CN105803053B

CN105803053B - Use of human RBM17 gene and related medicines

Info

Publication number: CN105803053B
Application number: CN201410852019.9A
Authority: CN
Inventors: 谭畅; 刘书童; 王庆亮; 杨敏; 徐述; 曹跃琼
Original assignee: Shanghai Genechem Technology Co ltd
Current assignee: Shanghai Ji Kai Gene Technology Co ltd
Priority date: 2014-12-31
Filing date: 2014-12-31
Publication date: 2021-03-16
Anticipated expiration: 2034-12-31
Also published as: CN105803053A

Abstract

The invention discloses the use of human RBM17 gene and related medicines. The invention discloses the application of human RBM17 gene in tumor treatment, tumor diagnosis and drug preparation. The present invention further constructs human RBM17 gene interfering RNA, human RBM17 gene interfering nucleic acid construct, and human RBM17 gene interfering lentivirus and discloses their uses. The siRNA or the nucleic acid construct comprising the siRNA sequence and the lentivirus provided by the present invention can specifically inhibit the expression of the human RBM17 gene, especially the lentivirus, which can efficiently infect target cells and efficiently inhibit the expression of the RBM17 gene in the target cells. , thereby inhibiting the growth of tumor cells and promoting tumor cell apoptosis, which is of great significance in tumor therapy.

Description

Application of human RBM17 gene and related medicine thereof

Technical Field

The invention relates to the technical field of biology, in particular to application of a human RBM17 gene and related medicines thereof.

Background

RNA interference (RNAi) is the post-transcriptional gene silencing by short double-stranded RNA (dsrna) composed of nucleotides. It can block the expression of specific gene in body efficiently and specially to result in its degradation, so that it can cause the silencing of specific gene in the body and make the cell show the deletion of some gene phenotype. Studies have shown that double-stranded RNA of 21-23nt in length is capable of specifically causing RNAi at the transcriptional and post-transcriptional levels (Tuschl T, Zamore PD, Sharp PA, Bartel DP. RNAi: double-stranded RNA direct the ATP-dependent cleavage of mRNA at 21to 23nucleotide intervals. cell 2000; 101: 25-33.). The five-year survival rate of tumor patients is very low although chemotherapy, radiotherapy and comprehensive treatment are carried out on the tumor patients, and if genes related to the tumor morbidity and progression can be intervened, a new way for treating tumors can be developed. In recent years, RNAi has become an effective strategy for gene therapy of tumors. RNAi technology can be used to inhibit the expression of proto-oncogenes, mutated anti-oncogenes, cell cycle-related genes, anti-apoptosis-related genes, etc., to inhibit tumor progression (Uplichard, Susan L. the therapeutic potential of RNA interference. FEBS Letters 2005; 579: 5996-.

In eukaryotic cells, the selective shearing of mRNA can regulate and control the multi-protein expression of a single gene, and the final composition of a protein product is influenced by splicing the exons in the transcribed mRNA precursor specifically, so that the diversity of functional proteins in the cells is ensured. Alternative splicing is involved in the regulation of 60-74% of human genome expression, and therefore the accuracy and effectiveness of mRNA splicing regulation is critical, and mRNA splicing defects are often associated with many human diseases (Modrek B, Resch A, Grasso C, Lee C.2001.genome-wide detection of alternative splicing in expressed sequences of human genes. nucleic Acids Res.29: 2850-2859). Alternative splicing is primarily regulated by the heterogeneous nuclear ribonucleoproteins (hnRNPs) and the Sr (serine-arginine-rich) family of splicing factor proteins, with the choice of splicing sites depending on the relative concentrations of these proteins and regulated by reversible phosphorylation (Hastings ML, Krainer AR.2001.functions of SR proteins in the U12-dependent AT-AC pre-mRNA splicing pathway. RNA 7: 471-482). RNA binding motif protein 17 (RBM), also known as alternative mRNA splicing factor 45 (SPF 45), encodes an RNA binding protein that is involved in the construction of the RNA spliceosome complex and that functions in the second catalytic step of mRNA splicing. RBM17 modulates alternative splicing of the precursor mRNA encoding the cell death receptor FAS, inducing exon 6 skipping comprising the transmembrane domain, which can produce soluble dominant negative FAS protein (Corsini L, et al 2007.U2AF-homology motif are required for alternative spaying regulation by SPF45.nat. Structure. mol. biol.14: 620) -629). RBM17 contains an unstructured N-terminal domain, a G-patch motif, RNA Recognition Motif (RRM) at the C-terminus that is involved in protein-protein and protein-nucleic acid interactions, and is essential for mRNA splicing (Lallena MJ, Chalmers KJ, Llamazares S, Lamond AI, Valcaccel J.2002.splicing regulation the second catalytic step by Sex-reactive tanks 3' splice site recognition by 45.cell 109: 285-. In humans, RBM17 is under-expressed in the ductal epithelium of normal breast, liver, prostate and pancreas, but is over-expressed in many human cancers, such as: bladder, breast, colon, lung, ovarian, pancreatic, and prostate cancer, among others; also, studies have shown that overexpression of RBM17 is associated with resistance in many cancers (Sampath J, Long PR, Shepard RL, et al. human SPF45, a fusing factor, has limited expression in normal tissues, is overexpressed in many tumors and can control multidrug resistance to cells. am J Pathol 2003; 163: 1781-90).

However, the current experimental report of the RBM17 gene in the tumor-related field is still a blank, especially in the research field of human glioma. The invention researches the role of RBM17 in the occurrence and development of glioma by taking RNAi as a means.

Disclosure of Invention

The invention aims to disclose a treatment method and a medicament related to a human RBM17 gene, and researches the action of the RBM17 gene in the survival and apoptosis process of tumor cells by taking RNA interference (RNAi) as a means.

In the first aspect of the invention, the function of RBM17 gene in tumorigenesis and development is researched by means of RNA interference, and a method for inhibiting or reducing the growth, proliferation, differentiation and/or survival of tumor cells is disclosed, which comprises the following steps: administering to the tumor cell a molecule capable of specifically inhibiting transcription or translation of the RBM17 gene, or capable of specifically inhibiting expression or activity of RBM17 protein, thereby inhibiting growth, proliferation, differentiation, and/or survival of the tumor cell.

The tumor cells are selected from tumor cells the growth of which is related to the expression or activity of RBM17 protein. Preferably, the tumor cell is selected from the group consisting of gliomas.

In the method of inhibiting or reducing tumor cell growth, proliferation, differentiation and/or survival, the molecule is administered in an amount sufficient to reduce transcription or translation of the RBM17 gene, or to reduce expression or activity of RBM17 protein. Further, the expression of the RBM17 gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

The molecule may be selected from, but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

Such nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (esiRNA) produced by endoribonuclease III, or short hairpin RNA (shRNA).

The double-stranded RNA, ribozyme, esiRNA or shRNA contains a promoter sequence of the RBM17 gene or an information sequence of the RBM17 gene.

Further, the double-stranded RNA is small interfering RNA (siRNA). The small interfering RNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is basically identical to 15-27 continuous nucleotide sequences in an RBM17 gene. The small interfering RNA can specifically bind to an mRNA fragment coded by a target sequence and specifically silence the expression of a human RBM17 gene.

Further, the first strand sequence of the small interfering RNA is substantially identical to the target sequence in the RBM17 gene. Preferably, the target sequence in the RBM17 gene comprises a sequence shown in SEQ ID NO. 1.

The target sequence in the RBM17 gene is a fragment in the RBM17 gene corresponding to an mRNA fragment complementarily combined with the small interfering RNA when the small interfering RNA specifically silences the expression of the RBM17 gene.

Preferably, the RBM17 gene is of human origin.

The invention also discloses the application of the separated human RBM17 gene in preparing or screening tumor treatment medicines or preparing tumor diagnosis medicines.

Further, the tumor is selected from glioma.

The application of the isolated RBM17 gene in preparing or screening tumor treatment medicines comprises two aspects: firstly, the RBM17 gene is used as a drug or preparation to be applied to the preparation of tumor treatment drugs or preparations aiming at the action target of tumor cells; secondly, the RBM17 gene is used as a drug or preparation to be applied to screening tumor treatment drugs or preparations aiming at the action target of tumor cells.

The application of the RBM17 gene as an action target of a medicine or a preparation for tumor cells in preparing a tumor treatment medicine or preparation specifically comprises the following steps: the RBM17 gene is used as a target of RNA interference effect to develop a medicament or a preparation aiming at tumor cells, so that the expression level of the RBM17 gene in the tumor cells can be reduced.

The application of the RBM17 gene as an action target of a medicine or a preparation for tumor cells in screening tumor treatment medicines or preparations specifically comprises the following steps: the RBM17 gene is used as an action object, and the drug or preparation is screened to find the drug which can inhibit or promote the expression of the human RBM17 gene and is used as a candidate drug for tumor treatment. The RBM17 gene small interfering RNA (siRNA) is obtained by screening a human RBM17 gene serving as an action object and can be used as a medicament with the effect of inhibiting the proliferation of tumor cells. In addition, the RBM17 gene and its protein can be used as targets of action, such as antibody drugs, small molecule drugs, etc.

The RBM17 gene is used for preparing tumor diagnosis medicines, and the RBM17 gene expression product is used as a tumor diagnosis index for preparing the tumor diagnosis medicines.

The expression level of the RBM17 gene in 4 glioma cells is detected by a Real-time Quantitative PCR method. The research finds that: the expression abundance of RBM17 in 4 glioma cells was high. The RBM17 is suggested to be possibly used as an oncogene and play an important role in the occurrence and development of tumors; the expression level of RBM17 gene can be used as a marker for tumor diagnosis.

The tumor treatment drug is a molecule which can specifically inhibit the transcription or translation of the RBM17 gene or specifically inhibit the expression or activity of the RBM17 protein, so that the expression level of the RBM17 gene in tumor cells is reduced, and the purposes of inhibiting the proliferation, growth, differentiation and/or survival of the tumor cells are achieved.

The tumor therapeutic drug or tumor diagnostic drug prepared or screened by the isolated RBM17 gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

The tumor treatment drug is administered in an amount sufficient to reduce transcription or translation of the human RBM17 gene, or to reduce expression or activity of the human RBM17 protein. Such that expression of the human RBM17 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

The method for treating the tumor by adopting the tumor treatment medicine mainly achieves the aim of treating by reducing the expression level of human RBM17 gene and inhibiting the proliferation of tumor cells. Specifically, in treatment, a substance effective in reducing the expression level of human RBM17 gene is administered to the patient.

In a second aspect, the present invention discloses an isolated nucleic acid molecule for reducing expression of RBM17 gene in a tumor cell, said nucleic acid molecule comprising:

a) double-stranded RNA containing a nucleotide sequence capable of hybridizing with the RBM17 gene under stringent conditions; or

b) shRNA which contains a nucleotide sequence capable of hybridizing with the RBM17 gene under stringent conditions.

Further, the double-stranded RNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is substantially identical to 15-27 consecutive nucleotide sequences in the RBM17 gene. Preferably, the sequence of the first strand is substantially identical to a 19-23 contiguous nucleotide sequence of the RBM17 gene; more preferably, the sequence of the first strand is substantially identical to a sequence of 19, 20 or 21 contiguous nucleotides of the RBM17 gene.

Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the RBM17 gene.

The length of the first strand and the second strand of the double-stranded RNA are both 15-27 nucleotides; preferably, the length is 19-23 nucleotides; most preferably, the length is 19, 20 or 21 nucleotides.

Further, the double-stranded RNA is small interfering RNA (siRNA). Further, the sequence of the first strand of the small interfering RNA is shown in SEQ ID NO: shown at 9, specifically 5'-AUACUUAAGUGUCCUACUAAA-3'.

SEQ ID NO: the siRNA shown in the 9 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at one strand of small interfering RNA of a human RBM17 gene, the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of an endogenous RBM17 gene in tumor cells.

Further, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is basically identical to 15-27 continuous nucleotide sequences in the RBM17 gene. The shRNA can become small interfering RNA (siRNA) after being processed, and further plays a role in specifically silencing the expression of an endogenous RBM17 gene in a tumor cell.

Further, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the RBM17 gene.

Preferably, the sense strand fragment is substantially identical to 19-23 contiguous nucleotide sequences in the RBM17 gene; more preferably, the sense strand segment is substantially identical to a 19, 20 or 21 contiguous nucleotide sequence of the RBM17 gene.

Further, the sequence of the stem-loop structure of the shRNA can be selected from any one of the following sequences: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC.

Furthermore, the sequence of the shRNA is shown as SEQ ID NO:14, and specifically comprises the following steps: 5'-AUACUUAAGUGUCCUACUAAAUUCAAGAGAUUUAGUAGGACACUUAAGUAU-3' are provided.

The shRNA can become siRNA after enzyme digestion processing, thereby playing a role in specifically silencing the expression of endogenous human RBM17 gene in tumor cells.

The interfering slow virus vector of the gene segment for encoding the shRNA contains a sequence shown in SEQ ID NO. 1 and a complementary sequence thereof.

The first strand of the double-stranded RNA or the sense strand segment of the shRNA is basically the same as a target sequence in an RBM17 gene, and the target sequence of the RBM17 gene is a segment in an RBM17 gene corresponding to an mRNA segment which is identified and silenced by the siRNA when the siRNA is used for specifically silencing the expression of the RBM17 gene.

Preferably, the target sequence in the RBM17 gene contains a sequence shown in SEQ ID NO. 1.

Further, the RBM17 gene is derived from human.

In a third aspect of the invention, an RBM17 gene interference nucleic acid construct is disclosed, which comprises a gene segment for encoding shRNA in the isolated nucleic acid molecule of the invention and can express the shRNA.

The human RBM17 gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human RBM17 gene shRNA into a known vector. Further, the RBM17 gene interference nucleic acid construct is an RBM17 gene interference lentiviral vector.

The RBM17 gene interference lentiviral vector is obtained by cloning a DNA fragment for coding the RBM17 gene shRNA into a known vector, the known vector is mostly a lentiviral vector, the RBM17 gene interference lentiviral vector is packaged into infectious viral particles by viruses, then the infectious viral particles infect tumor cells, the shRNA is transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the RBM17 gene.

Further, the RBM17 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence for coding a marker which can be detected in tumor cells; preferably, the detectable label is Green Fluorescent Protein (GFP).

Further, the lentiviral vector may be selected from the group consisting of: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.

The embodiment of the invention specifically discloses a human RBM17 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-RBM 17-siRNA.

The isolated nucleic acid molecule of the invention can be used for preparing a medicament for preventing or treating tumors, wherein the tumors are gliomas.

The RBM17 gene siRNA can be used for inhibiting the proliferation of tumor cells, and further can be used as a medicament or preparation for treating tumors. The RBM17 gene interference lentiviral vector can be used for preparing the RBM17 gene siRNA. When used as a medicament or formulation for treating tumors, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The fourth aspect of the invention discloses an RBM17 gene interference lentivirus, which is prepared by carrying out virus packaging on the RBM17 gene interference lentivirus vector under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect tumor cells and generate small interfering RNA aiming at RBM17 gene, thereby inhibiting the proliferation of glioma tumor cells. The RBM17 gene interference lentivirus can be used for preparing medicaments for preventing or treating tumors.

In the fifth aspect of the invention, a pharmaceutical composition for preventing or treating tumor is disclosed, the effective substance of which comprises one or more combinations of the isolated nucleic acid molecule, the RBM17 gene interfering nucleic acid construct or the RBM17 gene interfering slow virus.

Further, the pharmaceutical composition contains 1-99 wt% of the double-stranded RNA, shRNA, RBM17 gene interference nucleic acid construct or RBM17 gene interference lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

In preparing these compositions, the active ingredient is typically mixed with, or diluted with, excipients or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The preparation may further comprise a humectant, an emulsifier, a preservative (such as methyl and propyl hydroxybenzoate), a sweetener, etc.

The invention also discloses application of the pharmaceutical composition in preparing a medicament for treating glioma.

The application of the pharmaceutical composition provides a method for treating tumors, in particular to a method for preventing or treating tumors in a subject, which comprises the step of administering an effective dose of the pharmaceutical composition to the subject. Further, the tumor is selected from glioma.

When the pharmaceutical composition is used for preventing or treating tumors in a subject, an effective dose of the pharmaceutical composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited.

The subject of the method may be a human.

In a sixth aspect of the invention, a kit for reducing expression of RBM17 gene in tumor cells is disclosed, the kit comprising: the isolated nucleic acid molecule, RBM17 gene interfering nucleic acid construct, and/or the RBM17 gene interfering lentivirus present in the container.

In conclusion, the invention designs 1 RNAi target sequence aiming at the human RBM17 gene and constructs a corresponding RBM17RNAi vector, wherein the coding sequence is SEQ ID NO: the RNAi vector pGCSIL-GFP-RBM17-siRNA of 1 can obviously reduce the expression of RBM17 gene at mRNA level and protein level. Lentivirus (Lv) is used as a gene manipulation tool to carry an RNAi vector pGCSIL-GFP-RBM17-siRNA, so that the RNAi sequence aiming at the RBM17 gene can be efficiently introduced into glioma U251 cells in a targeted mode, the expression level of the RBM17 gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated silencing of RBM17 gene is therefore a potential clinical non-surgical treatment modality for malignancies.

The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the expression of the human RBM17 gene, particularly the lentivirus can efficiently infect target cells, efficiently inhibit the expression of the RBM17 gene in the target cells, promote apoptosis, reduce the invasion and transfer capacity of tumor cells and the like, further inhibit the growth of the tumor cells, promote the apoptosis of the tumor cells and have important significance in tumor treatment.

Drawings

FIG. 1: pGCSIL-GFP plasmid DNA map

FIG. 2: 5 days after the RBM17-RNAi lentivirus infects glioma U251 cells, the expression level of RBM17mRNA is obviously reduced

FIG. 3: 5 days after the RBM17-RNAi lentivirus infects glioma U251 cells, inhibition of cell proliferation is caused

Detailed Description

The invention relates to a group of small interfering RNA (siRNA) sequences, RNA interference vectors and RNA interference lentiviruses aiming at human RBM17 gene. The coding region sequence of human RBM17mRNA is selected as the target site of siRNA, and the siRNA target site sequence is designed according to the continuous 10-30 (preferably 15-27, more preferably 19-23) base sequences in the target site. Constructing a nucleic acid construct expressing the siRNA through gene cloning, and packaging the lentivirus expressing the siRNA. Cell experiments prove that the siRNA sequence can specifically silence the expression of an endogenous RBM17 gene in human tumor cells.

The inventor of the invention has found through extensive and intensive research that the RBM17 gene is obviously highly expressed in tumor tissues; the inventor finds that after the expression of the human RBM17 gene is down-regulated by an RNAi method, the proliferation of tumor cells can be effectively inhibited, the apoptosis is promoted, the invasion and the transfer capacity of the tumor cells are reduced, and the growth process of the tumor can be effectively controlled, and the research result shows that the RBM17 gene is a protooncogene and can be used as a target point for tumor treatment. The inventor further synthesizes and tests a plurality of siRNAs aiming at the RBM17 gene, screens out the siRNA which can effectively inhibit the expression of RBM17 and further inhibit the proliferation and growth of glioma U251 cells, and completes the invention on the basis.

The invention provides a series of small interfering RNA (siRNA) sequences interfering human RBM17 gene, and constructs a lentivirus capable of specifically silencing RBM17 gene expression. The research of the invention finds that the small interfering RNA and RNAi lentivirus designed aiming at the human RBM17 gene stably and specifically down-regulate the expression of the RBM17 gene and effectively inhibit the proliferation of human tumor cells. The invention shows that the RBM17 gene can promote the growth of tumor cells and is expected to become a target for early diagnosis and treatment of tumors. Further, silencing the expression of RBM17 gene by RNAi can be an effective means for inhibiting tumor development.

The design idea of the invention is as follows:

the invention obtains a human RBM17 gene RNAi lentivirus by screening through the following method: the sequence of human RBM17 gene is adjusted from Genbank; predicting the siRNA site; synthesizing an effective siRNA sequence aiming at the RBM17 gene and a double-stranded DNA Oligo with two ends containing sticky ends of enzyme cutting sites; after double enzyme digestion, the lentiviral vector is connected with a double-stranded DNA Oligo to construct an RNAi plasmid for expressing an RBM17 gene siRNA sequence; the RNAi plasmid and a helper vector (packaging Mix, Sigma-aldrich company) required by lentivirus packaging are cotransfected with a human embryonic kidney cell 293T to generate recombinant lentivirus particles, and the lentivirus capable of efficiently silencing the RBM17 gene can be prepared.

Based on the method, the invention provides 1 effective target (shown as SEQ ID NO: 1) for interfering the RBM17 gene, and constructs the lentivirus of the specific interfering human RBM17 gene.

Meanwhile, the invention also discloses an RNAi lentivirus (RBM17-RNAi) of the human RBM17 gene and preparation and application thereof.

The research discovers that the proliferation of the tumor cells can be effectively inhibited after the expression of the RBM17 gene in the tumor cells is reduced by utilizing a lentivirus-mediated RNAi method. The research shows that the RBM17 gene is a protooncogene, can promote the proliferation of tumor cells, has important biological functions in the occurrence and development of tumors, can be a target for tumor treatment by the RBM17 gene, and can be a new means for tumor treatment by lentivirus-mediated RBM17 gene specific silencing.

The invention is further illustrated by the following examples. It should be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In the examples, the experimental methods and reagents having no specific conditions described therein were performed under conventional conditions, such as those described in [ U.S. Sambrook.J.; huang Beitang, etc. Molecular cloning test guidelines, third edition. Beijing: the conditions described in scientific press 2002 or conditions suggested by the manufacturer.

Example 1 preparation of RNAi lentivirus against human RBM17 Gene

1. Screening effective siRNA target point aiming at human RBM17 gene

Calling RBM17 (NM-032905) gene information from Genbank; designing effective siRNA target points aiming at RBM17 gene. Table 1 lists 1 effective siRNA target sequences against RBM17 gene.

TABLE 1 siRNA target sequences targeting the human RBM17 gene

SEQ ID NO	TargetSeq
		1	ATACTTAAGTGTCCTACTAAA

2. Preparation of Lentiviral vectors

Synthesizing double-stranded DNA Oligo sequences (Table 2) containing Age I and EcoR I enzyme cutting sites at two ends aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes Age I and EcoR I were used to act on pGCSIL-GFP vector (supplied by Shanghai Jikai Gene chemistry, Ltd., FIG. 1), which was linearized, and the cleaved fragments were identified by agarose gel electrophoresis.

TABLE 2 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends

The vector DNA linearized by double digestion (digestion system shown in Table 4, 37 ℃, reaction 1h) was ligated to the purified double-stranded DNA Oligo by T4DNA ligase, ligated overnight at 16 ℃ in an appropriate buffer system (ligation system shown in Table 5), and the ligation product was recovered. The ligation product was transformed into calcium chloride prepared fresh E.coli competent cells (transformation protocol reference: molecular cloning protocols second edition, pages 55-56). Dipping the surface of the clone of the strain growing out of the connected transformation product, dissolving the surface in 10 mul LB culture medium, uniformly mixing and taking 1 mul as a template; designing universal PCR primers at the upstream and downstream of RNAi sequence in the lentiviral vector, wherein the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); the sequence of the downstream primer is as follows: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), and a PCR identification experiment was performed (PCR reaction system shown in Table 6-1, reaction conditions shown in Table 6-2). Sequencing and comparing the clones which are identified to be positive by the PCR, wherein the correctly compared clones are the clones which are successfully constructed and are directed at the nucleotide sequence shown in SEQ ID NO:1, named pGCSIL-GFP-RBM 17-siRNA.

pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When pGCSIL-GFP-Scr-siRNA negative control plasmids are constructed, double-stranded DNA Oligo sequences (table 3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at Scr siRNA targets, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-RBM 17-siRNA.

TABLE 3 double-stranded DNA Oligo with Age I and EcoR I cleavage sites sticky ends at both ends

The vector was linearized by T4DNA ligase (digestion system shown in Table 4, 37 ℃ C., reaction time 1h)

TABLE 4 pGCSIL-GFP plasmid digestion reaction System

Reagent	Volume (μ l)
		pGCSIL-GFP plasmid (1. mu.l)g/μl)	2.0
10×buffer	5.0
		100×BSA	0.5
Age I(10U/μl)	1.0
		EcoR I(10U/μl)	1.0
dd H₂O	40.5
		Total	50.0

TABLE 5 ligation reaction System of vector DNA and double-stranded DNA Oligo

Reagent	Positive control (μ l)	Self-contained control (μ l)	Connecting group (mu l)
				Linearized vector DNA (100 ng/. mu.l)	1.0	1.0	1.0
Annealed double stranded DNA Oligo (100 ng/. mu.l)	1.0	-	1.0
				10 XT 4 phage DNA ligase buffer	1.0	1.0	1.0
T4 phage DNA ligase	1.0	1.0	1.0
				dd H₂O	16.0	17.0	16.0
Total	20.0	20.0	20.0

TABLE 6-1 PCR reaction System

Reagent	Volume (μ l)
		10×buffer	2.0
dNTPs(2.5mM)	0.8
		Upstream primer	0.4
Downstream primer	0.4
		Taq polymerase	0.2
Form panel	1.0
		ddH₂O	15.2
Total	20.0

TABLE 6-2 PCR reaction System Programming

The DNA of RNAi plasmid pGCSIL-GFP-RBM17-siRNA was extracted using a plasmid extraction kit from Qiagen, Inc., and 100 ng/. mu.l of stock solution was prepared.

24h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were trypsinized and processed with DMEM containing 10% fetal bovine serumThe whole culture medium adjusted the cell density to 1.5X 10⁵Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator. The cell density can reach 70-80% to be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. Mu.l of Packing Mix (PVM), 12. mu.l of PEI, and 400. mu.l of serum-free DMEM medium were added to a sterilized centrifuge tube according to the instructions of the MISSION Lentiviral Packing Mix kit from Sigma-aldrich, and 20. mu.l of the above-mentioned extracted plasmid DNA was added to the above-mentioned PVM/PEI/DMEM mixture.

The transfection mixture was incubated at room temperature for 15min, transferred to medium of human embryonic kidney 293T cells at 37 ℃ with 5% CO₂Culturing for 16h in an incubator. The medium containing the transfection mixture was discarded, washed with PBS solution, 2ml of complete medium was added and incubation continued for 48 h. The cell supernatant was collected, and the lentivirus was purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) according to the following steps: (1) centrifuging at 4 deg.C and 4000g for 10min to remove cell debris; (2) filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; (3) centrifuging at 4000g for 10-15min to obtain the required virus concentration volume; (4) after the centrifugation is finished, separating the filter cup from the lower filtrate collecting cup, reversely buckling the filter cup on the sample collecting cup, and centrifuging for 2min until the centrifugal force is not more than 1000 g; (5) the centrifuge cup is removed from the sample collection cup, and the virus concentrate is obtained. Subpackaging the virus concentrated solution and storing at-80 ℃. The sequence of the first strand of siRNA contained in the virus concentrate is shown in SEQ ID NO 9. The packaging process of the control lentivirus was the same as that of the RBM17-siRNA lentivirus, except that pGCSIL-GFP-Scr-siRNA vector was used instead of pGCSIL-GFP-RBM17-siRNA vector.

Example 2 detection of silencing efficiency of RBM17 Gene by real-time fluorescent quantitative RT-PCR

Glioma U251 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (cell number approximately 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the value of the multiplicity of infection (MOI, U251:5), adding an appropriate amount of the virus prepared in example 1, culturing for 24h, replacing the culture medium, and collecting the virus after the infection time reaches 5 daysAnd (4) collecting cells. Total RNA was extracted according to the Trizol protocol of Invitrogen corporation. The RNA was reverse-transcribed to obtain cDNA according to the M-MLV protocol of Promega (reverse transcription reaction system shown in Table 7, reaction at 42 ℃ for 1 hour, followed by inactivation of the reverse transcriptase by water bath in a 70 ℃ water bath for 10 min).

Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (TAKARA). The primers for the RBM17 gene are as follows: an upstream primer 5'-CTGGGGCGAGGACTGTACT-3' (SEQ ID NO: 10) and a downstream primer 5'-AGTGGAGACCAGTGACTCAAA-3' (SEQ ID NO: 11). The housekeeping gene GAPDH is used as an internal reference, and the primer sequences are as follows: an upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 12) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 13). The reaction system was prepared in the proportions shown in Table 8.

TABLE 7 reverse transcription reaction System

Reagent	Volume (μ l)
		5×RT buffer	4.0
10mM dNTPs	2.0
		RNasin	0.5
M-MLV-RTase	1.0
		DEPC H₂O	3.5
Total	11.0

TABLE 8 Real-time PCR reaction System

Reagent	Volume (μ l)
		SYBR premix ex taq:	10.0
Upstream primer (2.5 μ M):	0.5
		downstream primer (2.5 μ M):	0.5
cDNA	1.0
		ddH₂O	8.0
Total	20.0

the program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 15 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 45 cycles were performed. Each time reading the absorbance value during the extension phase. After PCR was completed, denaturation was carried out at 95 ℃ for 1min, and then cooled to 55 ℃ to allowThe DNA double strands are well bound. Melting curves were prepared by increasing the temperature from 55 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By adopting 2-^ΔΔCtThe expression abundance of RBM17mRNA infected is calculated by an analytical method. Cells infected with a control virus (Lv-Scr-siRNA) served as controls. The results of the experiment (FIG. 2) show that the expression level of RBM17mRNA in glioma U251 cells was down-regulated by 67.1%.

Example 3 examination of the proliferative Capacity of tumor cells infected with RBM17-siRNA lentivirus

Glioma U251 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (cell number approximately 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number (MOI, U251:5), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. Complete medium resuspension into cell suspension (2X 10)⁴Per ml) at a cell density of about 2000 per well, 96-well plates were seeded. Each set of 5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO₂Culturing in an incubator. The plate readings were performed once a day with Cellomics apparatus (Thermo Fisher) for 5 consecutive days starting the second day after plating. The number of green fluorescent cells in the well plate for each scan was accurately calculated by adjusting the input parameters of the Cellomics arrayscan, and the data were statistically plotted to generate a cell proliferation curve (the results are shown in FIG. 3). The results show that after each tumor of the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of the tumor cells of the control group, the number of viable cells is respectively reduced by 83.6 percent, and the RBM17 gene silencing leads to the inhibition of the proliferation capacity of the tumor cells.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims

1. Use of an isolated human RBM17 gene as a target in screening tumor therapeutic drugs; the purposes specifically refers to: using the RBM17 gene as a target, screening double-stranded RNA or shRNA that can reduce the expression level of the RBM17 gene in tumor cells as a target. A tumor therapeutic drug, the target sequence of the double-stranded RNA or shRNA is shown in SEQ ID NO: 1, and the tumor is selected from glioma.

2. Use of an isolated nucleic acid molecule that reduces the expression of RBM17 gene in glioma cells in the preparation of a tumor therapeutic drug for treating glioma, the nucleic acid molecule comprising:

a) double-stranded RNA, the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the RBM17 gene; the double-stranded RNA includes a first strand and a second strand, and the first strand and the second strand are complementary Form RNA dimer together, and the sequence of described first strand is identical with the target sequence in RBM17 gene, the target sequence in described RBM17 gene is as shown in SEQ ID NO:1, or;

b) shRNA, the shRNA contains a nucleotide sequence capable of hybridizing with the RBM17 gene, the shRNA includes a sense strand fragment and an antisense strand fragment, and a stem-loop structure connecting the sense strand fragment and the antisense strand fragment, The sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is the same as the target sequence in the RBM17 gene, and the target sequence in the RBM17 gene is shown in SEQ ID NO: 1.

3 . The use according to claim 2 , wherein the double-stranded RNA is a small interfering RNA, and the sequence of the first strand of the small interfering RNA is shown in SEQ ID NO: 9. 4 .

4. The use according to claim 2, wherein the sequence of the shRNA is shown in SEQ ID NO: 14.

5. the purposes of a RBM17 gene interference nucleic acid construct in the preparation of a tumor therapeutic drug for the treatment of glioma, the RBM17 gene interference nucleic acid construct containing the nucleic acid molecule that is isolated as described in any of the claims 2-4 encoding The gene fragment of the shRNA in the shRNA can express the shRNA.

6. The use according to claim 5, wherein the RBM17 gene interference nucleic acid construct is an interference lentiviral vector.

7. The use according to claim 6, wherein the interfering lentiviral vector is obtained by cloning the gene fragment encoding the shRNA into a lentiviral vector selected from the group consisting of: pLKO.1-puro ,

pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV- TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC- TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA1. Any of 2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.

8. Use of an RBM17 gene interference lentivirus in the preparation of a tumor therapeutic drug for the treatment of glioma, wherein the RBM17 gene interference lentivirus is expressed in a lentivirus by the interference lentiviral vector described in any one of claims 6-7. With the help of packaging plasmids and cell lines, it is packaged by viruses.