CN105803053A - Uses and related drugs of human RBM17 gene - Google Patents

Abstract

The invention discloses uses and related drugs of a human RBM17 gene. The invention discloses the uses of the human RBM17 gene in tumor therapy, tumor diagnosis and drug preparation. The invention further discloses a human RBM17 gene small-molecular interference RNA, a human RBM17 gene interference nucleic acid construction body and a human RBM17 gene interference slow virus and uses thereof. The siRNA or the siRNA sequence-containing nucleic acid construction body and slow virus provided by the invention, especially the slow virus, can specifically inhibit expression of the human RBM17 gene, can efficiently infect target cells, efficiently inhibit expression of the RBM17 gene in the target cells, inhibit the growth of the tumor cells, promote the apoptosis of the tumor cells and have important significance in tumor therapy.

Description

Use of human RBM17 gene and related medicines

technical field

The present invention relates to the field of biotechnology, and more specifically relates to the use of human RBM17 gene and related medicines.

Background technique

RNA interference (RNAi) is short double-stranded RNA (dsRNA) composed of nucleotides for post-transcriptional gene silencing. It can efficiently and specifically block the expression of a specific gene in the body, leading to its degradation, thereby causing the silencing of a specific gene in the organism, and causing the cells to show the absence of a certain gene phenotype. It is a commonly used research gene emerging in recent years. Functional, laboratory techniques for finding cures for disease. Studies have shown that double-stranded RNA with a length of 21-23nt can specifically cause RNAi at the transcriptional and post-transcriptional levels (TuschlT, ZamorePD, SharpPA, BartelDP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to23nucleotide intervals. Cell2000; 101:25-33.) . Although cancer patients have undergone chemotherapy, radiotherapy and comprehensive treatment, their five-year survival rate is still very low. If the genes related to tumor onset and progression can be intervened, it will open up a new way for tumor treatment. In recent years, RNAi has become an effective strategy for gene therapy of tumors. The use of RNAi technology can inhibit the expression of proto-oncogenes, mutated tumor suppressor genes, cell cycle-related genes, and anti-apoptosis-related genes to inhibit tumor progression (Uprichard, Susan L. Therapeutic potential of RNA interference. FEBS Letters 2005; 579:5996-6007.).

In eukaryotic cells, alternative splicing of mRNA can regulate the polyprotein expression of a single gene. By splicing together the exon and exon of the pre-transcribed mRNA, it affects the final composition of the protein product and ensures Diversity of functional proteins in cells. Alternative splicing is involved in the regulation of 60-74% human genome expression, so the accuracy and effectiveness of mRNA splicing regulation is crucial, and mRNA splicing defects are often associated with many human diseases (ModrekB, ReschA, GrassoC, LeeC.2001.Genome -wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res. 29:2850–2859). Alternative splicing is mainly regulated by the heterogeneous nuclear ribonucleoproteins (hnRNP) and splicing factor protein Sr (serine-arginine rich) family, and the selection of splicing sites depends on the relative concentration of these proteins and is regulated by reversible phosphorylation (Hastings ML, Krainer AR. 2001. Functions of SR proteins in the U12-dependent AT-ACpre-mRNA splicing pathway. RNA 7:471-482). RNA binding motif protein 17 (RNAbindingmotifprotein, RBM), also known as alternative mRNA splicing factor 45 (splicingfactor45, SPF45) encodes an RNA binding protein, which is involved in the formation of the RNA splicing complex, and in the second step of mRNA splicing function in the catalytic step. RBM17 regulates alternative splicing of the pre-mRNA encoding the cell death receptor FAS, inducing the skipping of exon 6, which contains the transmembrane domain, which can generate soluble dominant negative Fas protein (CorsiniL, et al. 2007 . U2AF-homology motif interactions are required for alternative splicing regulation by SPF45. Nat. Struct. Mol. Biol. 14:620–629). RBM17 contains an unstructured N-terminal domain, a G-patch motif, involved in protein-protein and protein-nucleic acid interactions, and a C-terminal RNA recognition motif (RNA recognition motif) necessary for mRNA splicing. RRM) (Lallena MJ, Chalmers KJ, Llamazares S, Lamond AI, Valcarcel J. 2002. Splicing regulation at the second catalytic step by Sex-lethalin involves 3' splice site recognition by SPF45. Cell 109:285-296). In humans, RBM17 is low expressed in ductal epithelium of normal breast, liver, prostate and pancreas, but it is overexpressed in many human cancers, such as: bladder cancer, breast cancer, colon cancer, lung cancer, ovarian cancer , pancreatic cancer, and prostate cancer, etc.; at the same time, studies have proved that the overexpression of RBM17 is related to the drug resistance of many cancers (SampathJ, LongPR, ShepardRL, et al.HumanSPF45, asplicingfactor, haslimitedexpressioninnormaltissues, isoverexpressedinmanytumorsandcanconfermultidrugresistantphenotypetocells.AmJPathol2003: 116–3907); 116–3907); .

However, the current experimental reports on the RBM17 gene in tumor-related fields are still blank, especially in the field of human glioma research. The invention uses RNAi as a means to study the role of RBM17 in the occurrence and development of glioma.

Contents of the invention

The purpose of the present invention is to disclose the treatment method and medicine related to human RBM17 gene, and use RNA interference (RNAi) as a means to study the role of RBM17 gene in the survival and apoptosis process of tumor cells.

In the first aspect of the present invention, the role of the RBM17 gene in the occurrence and development of tumors is studied by means of RNA interference, and a method for inhibiting or reducing tumor cell growth, proliferation, differentiation and/or survival is disclosed, the method comprising: Administering a molecule that can specifically inhibit the transcription or translation of RBM17 gene, or the expression or activity of RBM17 protein, to tumor cells, so as to inhibit the growth, proliferation, differentiation and/or survival of tumor cells.

The tumor cells are selected from tumor cells whose growth is related to the expression or activity of RBM17 protein. Preferably, the tumor cells are selected from glioma.

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

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

The nucleic acid includes but is not limited to: antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, small interfering RNA (esiRNA) or short hairpin RNA (shRNA) prepared by endoribonuclease III.

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

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

Further, the first strand sequence of the small interfering RNA is basically the same as the target sequence in the RBM17 gene. Preferably, the target sequence in the RBM17 gene contains the sequence shown in SEQ ID NO:1.

The target sequence in the RBM17 gene is the fragment in the RBM17 gene corresponding to the mRNA fragment complementary to the small interfering RNA when the small interfering RNA specifically silences the expression of the RBM17 gene.

Preferably, the RBM17 gene is derived from human.

The first aspect of the present invention also discloses the use of an isolated human RBM17 gene in the preparation or screening of drugs for treating tumors, or in the preparation of drugs for diagnosing tumors.

Further, the tumor is selected from glioma.

The use of the isolated RBM17 gene for the preparation or screening of tumor therapeutic drugs includes two aspects: first, the use of the RBM17 gene as a target of the drug or preparation for tumor cells in the preparation of tumor therapeutic drugs or preparations; The RBM17 gene is used as the target of drugs or preparations for tumor cells to screen tumor treatment drugs or preparations.

The application of the RBM17 gene as the target of drugs or preparations for tumor cells in the preparation of tumor treatment drugs or preparations specifically refers to: using the RBM17 gene as a target of RNA interference to develop drugs or preparations for tumor cells, thereby reducing The expression level of RBM17 gene in tumor cells.

The application of the RBM17 gene as the target of drugs or preparations for tumor cells in the screening of tumor treatment drugs or preparations specifically refers to: using the RBM17 gene as the target of action, screening the drugs or preparations to find out the drugs or preparations that can inhibit or promote the human RBM17 gene. The expressed drug can be used as a candidate drug for tumor treatment. The RBM17 gene small molecule interfering RNA (siRNA) according to the present invention is obtained by screening the human RBM17 gene as an action object, and can be used as a drug capable of inhibiting tumor cell proliferation. In addition, such as antibody drugs, small molecule drugs, etc., can also target the RBM17 gene and its protein.

The use of the RBM17 gene in the preparation of tumor diagnostic drugs refers to the application of the RBM17 gene expression product as a tumor diagnostic index in the preparation of tumor diagnostic drugs.

The expression levels of RBM17 gene in four glioma cell lines were detected by Real-time Quantitative PCR. The study found that RBM17 was highly expressed in 4 glioma cell lines. It is suggested that RBM17 may be an oncogene and play an important role in the occurrence and development of tumors; the expression level of RBM17 gene may be a marker for tumor diagnosis.

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

The tumor therapeutic drug or tumor diagnostic drug obtained by preparing or screening 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 nucleic acid includes but is not limited to: antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, small interfering RNA (esiRNA) or short hairpin RNA (shRNA) prepared by endoribonuclease III.

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

The method of treating tumors with the aforementioned tumor therapeutic drugs mainly achieves the purpose of treatment by reducing the expression level of human RBM17 gene and inhibiting the proliferation of tumor cells. Specifically, during treatment, the substance that can effectively reduce the expression level of human RBM17 gene is administered to the patient.

The second aspect of the present invention discloses an isolated nucleic acid molecule that reduces RBM17 gene expression in tumor cells, the nucleic acid molecule comprising:

A) double-stranded RNA, which contains 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, and the first strand and the second strand are complementary to form an RNA dimer together, and the sequence of the first strand is the same as that of the 15-strand in the RBM17 gene. 27 consecutive nucleotide sequences are basically the same. Preferably, the sequence of the first strand is substantially identical to 19-23 consecutive nucleotide sequences in the RBM17 gene; more preferably, the sequence of the first strand is substantially identical to 19, 20 or 21 consecutive nucleotide sequences in the RBM17 gene The nucleotide sequences are basically identical.

Furthermore, the double-stranded RNA comprises a first strand and a second strand, and the first strand and the second strand are complementary to form an RNA dimer together, and the sequence of the first strand is the same as that in the RBM17 gene The target sequences are essentially identical.

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

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

The siRNA shown in SEQIDNO: 9 is a strand of small interfering RNA for the human RBM17 gene designed with the sequence shown in SEQIDNO: 1 as the RNA interference target sequence, and the other strand is the sequence of the second strand and the sequence of the first strand Complementary, the siRNA can specifically silence the expression of endogenous RBM17 gene in tumor cells.

Further, 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 positive sense strand fragment is basically the same as 15-27 consecutive nucleotide sequences in the RBM17 gene. The shRNA can be processed into a small interfering RNA (siRNA) to specifically silence the expression of the endogenous RBM17 gene in tumor cells.

Further, the shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, And the sequence of the positive sense strand fragment is basically the same as the target sequence in the RBM17 gene.

Preferably, the sense strand fragment is substantially identical to 19-23 consecutive nucleotide sequences in the RBM17 gene; more preferably, the sense strand fragment is identical to 19, 20 or 21 consecutive nucleotide sequences in the RBM17 gene The sequences are basically the same.

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

Furthermore, the sequence of the shRNA is shown in SEQ ID NO: 14, specifically: 5'-AUACUUAAGUGUCCUACUAAAUUCAAGAGAUUUAGUAGGACACUUAAGUAU-3'.

The shRNA can become siRNA after digestion and processing, and then play a role in specifically silencing the endogenous human RBM17 gene expression in tumor cells.

The interfering lentiviral vector encoding the shRNA gene fragment of the present invention contains the sequence shown in SEQ ID NO: 1 and its complementary sequence.

The first strand of the double-stranded RNA or the positive-sense strand fragment of the shRNA is basically the same as the target sequence in the RBM17 gene, and the target sequence of the RBM17 gene is when siRNA is used to specifically silence the expression of the RBM17 gene The fragment in the RBM17 gene corresponding to the mRNA fragment recognized and silenced by siRNA.

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

Further, the RBM17 gene is derived from human.

The third aspect of the present invention discloses an RBM17 gene interference nucleic acid construct, which contains a gene segment encoding the shRNA in the isolated nucleic acid molecule of the present invention and can express the shRNA.

The human RBM17 gene interference nucleic acid construct can be obtained by cloning the gene fragment encoding the aforementioned 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 of the present invention is obtained by cloning the DNA fragment encoding the aforementioned RBM17 gene shRNA into a known carrier. After infecting virus particles, tumor cells are infected, and then the shRNA of the present invention is transcribed, and the siRNA is finally obtained through steps such as enzyme digestion and processing, 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 encoding a detectable marker in tumor cells; preferably, the detectable marker such as green fluorescent protein (GFP).

Further, the lentiviral vector can be selected from: 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, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/ Either of V5-GW/lacZ.

The embodiment of the present invention specifically lists the human RBM17 gene interference lentiviral vector constructed with pGCSIL-GFP as the vector, named as pGCSIL-GFP-RBM17-siRNA.

The isolated nucleic acid molecules of the present invention can be used to prepare drugs for preventing or treating tumors, and the tumors are gliomas.

The RBM17 gene siRNA of the present invention can be used to inhibit the proliferation of tumor cells, and further can be used as a drug or preparation for treating tumors. The RBM17 gene interference lentiviral vector can be used to prepare the RBM17 gene siRNA. When used as a drug or preparation for treating tumors, a safe and effective amount of the nucleic acid molecule is administered to mammals. The specific dosage should also consider factors such as the route of administration, the health status of the patient, etc., all of which are within the skill of skilled physicians.

In the fourth aspect of the present invention, an RBM17 gene interference lentivirus is disclosed, which is packaged by the aforementioned RBM17 gene interference lentivirus vector with the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect tumor cells and produce small molecule interference RNA targeting RBM17 gene, thereby inhibiting the proliferation of glioma tumor cells. The RBM17 gene interference lentivirus can be used to prepare medicaments for preventing or treating tumors.

The fifth aspect of the present invention discloses a pharmaceutical composition for preventing or treating tumors, the effective substance of which contains one or more of the aforementioned isolated nucleic acid molecule, RBM17 gene interference nucleic acid construct or RBM17 gene interference lentivirus combination of species.

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 pharmaceutically acceptable carriers, diluents or excipients.

In preparing these compositions, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient acts as a diluent, it can be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Thus, the composition can 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 formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl and propylparabens), sweeteners, and the like.

The invention also discloses the application of the pharmaceutical composition in the preparation of therapeutic drugs for treating glioma.

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

When the pharmaceutical composition is used to prevent or treat 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 said tumor is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the tumor part is suppressed.

The subject of the method can be a human.

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

In summary, the present invention designs an RNAi target sequence for the human RBM17 gene, and constructs a corresponding RBM17 RNAi vector, wherein the RNAi vector pGCSIL-GFP-RBM17-siRNA with the coding sequence SEQ ID NO: 1 can significantly down-regulate the RBM17 gene in mRNA Expression at the level and protein level. Using lentivirus (abbreviated as Lv) as a gene manipulation tool to carry the RNAi vector pGCSIL-GFP-RBM17-siRNA can efficiently introduce the RNAi sequence targeting the RBM17 gene into glioma U251 cells, reducing the expression level of the RBM17 gene, Significantly inhibit the proliferation ability of the above tumor cells. Therefore, lentivirus-mediated RBM17 gene silencing is a potential clinical non-surgical treatment for malignant tumors.

The siRNA provided by the present invention or the nucleic acid construct containing the siRNA sequence, and the lentivirus 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 , promote cell apoptosis, reduce the invasion and metastasis ability of tumor cells, etc., and then inhibit the growth of tumor cells and promote tumor cell apoptosis, which is of great significance in tumor treatment.

Description of drawings

Figure 1: DNA map of pGCSIL-GFP plasmid

Figure 2: After 5 days of RBM17-RNAi lentivirus infection of glioma U251 cells, the expression level of RBM17mRNA was significantly reduced

Figure 3: 5 days after RBM17-RNAi lentivirus infects glioma U251 cells, it causes cell proliferation inhibition

detailed description

The invention relates to a group of small molecule interference RNA (siRNA) sequences, RNA interference vectors and RNA interference lentiviruses targeting human RBM17 gene. The human RBM17mRNA coding region sequence 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. Through gene cloning, a nucleic acid construct expressing the above siRNA is constructed, and a lentivirus expressing the above siRNA is packaged. Cell experiments have proved that the above siRNA sequence can specifically silence the expression of endogenous RBM17 gene in human tumor cells.

The inventors of the present invention have found through extensive and in-depth research that the RBM17 gene is significantly highly expressed in tumor tissues; the inventors have found that down-regulating the expression of the human RBM17 gene by RNAi can effectively inhibit the proliferation of tumor cells and promote apoptosis. It can effectively control the growth process of tumors by reducing the ability of tumor cell death, invasion and metastasis, etc. This research result shows that RBM17 gene is a proto-oncogene and can be used as a target for tumor therapy. The inventors further synthesized and tested a variety of siRNAs against the RBM17 gene, screened out siRNAs that can effectively inhibit the expression of RBM17 and thereby inhibit the proliferation and growth of glioma U251 cells, and completed the present invention on this basis.

The invention provides a series of small interfering RNA (siRNA) sequences that interfere with the human RBM17 gene, and constructs a lentivirus capable of specifically silencing the expression of the RBM17 gene. The research of the present invention finds that the small interfering RNA and RNAi lentivirus designed against the human RBM17 gene can 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. Moreover, silencing the expression of RBM17 gene by RNAi can be used as an effective means to inhibit tumor development.

Design idea of the present invention is:

The present invention screens and obtains a human RBM17 gene RNAi lentivirus through the following methods: transfer the human RBM17 gene sequence from Genbank; predict the siRNA site; synthesize an effective siRNA sequence for the RBM17 gene, with enzyme cleavage sites at both ends The double-stranded DNA Oligo at the end; the lentiviral vector was double-digested and connected with the double-stranded DNA Oligo to construct the RNAi plasmid expressing the siRNA sequence of the RBM17 gene; the RNAi plasmid and the auxiliary vector (PackingMix, Sigma-aldrich company) required for lentiviral packaging were co-transfected Human embryonic kidney cell 293T produces recombinant lentiviral particles, which can produce a lentivirus that efficiently silences the RBM17 gene.

Based on the above method, the present invention provides an effective target for interfering with the RBM17 gene (specifically as shown in SEQ ID NO: 1), and constructs a lentivirus that specifically interferes with the human RBM17 gene.

At the same time, the invention also discloses a human RBM17 gene RNAi lentivirus (RBM17-RNAi) and its preparation and application.

This study found that the use of lentivirus-mediated RNAi method can effectively inhibit the proliferation of tumor cells after reducing the expression of RBM17 gene in tumor cells. This study shows that the RBM17 gene is a proto-oncogene that can promote tumor cell proliferation and has important biological functions in the occurrence and development of tumors. The RBM17 gene can be a target for tumor therapy. Lentivirus-mediated RBM17 gene-specific silencing It can be used as a new method for tumor treatment.

The present invention is further set forth below in conjunction with embodiment. It should be understood that the examples are only used to illustrate the present invention, not to limit the scope of the present invention. The experimental method and the reagent of unspecified formula are all according to conventional conditions in the embodiment, such as [US] Sambrook.J et al.; Huang Peitang etc. translation. Molecular Cloning Experimental Guide, Third Edition. Beijing: The conditions described in Science Press 2002 or the conditions suggested by the manufacturer were performed or configured.

Example 1 Preparation of RNAi lentivirus for human RBM17 gene

1. Screening for effective siRNA targets against the human RBM17 gene

Retrieve RBM17 (NM_032905) gene information from Genbank; design effective siRNA targets for RBM17 gene. Table 1 lists one of the effective siRNA target sequences for the RBM17 gene.

Table 1 is targeted at the siRNA target sequence of human RBM17 gene

SEQ ID NO TargetSeq 1 ATACTTAAGTGTCCTACTAAA

2. Preparation of lentiviral vector

Aiming at the siRNA target (taking SEQIDNO: 1 as an example) synthetic double-stranded DNA Oligo sequence (table 2) containing AgeI and EcoRI restriction endonuclease at both ends; Acting on the pGCSIL-GFP vector ( Provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., Figure 1), it was linearized, and the digested fragments were identified by agarose gel electrophoresis.

Table 2 Double-stranded DNA Oligo with sticky ends containing AgeI and EcoRI restriction sites at both ends

Ligate the vector DNA and the purified double-stranded DNA Oligo with the double-digested linearized (enzyme digestion system as shown in Table 4, 37°C, reaction for 1 h) by T4 DNA ligase, and in an appropriate buffer system (the ligation system is as shown in Table 5) Shown) at 16°C for overnight ligation, and the ligated product was recovered. The ligation product was transformed into fresh Escherichia coli competent cells prepared with calcium chloride (transformation operation reference: Molecular Cloning Experiment Guide, Second Edition, pages 55-56). Dip it on the surface of the bacterial clone grown by the connection transformation product, dissolve it in 10 μl LB medium, mix well and take 1 μl as a template; design general PCR primers upstream and downstream of the RNAi sequence in the lentiviral vector, the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA -3' (SEQ ID NO: 6); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), PCR identification experiment was carried out (PCR reaction system is shown in Table 6-1, and reaction conditions are shown in Table 6-2). The positive clones identified by PCR were sequenced and compared for analysis, and the clones with correct comparisons were successfully constructed vectors expressing RNAi against SEQ ID NO: 1, named pGCSIL-GFP-RBM17-siRNA.

The pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed, and the negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When constructing the pGCSIL-GFP-Scr-siRNA negative control plasmid, synthesize a double-stranded DNA Oligo sequence with sticky ends containing AgeI and EcoRI restriction sites at both ends for the ScrsiRNA target (Table 3), and the rest of the construction methods, identification methods and conditions are the same as pGCSIL - GFP-RBM17-siRNA.

Table 3 Double-stranded DNA Oligo with sticky ends containing AgeI and EcoRI restriction sites at both ends

Carrier linearized by double enzyme digestion with T4 DNA ligase (enzyme digestion system is shown in Table 4, 37°C, reaction 1h)

Table 4 pGCSIL-GFP plasmid digestion reaction system

Reagent Volume (μl) pGCSIL-GFP plasmid (1 μg/μl) 2.0 10×buffer 5.0 100×BSA 0.5 Age I (10U/μl) 1.0 EcoR I (10U/μl) 1.0 dd H ₂ O 40.5 Total 50.0

Table 5 Carrier DNA and double-stranded double-stranded DNA Oligo ligation reaction system

Reagent Positive control (μl) Self-linked control (μl) Connectome (μl) Linearized carrier DNA (100ng/μl) 1.0 1.0 1.0 Annealed dsDNA Oligo (100ng/μl) 1.0 - 1.0 10x T4 phage DNA ligase buffer 1.0 1.0 1.0 T4 bacteriophage DNA ligase 1.0 1.0 1.0 dd H ₂ O 16.0 17.0 16.0 Total 20.0 20.0 20.0

Table 6-1 PCR reaction system

Reagent Volume (μl) 10×buffer 2.0 dNTPs (2.5mM) 0.8 upstream primer 0.4 downstream primer 0.4 Taq polymerase 0.2 template 1.0 ddH ₂ O 15.2 Total 20.0

Table 6-2 PCR reaction system program setting

The DNA of the RNAi plasmid pGCSIL-GFP-RBM17-siRNA was extracted with a plasmid extraction kit from Qiagen, and prepared into a 100ng/μl storage solution.

24 hours before transfection, human embryonic kidney cells in logarithmic growth phase 293T cells were digested with trypsin, and the cell density was adjusted to 1.5×10 ⁵ cells/ml with DMEM complete medium containing 10% fetal bovine serum, and seeded in 6-well plates , 37°C, 5% CO ₂ incubator. When the cell density reaches 70%-80%, it can be used for transfection. 2 hours before transfection, aspirate the original medium and add 1.5ml fresh complete medium. According to the instructions of the MISSIONLentiviralPackagingMix kit from Sigma-aldrich, add PackingMix (PVM) 20μl, PEI 12μl, serum-free DMEM medium 400μl to a sterilized centrifuge tube, take 20μl of the plasmid DNA extracted above, and add it to the above PVM/PEI /DMEM mixture.

The above transfection mixture was incubated at room temperature for 15 min, transferred to the culture medium of human embryonic kidney cell 293T cells, and cultured in a 5% CO ₂ incubator at 37° C. for 16 h. Discard the culture medium containing the transfection mixture, wash with PBS solution, add 2ml of complete medium, and continue to cultivate for 48h. The cell supernatant was collected, and the CentriconPlus-20 centrifugal ultrafiltration device (Millipore) was used to purify and concentrate the lentivirus. The steps were as follows: (1) 4°C, 4000g centrifugation for 10 min to remove cell debris; (2) filter the supernatant with a 0.45 μm filter in In a 40ml ultracentrifuge tube; (3) Centrifuge at 4000g for 10-15min to reach the required virus concentration volume; (4) After centrifugation, separate the filter cup from the filtrate collection cup below, and place the filter cup upside down on the sample On the collection cup, centrifuge for 2 minutes and the centrifugal force does not exceed 1000g; (5) Remove the centrifuge cup from the sample collection cup, and the virus concentrate is in the sample collection cup. Store the virus concentrate at -80°C after aliquoting. 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 is the same as that of the RBM17-siRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector is used instead of the pGCSIL-GFP-RBM17-siRNA vector.

Example 2 Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of RBM17 gene

The glioma U251 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 ⁴ /ml) and inoculated in a 6-well plate, and cultured until the cell confluence reached about 30%. According to the multiplicity of infection (MOI, U251:5) value, an appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, and the cells were collected after the infection time reached 5 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 7 for the reverse transcription reaction system, react at 42° C. for 1 h, and then bathe in a water bath at 70° C. for 10 min to inactivate reverse transcriptase).

TP800 Realtime PCR instrument (TAKARA) was used for real-time quantitative detection. The primers of RBM17 gene are as follows: upstream primer 5'-CTGGGGCGAGGACTGTACT-3' (SEQ ID NO: 10) and downstream primer 5'-AGTGGAGACCAGTGACTCAAA-3' (SEQ ID NO: 11). The housekeeping gene GAPDH was used as an internal reference, and the primer sequences were as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 12) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 13). Configure the reaction system according to the ratio in Table 8.

Table 7 Reverse transcription reaction system

Reagent Volume (μl) 5×RT buffer 4.0 10mM dNTPs 2.0 RNasin 0.5 M-MLV-RTase 1.0 DEPC H ₂ O 3.5 Total 11.0

Table 8 Real-timePCR reaction system

Reagent Volume (μl) SYBR premix ex taq: 10.0 Upstream primer (2.5μM): 0.5 Downstream primer (2.5μM): 0.5 cDNA 1.0 ddH ₂ O 8.0 Total 20.0

The program was set as two-step Real-timePCR: pre-denaturation at 95°C for 15s; subsequent denaturation at 95°C for 5s; annealing extension at 60°C for 30s; a total of 45 cycles. Read the absorbance each time during the extension phase. After PCR, denature at 95°C for 1 min, then cool to 55°C to fully bind the DNA double strands. From 55°C to 95°C, increase by 0.5°C in each step, hold for 4s, read the absorbance value at the same time, and make the melting curve. The expression abundance of infected RBM17 mRNA was calculated by 2- ^ΔΔCt analysis. Cells infected with a control virus (Lv-Scr-siRNA) served as a control. The experimental results ( FIG. 2 ) showed that the expression level of RBM17 mRNA in glioma U251 cells was down-regulated by 67.1%.

Example 3 Detection of proliferation ability of tumor cells infected with RBM17-siRNA lentivirus

The glioma U251 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 ⁴ /ml) and inoculated in a 6-well plate, and cultured until the cell confluence reached about 30%. According to the multiplicity of infection (MOI, U251:5), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 ⁴ /ml), and seeded in a 96-well plate at a cell density of about 2000 cells/well. 5 replicate wells in each group, 100 μl per well. After laying the plates, culture them in a 37°C, 5% CO ₂ incubator. From the second day after plating, the plate was detected and read once a day with a Cellomics instrument (ThermoFisher), and the plate was continuously detected and read for 5 days. By adjusting the input parameters of Cellomicsarrayscan, the number of cells with green fluorescence in each scanning well plate was accurately calculated, the data was statistically plotted, and the cell proliferation curve was drawn (the results are shown in Figure 3). The results showed that the growth rate of each tumor in the lentivirus infection group was significantly slowed down after the cells were cultured in vitro for 5 days, which was much lower than that of the control group, and the number of viable cells decreased by 83.6%, indicating that RBM17 gene silencing led to tumor cell proliferation Ability is inhibited.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any form and in essence. Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those who are familiar with this profession, without departing from the spirit and scope of the present invention, when they can use the technical content disclosed above to make some changes, modifications and equivalent changes of evolution, are all included in the present invention. Equivalent embodiments; at the same time, all changes, modifications and evolutions of any equivalent changes made to the above-mentioned embodiments according to the substantive technology of the present invention still belong to the scope of the technical solution of the present invention.

Claims (14)

1. The use of an isolated human RBM17 gene in the preparation or screening of tumor therapeutic drugs, or in the preparation of tumor diagnostic drugs. 2. The use according to claim 1, wherein the tumor is selected from glioma. 3. An isolated nucleic acid molecule that reduces RBM17 gene expression in tumor cells, said nucleic acid molecule comprising: A) double-stranded RNA, which contains 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. 4. The isolated nucleic acid molecule of claim 3, wherein the double-stranded RNA comprises a first strand and a second strand, and the first strand and the second strand are complementary to form an RNA dimer , and the sequence of the first strand is substantially identical to the target sequence in 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, so The sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is substantially identical to the target sequence in the RBM17 gene. 5. The isolated nucleic acid molecule according to claim 3 or 4, wherein the target sequence of the RBM17 gene contains the sequence shown in SEQ ID NO:1. 6. The isolated nucleic acid molecule according to claim 3 or 4, 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. 7. The isolated nucleic acid molecule according to claim 3 or 4, wherein the shRNA has a sequence as shown in SEQ ID NO:14. 8. A RBM17 gene interference nucleic acid construct, comprising a gene segment encoding the shRNA in the isolated nucleic acid molecule of any one of claims 3-7, capable of expressing the shRNA. 9. The RBM17 gene interference nucleic acid construct according to claim 8, wherein the RBM17 gene interference nucleic acid construct is an interference lentiviral vector. 10. RBM17 gene interference nucleic acid construct as claimed in claim 9, is characterized in that, described interference lentiviral vector is obtained after the gene fragment of encoding described shRNA is cloned into lentiviral vector, and described lentiviral vector is selected from: 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/ Any of U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP, or pLenti6.2/N-Lumio/V5-GW/lacZ. 11. A RBM17 gene interference lentivirus, which is formed by virus packaging the interference lentiviral vector of any one of claims 9-10 with the assistance of lentiviral packaging plasmids and cell lines. 12. A pharmaceutical composition for preventing or treating tumors, the effective substance of which contains the isolated nucleic acid molecule according to any one of claims 3-7, and the RBM17 gene interference according to any one of claims 8-10. Nucleic acid construct, and/or the RBM17 gene interference lentivirus according to claim 11, and a pharmaceutically acceptable carrier, diluent or excipient. 13. The application of the pharmaceutical composition according to claim 12 in the preparation of tumor therapeutic drugs for the treatment of glioma. 14. A kit for reducing RBM17 gene expression in tumor cells, characterized in that the kit comprises: the isolated nucleic acid molecule of any one of claims 3-7 present in a container, The RBM17 gene interference nucleic acid construct according to any one of claims 8-10, and/or the RBM17 gene interference lentivirus according to claim 11.