to investigate the mechanism by which chemerin affects the migration ability of hepatoma cells, we examined the relevant signaling pathways and found that the phosphorylation level of Akt was significantly decreased in hepatoma cells with high expression of chemerin (Fig. 4A, Fig. 6C). In HepG2 cells that knocked down chemerin by RNAi, the phosphorylation level of Akt increased significantly (Fig. 4A). Akt is an important molecule involved in the regulation of cell migration. This result suggests that the inhibitory effect of chemerin on cell migration is likely to be achieved by down-regulating the level of Akt activity. Previous studies have shown that chemerin binds to the receptor and activates downstream Akt, causing an increase in Akt phosphorylation, contrary to what we have observed. To explain this difference, we used commercial, active chemerin recombinant proteins for short- and long-term treatment of liver cancer cells. We found that short-term treatment (15 minutes) did increase the phosphorylation level of Akt, but if long-term treatment was performed, the phosphorylation level of Akt was It started to drop from 30 minutes, and fell to the lowest level in about 2 hours. After 4 hours, it returned (because the chemerin added to the medium has been metabolized at this time, if the fresh chemerin is continuously added, the phosphorylation of Akt can still be maintained. Inhibition effect) (Fig. 4B). Therefore, our work reveals that long-lasting chemerin stimulation inhibits the activity of Akt in liver cancer cells, which is consistent with its inhibition of liver cancer cell metastatic ability.

 To further clarify the role of Akt in the inhibition of hepatocarcinoma cell migration by chemerin, we transferred a plasmid expressing a continuously activated Akt (CA-Akt) into a liver cancer cell overexpressing chemerin. As shown in Figure 13, sustained activation of Akt can effectively reverse the inhibition of chemerin on the migration of hepatoma cells, combined with previous experiments: the phosphorylation level of Akt is decreased in hepatoma cells overexpressing exogenous chemerin; Chemerin treatment of liver cancer cells for a long time also causes a decrease in Akt phosphorylation. We conclude that chemerin inhibits the migration of hepatoma cells by negatively regulating the activity of Akt.

 In addition, we knocked chemerin into 7404 cell clone 7404/chemerin H with high expression of chemerin by RNAi, and screened for control clones by FACS: 7404/chemerin H icon and expression levels of two chemerins were inhibited to varying degrees Clone: 7404/chemerin H il and 7404/chemerin H i2. The results of the Boyden chamber assay showed that when the expression level of chemerin was inhibited in clones overexpressing chemerin, the in vitro migration ability of the cells was restored, and the degree of recovery was inhibited in a dose-dependent manner with the inhibition of chemerin expression (Fig. 6). At the same time, the level of Akt phosphorylation has also rebounded, consistent with the trend of in vitro migration capacity recovery. This result demonstrates that chemerin does affect the ability of hepatoma cells to migrate in vitro by regulating the phosphorylation level and activation of Akt.

4. chemerin down-regulates VEGF mRNA and protein levels by inhibiting the transcriptional activity of the VEGF promoter region

The transfer of tumor cells in the body is affected by a variety of factors. In addition to the migration ability of tumor cells themselves, the influence of tumor cells on angiogenesis is also a key factor that determines the formation and development of metastases. VEGF is a key molecule regulating angiogenesis. Therefore, we examined the effect of chemerin on the expression of VEGF in hepatoma cells. We found that high expression of chemerin significantly inhibited the mRNA and protein levels of VEGF (Fig. 5A-5B, Fig. 6C). Moreover, the tubule formation experiment The results showed that conditioned medium of hepatin cells highly expressing chemerin had a significant inhibitory effect on the in vitro angiogenic ability of HUVEC cells compared to control cells (Fig. 5C, Fig. 7A). When we knocked out the exogenous chemerin in 7404/chemerin H, the conditioned medium of 7404/chemerin H il and 7404/chemerin H i2 significantly increased the angiogenic ability of HUVEC compared with the conditioned medium of the control cells. (Fig. 7B), and the VEGF protein level of the chemerin RNAi clone was also increased (Fig. 6C). These results suggest that chemerin not only directly inhibits the metastatic ability of hepatoma cells, but also the presence of chemerin, which also inhibits the development of liver cancer and angiogenesis during metastasis, which is consistent with the down-regulation of chemerin in hepatoma cells. Further supports and demonstrates the role of chemerin as a tumor suppressor molecule in liver cancer.

 To further explore the relevant mechanisms, we used a luciferase reporter vector containing the VEGF promoter region for detection. As shown in Figure 14, chemerin significantly down-regulated the luciferase activity of the VEGF promoter in three different liver cancer cells, suggesting that chemerin down-regulates its mRNA level by inhibiting the transcriptional activity of the VEGF promoter. Consistent with the above findings.

5. chemerin inhibits the distant metastasis and intrahepatic metastasis of hepatoma cells in vivo

 We used a left ventricular injection model to examine the effect of chemerin on the ability of distant metastasis of liver cancer cells. The results of the experiment showed that overexpression of chemerin resulted in a decrease in the ability of liver cancer cells to metastasize in vivo (Fig. 8A).

 As a result of liver cancer tissue microarray, there is a significant correlation between chemerin and the development and progression of portal vein tumor thrombus. Therefore, we used an intrahepatic injection model that favors intrahepatic metastasis to study chemerin versus liver. The effect of internal transfer.

We injected the same amount of luciferase-labeled PVTT-1 con and PVTT-1 chemerin (5x10 ⁵ cells) into the left hepatic lobe of nude mice, and then injected the substrate weekly to detect the fluorescence signal. After 8 weeks of surgery, the two groups of experimental mice were sacrificed, dissected, and the white foci on the liver surface were counted (into the injected leaves and non-injected leaves/transfer leaves), and then the liver was fixed and covered with paraffin. Buried, sectioned, and subjected to HE staining. As shown in Fig. 8C, the number of lesions on the surface of the liver showed that in the PVTT-1 con group, both the injected leaf and the non-injected leaf/transferred leaf, the number of cancers far exceeded the PVTT-1 chemerin group. Figure 8B is a photograph showing a representative, white surface of many cancerous lesions. The liver of the mouse in the PVTT-1 con group, and the white primary tumor in addition to the injected leaf, the liver of the mouse in the PVTT-1 chemerin group of the cancerous area was rarely seen in other parts of the surface. The results of HE staining of the corresponding sections of the liver showed that there were many pink lesions in the liver of the PVTT-1 con group, and the surrounding tumors showed different morphology of the normal hepatocytes, which was a white cancerous foci. The staining results on the slices are reflected. In the liver of the PVTT-1 chemerin group, uniform normal hepatic parenchymal cells were present, and no tumor formation was observed, which was consistent with the almost normal liver surface condition we observed (Fig. 8B). These data indicate that chemerin effectively inhibits intrahepatic metastasis of liver cancer cells. 6. Chemerin induces macrophage polarization to Ml type in liver cancer microenvironment

 Chemerin was discovered and reported as a chemokine, and chemerin's recruitment of macrophages was revealed at the beginning of its research. Our study explored the effects of chemerin on macrophage subtypes. We treated THP-1 cells that had been induced to differentiate into macrophages using control cells and conditioned medium of hepatoma cells with high expression of chemerin, respectively. By real-time PCR, we examined the expression of the marker molecules TNF-alpha, IL-6, IL-12a of Ml-type macrophages and the expression of the marker molecule CCL24 of M2-type macrophages. As shown in Figure 9, conditioned medium of hepatoma cells overexpressing chemerin significantly promoted the polarization of Ml-type macrophages compared to conditioned medium treatment of control cells. At the same time, it inhibits the polarization of M2 macrophages. Because Ml type macrophages have the effect of tumor killing, M2 type has the effect of promoting tumor development. Therefore, this result suggests that chemerin can promote the polarization of tumor-killing M1 macrophages in the microenvironment by changing the secretion profile of hepatoma cells, thereby promoting the formation of a tumor suppressor microenvironment, which is the tumor suppressor of chemerin in liver cancer. The role of the role in the tumor microenvironment.

7. Explore the possibility of applying chemerin to liver cancer treatment by using the purified chemerin protein obtained from the purified form and a mouse model injected intrahepatically.

We draw on the method of Xiang D et al. (Expressions and purification of a mature form of recombinant human chemerin in Escherichia coli. Protein Expr. Purif. 2010; 69(2): 153-8) and according to its own experimental conditions. Improved and optimized (see the Materials and Methods section above) to obtain a biologically active mature chemerin recombinant protein (Fig. 10A). As shown in Fig. 10B, when the purified protein was subjected to long-term treatment, the phosphorylation level of Akt was inhibited for a long period of time.

 As shown in Figure 11, chemerin treatment significantly prolonged the survival time of liver-bearing mice compared with PBS control, and also effectively alleviated the weight loss caused by the development of liver cancer, which indicates that chemerin has been applied to liver cancer treatment. Great potential.

8. Comparison of chemerin and chemerin-9 on the inhibition of hepatoma cell metastasis and Akt phosphorylation

According to the literature, the 9-peptide of chemerin C-ter also binds and activates ChemR23. Therefore, we commissioned Shanghai according to the literature (Wittamer V et al., The C-terminal Nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency, J Biol. Chem. 2004, 12; 279(11): 9956-62). Aibo Biotechnology Co., Ltd. synthesized the C-terminal 9-peptide sequence of chemerin ( ¹⁴⁹ YFPGQFAFS ¹⁵⁷ , chemerin-9) and examined its effect on liver cell migration and Akt phosphorylation in vitro according to the aforementioned method. As shown in Figure 15, we found that the same concentration of chemerin-9 inhibited the migration of hepatoma cells and the phosphorylation of Akt to some extent compared to the active form of chemerin. While the invention has been described in terms of the embodiments of the present invention, it is understood that modifications and changes may be made without departing from the spirit and scope of the invention. within.

Claims

Claim Use of a polypeptide having anticancer activity for the preparation of a medicament for treating or preventing cancer, characterized in that the amino acid sequence of the polypeptide comprises:  (1) YFPGQF AFS; or  (2) Variants in YFPGQF AFS that have been substituted, deleted, added or chemically modified with one or several amino acid residues and retain the biological activity or function of YFPGQFAFS. The use according to claim 1, wherein the variant of YFPGQFAFS is: XJX2PGQFAFX9, wherein Χ, X ₂ is an aromatic residue or a hydrophobic residue, and X ₉ is a serine residue or C An aspartic acid residue (D-CONH ₂ ) in which a hydroxyl group in a terminal carboxyl group is substituted with an amino group. The use according to any one of claims 1 to 2, wherein the variant of YFPGQFAFS is selected from the group consisting of: AFPGQFAFS, YAPGQFAFS, YFAGQFAFS, YFPAQFAFS, YFPGAFAFS, YFPGQAAFS, YFPGQF AAS > YFPGQF AFA LFPGQFAFS > IFPGQFAFS , FLPGQFAFS, YLPGQFAFS, YVPGQFAFS, YFPGQF AFD-CONH ₂ , QRAGEDPHSFYFPGQFAF or FPGQFAFS.  The use according to any one of claims 1 to 3, wherein the polypeptide is selected from the group consisting of -(1) YFPGQFAFS, AFPGQFAFS, YAPGQFAFS, YFAGQFAFS, YFPAQFAFS, YFPGAFAFS, YFPGQAAFS YFPGQF AAS, YFPGQF AFA LFPGQFAFS, IFPGQFAFS FLPGQFAFS, YLPGQFAFS > YVPGQFAFS, YFPGQF AFD-CONH2 QRAGEDPHSFYFPGQFAF or FPGQFAFS;  (2) the amino acid sequence set forth in SEQ ID NO: 2, or the substitution, deletion, addition or chemical modification of one or several amino acid residues in SEQ ID NO: 2 and retaining the biological activity of SEQ ID NO: 2 or Functional amino acid sequence; and  (3) an amino acid sequence set forth in SEQ ID NO: 49, or a substitution, deletion, addition or chemical modification of one or several amino acid residues in SEQ ID NO: 49 and retaining the biological activity of SEQ ID NO: 49 or Functional amino acid sequence.  5. Use of a coding sequence for a polypeptide according to any one of claims 1 to 4 or a polynucleotide construct comprising the coding sequence for the preparation of a medicament for the treatment or prevention of cancer. The use according to any one of claims 1 to 5, wherein the polypeptide is used in a preparation WO 2012/152063

A drug that inhibits the ability of cancer cells to metastasize.  The use according to any one of claims 1 to 5, wherein the polypeptide is used for the preparation of a medicament for anti-tumor angiogenesis.  The use of the polypeptide of any one of claims 1 to 4, the coding sequence of the polypeptide or the polynucleotide construct comprising the coding sequence for the preparation of a medicament for inhibiting the Akt signaling pathway.  The use according to any one of claims 1 to 8, wherein the cancer is selected from the group consisting of thyroid cancer, pancreatic cancer, breast cancer, head and neck squamous cell carcinoma, liver cancer, and squamous cell carcinoma of the skin. , adrenal cortical cancer or prostate cancer.  The use according to any one of claims 1 to 8, wherein the cancer is liver cancer.  A diagnostic kit for cancer prognosis, the kit comprising an agent for detecting the level of retinoic acid receptor response protein 2 in a sample.  The diagnostic kit according to claim 1, wherein the kit comprises a nucleotide primer that specifically binds to a coding sequence of a retinoic acid receptor-reactive protein 2 coding sequence or a receptor thereof.  The diagnostic kit according to claim 1, wherein the kit comprises an antibody that specifically binds retinoic acid receptor-responsive protein 2 or a receptor thereof.  The diagnostic kit according to any one of claims 11 to 13, characterized in that the kit further comprises instructions for guiding diagnosis of cancer.  15. Use of an agent capable of detecting retinoic acid receptor response protein 2 in the preparation of a kit for the diagnosis of cancer prognosis.