CN111487408B - CALB2 and application and tool of reagent for quantitatively detecting CALB2 - Google Patents
CALB2 and application and tool of reagent for quantitatively detecting CALB2 Download PDFInfo
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Abstract
The invention relates to an application of CALB2 as a liver cancer metastasis detection molecule target spot. The invention discovers that the expression level of CALB2 in a high-metastasis hepatoma cell strain is up-regulated, and the quantitative detection of CALB2 can be used for detecting the metastasis of the hepatoma. The research result of the invention provides a theoretical basis for the clinician to formulate a personalized treatment scheme, and can provide a new drug target for the development of the anti-liver cancer metastasis drug.
Description
Technical Field
The present invention is in the field of molecular biology and liver cancer prevention and treatment, and more specifically, the present invention relates to the field of predicting liver cancer metastasis. The invention provides an application of CALB2 as a reagent for detecting liver cancer metastasis, and a reagent for predicting liver cancer metastasis can be designed by quantitatively detecting CALB2 molecular expression.
Background
The liver is the central and leading organ for metabolism, energy conversion and supply of human body. Cancer is the second leading cause of death in the population. The incidence rate and death rate of liver cancer are high, and the 5-year survival rate is only 5% -6%. At present, primary liver cancer has risen to be the second cancer killer in China. Because primary liver cancer does not have any symptoms in the early stage generally, once clinical manifestations appear, most of the cases enter the middle stage or the late stage, and the degree of malignancy is high, the prognosis is poor, and the invasion and metastasis are strong. Clinical data show that the high invasive metastatic capacity and postoperative high recurrence and metastasis rate of liver cancer are important factors affecting the long-term survival of liver cancer patients. Therefore, liver cancer metastasis is a problem that life sciences need to solve urgently.
Liver cancer metastasis is classified into distant metastasis and intrahepatic metastasis. The metastatic process generally involves the ability of tumor cells to gain invasion outward such that the cells escape the primary organ, invade surrounding tissues and penetrate into lymphatic and blood vessels; the water flows along with the circulation system and stays; tumor cells proliferate and form metastases. The change of chromosome level, the increase of oncogene expression, the inactivation of cancer suppressor gene, epithelial cell-mesenchymal transition, the synthesis of proteolytic enzyme between tumor cells, the adhesion of cells and basement membrane, the low immune recognition of immune system, and the change of some cell factors and receptors can promote the transfer of liver cancer. The increased cell migration and motility are key factors that cause tumors to invade surrounding tissues and to undergo distant metastasis. However, the molecular mechanism for regulating the movement invasion capacity of liver cancer cells is still unclear, and it is difficult to develop effective drugs for inhibiting liver cancer metastasis, so that the search and discovery of liver cancer metastasis-related proteins for metastasis and recurrence research and drug target design are of great significance for realizing the improvement of survival rate of liver cancer patients.
CALB2 is a calcium binding protein and is abundant in auditory neurons. CALB2 protein plays a role in a variety of cellular functions, including message targeting and intracellular calcium buffering, and is involved in calcium signaling pathways. Ca 2+ Is a messenger that coordinates the endoplasmic reticulum-mitochondria interactions and regulates apoptosis. Mitochondrial Ca 2+ Kinetics are also involved in the regulation of cellular energy metabolism and in processes such as cellular movement and neurotransmitter release. CALB2 is also a diagnostic Marker for mesothelioma (Doglioni C, et al. Calretinin: A Novel immunochemical Marker for medical Pathology. American Journal of scientific Pathology,1996,20 (9): 1037-1046 Chu A Y, et al, utility of D2-40, a Novel medical Marker, in the diagnosis of malignant tumor model Pathology,2005,18 (1): 105-110.). Studies have shown that there is no expression of CALB2 in normal colonic epithelial cells, but expression is found in primary colon cancer cells (Gotzos V, et al. Selective distribution of calretinin in acquired tissues of the human colon and acquired tissues, american Journal of scientific Pathology,1999,23 (6): 701-711zos V, et al, heterocyclic of expression of the calcium-binding protein in human collagen cancer cell lines, anticancer Research,1996,16 (6B): 3491-3498.). Following treatment of Colorectal Cancer cells with 5-FU, CALB2 may be involved in apoptosis induction through the Intrinsic mitochondrial pathway (Stevenson L, et al, calbindin 2 (CALB 2) regulations 5-fluorogenic sensing in the scientific Cancer by Modulating the Intrinsic apoptosis pathway Ploss One,2011,6 (5): 1575-1581.). Calb2 has also been reported to act as a factor in mesenchymal cells via PLC-Ca 2+ The PKC signaling Pathway is involved in the regulation of steroid hormone synthesis and may promote stromal cell proliferation by potentiating PI3K-AKT and ERK l/2 signals (Xu W, et al.Calretin ligands in Regulating Steroidogenesis by PLC-Ca2+ -PKC Pathway in Leydig cells. Scientific Reports,2018, 8.). However, there is no report on whether CALB2 can regulate the growth, migration and invasion of hepatoma cells and how to regulate them.
Disclosure of Invention
The invention provides an application of a product for detecting CALB2 gene expression in preparing a tool for diagnosing liver cancer metastasis.
Furthermore, the product for detecting CALB2 gene expression comprises a product for detecting CALB2 gene mRNA level and/or a product for detecting CALB2 protein level.
Further, the product for detecting CALB2 gene expression comprises: products of liver cancer metastasis are diagnosed by detecting the expression level of CALB2 gene and its expression products through RT-PCR, real-time quantitative PCR, immunodetection, in-situ hybridization or chip detection.
Furthermore, the product for diagnosing liver cancer metastasis by RT-PCR at least comprises a pair of primers for specifically amplifying CALB2 gene; the product for diagnosing liver cancer metastasis by using real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CALB2 genes; the product for diagnosing liver cancer metastasis by immunoassay comprises: an antibody that specifically binds to CALB2 protein; the product for diagnosing liver cancer metastasis by in situ hybridization comprises: a probe that hybridizes to a nucleic acid sequence of the CALB2 gene; the product for diagnosing liver cancer metastasis by using the chip comprises: protein chips and gene chips; the protein chip comprises an antibody specifically combined with CALB2 protein, and the gene chip comprises a probe hybridized with the nucleic acid sequence of CALB2 gene.
The product for diagnosing liver cancer metastasis by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CALB2 gene, which are shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention also provides a tool for diagnosing liver cancer metastasis, which comprises a reagent for detecting CALB2 gene expression; the reagent comprises a primer and/or a probe for detecting CALB2 gene mRNA and an antibody for detecting CALB2 protein.
The diagnosis of liver cancer metastasis according to the present invention includes determining whether the subject has developed liver cancer metastasis, is at risk of liver cancer metastasis, or has relapsed metastasis.
The invention has the following advantages:
because liver cancer metastasis has become the important factor that influences the prognosis of liver cancer patients at present, the invention discloses a specific molecular marker CALB2, and the quantitative detection of CALB2 expression can be used as an important molecular marker for predicting liver cancer metastasis.
Description of the drawings:
FIG. 1.CALB2 gene differential expression in liver cancer tissue and para-cancer tissue
Detailed Description
The embodiments of the invention will be described in detail below with reference to the drawings, but the invention can be implemented in many different ways as defined and covered by the claims. The experimental procedures in the present invention are generally carried out according to conventional experimental conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) can also be performed according to the experimental conditions in the examples, and the reagents can be used according to the instructions attached to the manufacturer.
Example 1 differential expression of CALBB 2 Gene in liver cancer tissue and paracancerous tissue
1, 50 cases of normal tissues with metastatic liver cancer tissues and paracancer (at least 5cm away from the cancer tissues, specifically, see sampling standards) and 50 cases of normal tissues without metastatic liver cancer tissues and paracancer tissues are collected. Sampling a tissue sample by a pathologist, wherein the sampling standard is (1) primary liver cancer, no other diseases exist, and the patient does not undergo radiotherapy and chemotherapy for cancer before operation; (2) The sample is diagnosed as liver cancer by a pathology department, and no tumor cells are detected in a normal tissue beside the cancer; (3) In order to avoid unnecessary cross contamination, two sets of equipment are adopted during sampling, firstly, a normal tissue sample beside cancer without obvious lesion is observed by naked eyes which is 5cm away from cancer tissues at least, then, a whole layer of liver cancer tissue sample is taken and labeled, then, the whole layer of liver cancer tissue sample is immediately subpackaged and put into an EP (EP) tube added with 1ml of RNAlater for overnight at 4 ℃, and then, the mixture is stored in a refrigerator at-80 ℃.
2, the RNA Extraction Kit TaKaRa MiniBEST Universal RNA Extraction Kit (Code No. 9767) from Takara MiniBEST, co., ltd was used to extract RNA from the tissue sample. The specific operation steps can be realized by referring to the specification.
3, measuring the concentration and purity of the extracted RNA
The concentration and purity of the extracted RNA were measured using an ultraviolet spectrophotometer NanoDrop 1000. OD260/OD280 (R) shows the pollution degree of protein and other organic matters in RNA, the R value of RNA with good quality is 1.8-2.0, and when R is less than 1.8, the pollution of the protein and other organic matters in solution is obvious; when R >2.2, it indicates that the RNA has been hydrolyzed to a single nucleotide. After recording the concentration and purity of the RNA assay, the RNA was stored at-80 ℃.
The measured concentration of the para-carcinoma tissue was 906.7 (ng/. Mu.l), A260/280 was 1.96, the measured concentration of the carcinoma tissue was 890.0 (ng/. Mu.l), and A260/280 was 1.97.
4, primer design Synthesis
The primer sequence for detecting CALB2 gene expression by qRT-PCR and the primer sequence for amplifying internal reference beta-actin by qRT-PCR are designed and synthesized by Taobao bioengineering (Dalian) Co.
Oligo name base sequence (5 'to 3' orientation)
β-actin-F ACCCCGTGCTGCTGACCGAG
β-actin-R TCCCGGCCAGCCAGGTCCA
CALB2-F GCAGAGCTGGCGCAGATC(SEQ ID NO.1)
CALB2-R GCTCATCGTACGGCCGGTTCG(SEQ ID NO.2)
5, reverse transcription reaction
Reverse transcription kit TaKaRa PrimeScript using quantitative RT-PCR purchased from Takara Bio-engineering (Dalian) Ltd TM RT Master Mix (Perfect Real Time) (Code No. RR036A). The specific operation steps can be realized by referring to the specification.
6，Real Time PCR
Adopts a kit TaKaRa TB Green purchased from Takara Bio-engineering (Dalian) Co Ltd TM Premix Ex Taq TM II (Tli RNaseH Plus) (Code No. RR820A), the operation method of CFX96Real-Time PCR Detection System was applied. The specific operation steps can be realized by referring to the specification.
The assay was repeated 3 times for each sample, averaged, and the data analyzed using CFX Manager software (BioRad). Statistical analysis adopts the sps 22.0 software to carry out T test to judge whether the expression of CALB2 in the sample of the liver cancer tissue and the tissue beside the cancer has statistical significance difference. P <0.05 is statistically different.
7, the results are shown in figure 1, in the group with liver cancer metastasis, the expression level of CALB2 gene mRNA is increased compared with that of the tissues beside the cancer, and the difference has statistical significance; in the non-metastatic group of liver cancer, compared with the tissues beside the cancer, the CALB2 gene mRNA expression level has no significant difference. There was no significant difference in CALB2 gene mRNA expression levels in the paracarcinoma tissues in both groups. The results show that CALB2 gene is involved in the metastasis of liver cancer tissue.
Example 2 Effect of CALBB 2 Gene expression on migration ability of liver cancer cells
1,siRNA design Synthesis
siRNA was designed and synthesized by jima pharmaceutical technologies ltd, shanghai according to the CALB2 gene sequence, and at the same time, negative control siRNA (siRNA NC) having no sequence homology with the CALB2 gene was provided.
siRNA NC (sequence 5'to 3')
Forward:UUCUCCGAACGUGUCACGUTT
Reverse:ACGUGACACGUUCGGAGAATT
siRNA CALB2 (sequence 5'to 3')
Forward:GGAAAUAUGGAAGCACUUUTT
Reverse:AAAGUGCUUCCAUAUUUCCTT
2, cell transfection and cell scratch test
And (3) spreading the HCCLM3 cells which grow well and are in the logarithmic phase to a six-hole plate, adopting liposome Lipofectamine2000 as a transfection reagent when the cell confluence rate reaches 50%, transfecting siRNA NC in a control group, transfecting CALB2 in an experimental group, and carrying out experimental operation according to the Lipofectamine2000 reagent instruction. Protein expression levels were determined using western blot (ref. Molecular cloning). CALB2 antibody was purchased from Sigma, USA under the designation HPA007305.Western blot results show that after the experimental group is transfected with siRNA CALB2, the expression level of CALB2 is reduced compared with that of the control group, which indicates that the transfection is successful.
Transfecting cells of a control group and cells of an experimental group according to the cell transfection method in the steps, and placing the cells in
5% by weight the culture was continued in the CO2 incubator. Scratching is carried out when the cell confluence rate reaches 90%, a 200-mu-L gun head is taken, a cross is scratched uniformly, the action is slow, the force is uniform, after scratching is finished, PBS (Gibco company, USA) is cleaned for 2 times, the liquid is changed into a serum-free DMEM medium (Gibco company, USA), the influence of cell proliferation on migration is eliminated, and the scar state is observed and photographed for 0h under an inverted microscope. 24h after scratching, PBS was washed once, the solution was changed to serum-free DMEM medium, and the healing state of the scar at the same position was recorded by photographing. Statistically analyzing the healing rate of the scratches of the two groups of cells.
The results showed that the healing rate of siRNA NC group was 31% +4% and that of siRNA CALB2 group was 9% +3%. Compared with a control group, after the expression of CALB2 is knocked down in HCCLM3 cells with high transfer capacity, the transfer capacity of the cells is reduced, which indicates that CALB2 has a promoting effect on the transfer of liver cancer cells. The difference was statistically significant (. P < 0.05).
Example 3 Effect of CALBB 2 Gene expression on the migration and invasion Capacity of hepatoma cells
1,siRNA design Synthesis
siRNA was designed and synthesized by jima pharmaceutical technologies ltd, shanghai according to the CALB2 gene sequence, and at the same time, negative control siRNA (siRNA NC) having no sequence homology with the CALB2 gene was provided.
siRNA NC (sequence 5'to 3')
Forward:UUCUCCGAACGUGUCACGUTT
Reverse:ACGUGACACGUUCGGAGAATT
siRNA CALB2 (sequence 5'to 3')
GGCUCUGGCAUGAUGUCAATT
UUGACAUCAUGCCAGAGCCTT
2, cell transfection
And (3) spreading the HCCLM3 cells which grow well and are in the logarithmic phase to a 10cm culture dish, adopting liposome Lipofectamine2000 as a transfection reagent when the cell confluence rate reaches 50%, transfecting siRNA NC in a control group, transfecting CALB2 in an experimental group, and carrying out experimental operation according to the Lipofectamine2000 reagent instruction.
Protein expression levels were determined using western blot (ref. Molecular cloning). CALB2 antibody was purchased from Sigma, USA under the designation HPA007305.Western blot results show that after the experimental group is transfected with siRNA CALB2, the expression level of CALB2 is reduced compared with that of the control group, which indicates that the transfection is successful.
3, transwell experiment
(1) Transwell migration experiment
Transfecting cells of a control group and an experimental group according to the cell transfection method in the step 2, and placing the cells in
5% by weight the culture was continued in the CO2 incubator. Starving for 24h when the cell confluence rate reaches 80%. Washing with PBS for 2 times, digesting with 500 μ L of 0.25% pancreatin for 2min, preparing into single cell suspension with serum-free DMEM medium, centrifuging for 3min at 500g, discarding the medium, blowing and beating with serum-free DMEM medium, mixing, counting, and adjusting cell concentration to 1.0 × 10 6 cell/ml. 250 μ L of cell suspension was added to the upper chamber of the Transwell. In the Transwell lower chamber was added 500. Mu.L of DMEM medium containing fetal bovine serum at a volume concentration of 10%. Placing the upper chamber into a lower chamber, avoiding air bubbles between the culture medium in the upper chamber and the lower chamber, into->
5% CO2 incubator for 48h. The chamber was removed, the liquid in the upper chamber was discarded, and the chamber was washed 2 times in the lower chamber containing PBS. 200 μ L of crystal violet (containing 10% methanol by volume) with a mass concentration of 0.1% was added to the lower chamber and stained for 20 minutes. Non-migrated cells on the upper chamber side were gently scraped off with a cotton swab and washed 3 times with PBS. After drying, the number of migrated cells was observed under an inverted microscope and recorded by photography. Each group was counted randomly for 5 fields and statistically analyzed. The cell migration number counted by the control group is 329+26, the cell migration number counted by the experimental group is 156+17, and the difference has statistical significance (. + > p)<0.05)。
(2) Transwell invasion test
Placing Transwell Matrigel gel at room temperature for 30min, adding 300 μ L serum-free DMEM medium preheated in advance to the upper chamber, and placing
5% CO2 incubator incubation for 1h. The medium was then removed to leave approximately 50. Mu.L. 250 μ L of the above cell suspension was added to the upper chamber of the Transwell. 500 μ L of complete medium containing 10% fetal bovine serum by volume was added to the lower chamber of the Transwell. Placing the upper chamber into the lower chamber, avoiding air bubbles between the culture medium in the upper chamber and the lower chamber, into the chamber>
5% by volume in CO2 incubator for 48h. The chamber was removed, the liquid in the upper chamber was discarded, and the chamber was washed 2 times in the lower chamber containing PBS. 200 μ L of crystal violet (containing 10% by volume methanol) with a mass concentration of 0.1% was added to the lower chamber and stained for 20 minutes. Non-migrated cells on the upper chamber side were gently scraped off with a cotton swab and washed 3 times with PBS. After drying, the number of migrated cells was observed under an inverted microscope and recorded by photography. Each group was counted randomly for 5 fields and statistically analyzed. The number of cell invasion counted by the control group is 108+16, the number of cell invasion counted by the experimental group is 36+8, and the difference has statistical significance ([ p ])<0.05)
The results of Transwell experiments show that, compared with a control group, after the expression of CALB2 is reduced in HCCLM3 cells with high transfer capacity, the migration and invasion capacities of the cells are reduced, and the promotion effect of CALB2 on the migration and invasion of liver cancer cells is prompted. The difference was statistically significant (. P < 0.05).
The above description is only a preferred embodiment of the present invention and is not intended to limit the practice of the present invention, and various modifications may be made by those skilled in the art. Any modification, improvement, combination, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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