Application of RNA polymerase II subunit C as gastric cancer treatment target

Abstract

The invention relates to application of RNA polymerase II subunit C as a gastric cancer treatment target. Specifically, the invention provides an application of an inhibitor of RNA polymerase II subunit C (POLR2C) in preparation of a product for inhibiting proliferation, metastasis and / or invasion of gastric cancer cells, in particular an application in a drug for treating gastric cancer. Also provided herein are products that inhibit gastric cancer cell proliferation, metastasis and / or invasion.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and medicine. Specifically, the present invention relates to the application of RNA polymerase II subunit C (POLR2C) as a target for gastric cancer treatment, and also relates to the application of an inhibitor of RNA polymerase II subunit C (POLR2C) in the preparation of a product for gastric cancer treatment. Background Art

[0002] Cancer is one of the major diseases that seriously affects the quality of life, health and life expectancy of human beings, and also brings great burden to society and economy. With the aging of population and the continuous growth of population, the burden of cancer incidence and mortality is growing rapidly around the world.

[0003] The Global Cancer Statistics 2020, released by CACancer J Clin on February 4, 2021, counted the incidence and mortality rates of 36 types of cancer in 185 countries around the world. According to the report, 19.3 million new cases of cancer were diagnosed worldwide in 2020, a further increase from 18.1 million in 2018; the number of cancer deaths is also expected to reach 10 million, surpassing the 9.6 million in 2018. Among these cancers, gastric cancer has a higher incidence and mortality rate than malignant tumors.

[0004] One of the main reasons for the high mortality rate of gastric cancer is that its metastasis mechanism is complex, it has high invasive activity, and it is easy to metastasize early, which is also a key factor that seriously affects the prognosis of patients. The early symptoms of gastric cancer are not obvious, making it difficult to detect and treat early, which ultimately affects the prediction and prognosis of the disease. Therefore, the study of gastric cancer, especially its metastasis and invasion, has important clinical significance, and it is extremely important to explore more effective treatment methods.

[0005] Due to the individual differences between different gastric cancer patients, the discovery of tumor-specific targets has become a current research hotspot. The use of specific inhibitors for patients with metastatic gastric cancer with high expression of VEGF, PD-L1, HER2, etc. can improve the survival of patients to a certain extent. However, the clinical response rate of these specific inhibitors is generally low. Therefore, some researchers have improved the treatment effect of gastric cancer by targeting multiple targets at the same time. However, this combined treatment undoubtedly increases the toxic side effects and economic burden on patients. Therefore, screening new and more effective markers and targets will help in the precise diagnosis and treatment of cancer.

[0006] RNA polymerase II subunit C (POLR2C) is classified as a transcription machinery. This gene encodes the third largest subunit of RNA polymerase II, which is responsible for the synthesis of messenger RNA in eukaryotes. The product of this gene contains a cysteine-rich region and exists as a heterodimer with another polymerase subunit POLR2J. These two subunits constitute the core subunit of the polymerase. POLR2C is produced in the cytoplasm and participates in the regulation of tissue-specific transcription.

[0007] At present, there are few reports on the correlation between POLR2C and tumors. A study analyzed genes and miRNAs related to gastric cancer resistance through bioinformatics, and found that genes such as POLR2L, POLR2C, POLR2F, APRT and LMAN2 may be related to gastric cancer resistance, and miR-604 related to these genes may become an indicator for the diagnosis of resistant gastric cancer [Zhou D, LiX, Zhao H, et al. Combining multi-dimensional data to identify a key signature (gene and miRNA) of cisplatin-resistant gastric cancer [J]. J Cell Biochem, 2018, 119 (8): 6997-7008.]. However, this document is only related to drug resistance, and no further study was conducted on the correlation between POLR2C itself and gastric cancer treatment. In addition, it has been reported that POLR2C is upregulated in hepatocellular carcinoma (HCC), and increased POLR2C expression is also significantly correlated with advanced HCC stage and shortened disease-free survival of patients. The expression of POLR2C in normal tissues and various cancer tissues was compared in the GEPIA database (Gene Expression Profiling Interactive Analysis). The results showed that POLR2C expression was different in different cancer tissues. Compared with adjacent normal tissues, POLR2C expression was increased in some cancer tissues, but decreased or no significant change was found in some cancer tissues (for example, see Figure 1 A); in gastric cancer tissues, the expression of POLR2C is higher than that in normal tissues (see Figure 1 B) There is no disclosure or suggestion in the art regarding the relevance of POLR2C to gastric cancer treatment.

[0008] In summary, there is no report on the effect of POLR2C on gastric cancer treatment, especially its effect on gastric cancer cell proliferation, metastasis and / or invasion. Therefore, revealing the function of POLR2C in gastric cancer has guiding significance for clinical anti-tumor research. Summary of the invention

[0009] In order to solve the deficiencies mentioned in the above background technology, the purpose of the present invention is to provide the use of POLR2C as a target in the prevention and treatment of gastric cancer, and to provide a new method and target for the treatment of gastric cancer.

[0010] In some aspects of the present application, provided is the use of an inhibitor of RNA polymerase II subunit C (POLR2C) or a vector, cell or organism for producing the inhibitor in the preparation of a product for inhibiting gastric cancer cell proliferation, metastasis and / or invasion.

[0011] In other aspects of the present application, a product for inhibiting the proliferation, metastasis and / or invasion of gastric cancer cells is provided, which comprises an inhibitor of RNA polymerase II subunit C (POLR2C) or a vector, cell or organism for producing the inhibitor.

[0012] In some embodiments, the product of the present application is used for the prevention and / or treatment of gastric cancer.

[0013] In some embodiments, the inhibitor of the present application is one or more selected from the following groups: substances that knock down or knock out POLR2C gene expression, substances that reduce the level of POLR2C nucleic acid and / or polypeptide, and substances that weaken the function of POLR2C nucleic acid and / or polypeptide.

[0014] In some embodiments, the inhibitor of the present application is one or more selected from the following groups: antibodies against POLR2C, interfering RNA (siRNA) against POLR2C, antisense oligonucleotides (ASO) against POLR2C, microRNA (miRNA) against POLR2C, nucleic acid aptamers against POLR2C, decoys against POLR2C, guide RNA (gRNA) against POLR2C, POLR2C antagonists, and POLR2C blockers.

[0015] In some embodiments, the inhibitor of the present application is selected from: siRNA of SEQ ID NO:3, siRNA of SEQ ID NO:4, and siRNA of SEQ ID NO:5.

[0016] In some embodiments, the POLR2C of the present application is a polypeptide molecule having an amino acid sequence shown in SEQ ID NO: 2, or a homologous sequence thereof.

[0017] In some embodiments, the coding molecule of the POLR2C of the present application has the nucleotide sequence shown in SEQ ID NO: 1, or a homologous sequence thereof.

[0018] In some embodiments, the form of the product of the present application is suitable for an administration route selected from the group consisting of enteral administration, parenteral administration, such as intravenous, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal (such as iontophoresis).

[0019] In some embodiments, the form of the product of the present application is suitable for an administration route selected from the following group: direct injection of liposome-encapsulated DNA, gold-coated DNA gene gun bombardment, replication-defective bacteria carrying plasmid DNA, and replication-defective adenovirus carrying target DNA.

[0020] In some embodiments, the subject of administration of the product of the present application is a mammal, such as primates, rodents, livestock, pets, etc., preferably humans, rats, mice, dogs, horses, cows, rabbits or monkeys.

[0021] In some embodiments, the product of the present application is selected from: medicines, health products, kits, medical devices, or combinations thereof.

[0022] In some embodiments, the product of the present application further comprises a pharmaceutically acceptable carrier.

[0023] In some embodiments, the product of the present application is combined with one or more other tumor immunotherapy drugs or therapies, such as tumor vaccines, adoptive cell immunotherapy, T-cell targeted immunomodulatory drugs, immune checkpoint inhibitors (ICI), such as PD1 / PDL1 inhibitors (such as PD1 antibodies, pembrolizumab).

[0024] In some aspects of the present application, a method for inhibiting the proliferation, metastasis and / or invasion of gastric cancer cells is provided, which comprises administering to a subject in need thereof an effective amount of an inhibitor of RNA polymerase II subunit C (POLR2C) or a vector, cell or organism for producing the inhibitor and / or a product of the present application.

[0025] Those skilled in the art may arbitrarily combine the above technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention are obvious to those skilled in the art due to the disclosure of this article. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below in conjunction with the accompanying drawings, wherein the accompanying drawings are only for illustrating the embodiments of the present invention rather than for limiting the scope of the present invention.

[0027] Figure 1: Comparison of POLR2C expression levels recorded in the GEPIA database:

[0028] Figure 1 A: Comparison of POLR2C expression in various tumor tissues and normal tissues, where STAD is gastric cancer;

[0029] Figure 1 B: Comparison of POLR2C expression levels in gastric cancer tissues (left) and normal tissues (right), where num(T) is the number of gastric cancer tissues and num(N) is the number of normal tissues.

[0030] Figure 2 :Immunohistochemical staining analysis of POLR2C expression in gastric cancer tissues and adjacent tissues:

[0031] Figure 2 A: Immunohistochemical staining photos of POLR2C in adjacent paracancerous tissues and gastric cancer tissues (magnification: 200X);

[0032] Figure 2 B: Comparison of immunohistochemical expression of POLR2C in adjacent paracancerous tissues and gastric cancer tissues.

[0033] Among them, ***, p<0.001.

[0034] Figure 3 :Comparison of POLR2C levels in normal gastric tissue cells (GES1) and six gastric cancer cells (BGC823, HGC27, MGC803, AGS, SGC7901, MKN45):

[0035] Figure 3 A: q-PCR was used to compare the POLR2C mRNA levels in different cells;

[0036] Figure 3 B: Western blot results of POLR2C in different cells;

[0037] Figure 3 C: Comparison of protein expression levels of POLR2C in different cells by Western blot assay;

[0038] Among them, *, p<0.05; **, p<0.01; ***, p<0.001.

[0039] Figure 4 : Identification of POLR2C overexpressing gastric cancer stable cell lines, where the mock was the stable cell line monoclonal #4 screened after AGS cells were transfected with the blank vector pcDNA3.1, and the overexpression was the overexpression stable cell line monoclonal #1, #3, #4 and #5.

[0040] Figure 5 : POLR2C-siRNA interference efficiency determination:

[0041] Figure 5 A: Relative expression level of POLR2C after interference with POLR2C using siRNA-1, -2 and -3;

[0042] Figure 5 B: Western blot photo after POLR2C interference;

[0043] The control was MGC803 cells transfected with negative control siRNA (sequence: 5-UUCUCCGAACGUGUCACGUTT-3); ***, p<0.001.

[0044] Figure 6 :Effect of POLR2C on the growth of gastric cancer cells in vitro:

[0045] Figure 6 A: Effect of POLR2C overexpression on gastric cancer cell proliferation, where the negative control is a stable cell line (monoclonal cell #4) screened after AGS cells were transfected with blank vector pcDNA3.1;

[0046] Figure 6 B: Effect of POLR2C knockdown on gastric cancer cell proliferation, where the control is MGC803 cells transfected with negative control siRNA;

[0047] Among them, ***, p<0.001.

[0048] Figure 7 :Effect of POLR2C on the invasion of gastric cancer cells AGS:

[0049] Figure 7 A: Invasion photos of POLR2C overexpressing cells and negative control cells (200×);

[0050] Figure 7 B: Effect of POLR2C overexpression on the invasion of gastric cancer cells;

[0051] Among them, the negative control was a stable cell line (monoclonal cell #4) screened after AGS cells were transfected with blank vector pcDNA3.1; ***, p<0.001.

[0052] Figure 8 :Effect of POLR2C on the invasion of gastric cancer cell MGC803:

[0053] Figure 8 A: Invasion photos of POLR2C knockdown cells and control cells (200×);

[0054] Figure 8 B: Effect of POLR2C knockdown on invasion of gastric cancer cells;

[0055] The control was MGC803 cells transfected with negative control siRNA; **, p<0.01.

[0056] Fig. 9 :Effect of POLR2C on the migration of gastric cancer cell MGC803:

[0057] Fig. 9 A: Migration photos of POLR2C knockdown cells and control cells (40×);

[0058] Fig. 9 B: Effect of POLR2C knockdown on gastric cancer cell migration;

[0059] The control was MGC803 cells transfected with negative control siRNA; **, p<0.01. DETAILED DESCRIPTION

[0060] The present application discloses a new target, POLR2C, which can be used for the prevention and / or treatment of gastric cancer. The present application also discloses the use of an inhibitor of POLR2C in inhibiting the proliferation, invasion and / or metastasis of gastric cancer cells. The present application also provides the use of an inhibitor of POLR2C in the prevention and / or treatment of gastric cancer.

[0061] Specifically, the applicant discovered the correlation between POLR2C and gastric cancer cell proliferation, invasion and metastasis through long-term and in-depth research: the expression of POLR2C in gastric cancer tissue and adjacent tissue was analyzed by immunohistochemical staining, and the results showed that POLR2C was highly expressed in gastric cancer tissue; q-PCR and Western blot methods were used to detect and determine the high expression of POLR2C at the mRNA and protein levels in gastric cancer cells; CCK8 experiments proved that high expression of POLR2C can promote the proliferation of gastric cancer cells, while low levels of POLR2C can inhibit the proliferation of gastric cancer cells; Transwell and scratch experiments respectively proved that high expression of POLR2C in gastric cancer cells enhances the invasion and migration ability of cells, while the invasion and migration ability of gastric cancer cells is significantly reduced after POLR2C is inhibited. On this basis, the new target for the prevention and / or treatment of gastric cancer, POLR2C, is disclosed for the first time in this application, and the role of inhibiting POLR2C in inhibiting the proliferation, invasion and / or metastasis of gastric cancer cells is disclosed, thereby providing a new way to prevent and treat gastric cancer.

[0062] All numerical ranges provided herein are intended to clearly include all values ​​falling between the range endpoints and the numerical ranges therebetween. Features mentioned in the present invention or in the embodiments may be combined. All features disclosed in this specification may be used in combination with any form of composition, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0063] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0064] POLR2C protein (polypeptide) and its coding sequence

[0065] As used herein, the terms "POLR2C (polypeptide)", "POLR2C protein (polypeptide)", and "POLR2C" are used interchangeably and refer to a class of RNA polymerase II subunit C protein, which contains a cysteine-rich region and can exist as a heterodimer with another polymerase subunit POLR2J to form the core subunit of the polymerase.

[0066] The POLR2C protein herein may be a human or non-human mammal POLR2C protein, such as a protein encoded by the sequence of SEQ ID NO: 1 (full-length human sequence) or a homologous sequence (such as a homologous sequence of POLR2C that can be obtained through a database or alignment software known in the art), variant or modified form of these proteins that have the same or similar RNA polymerase subunit function. For example, the POLR2C protein may be selected from: (a) the amino acid sequence shown in SEQ ID NO: 2; or (b) a protein or polypeptide derived from (a) that has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (a) and has the function of an RNA polymerase subunit. The POLR2C protein or polypeptide in the present invention is preferably encoded by the human POLR2C gene or its homologous gene or family gene.

[0067] As used herein, the terms "POLR2C gene", "POLR2C coding sequence / molecule" or "POLR2C protein coding sequence / molecule" are used interchangeably, and all refer to a sequence or molecule encoding the POLR2C protein or polypeptide described in the present invention. In the present application, the POLR2C coding molecule may be a nucleotide sequence shown in SEQ ID NO: 1 (human full length), a molecule that hybridizes with these sequences under stringent conditions, or a family gene molecule that is highly homologous to the above-mentioned molecule, and the expression of the gene produces a functional POLR2C protein. The POLR2C gene of the present invention may be selected from: (i) a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO: 1; (ii) a codon-optimized or degenerate sequence of the sequence in (i); or (iii) a molecule that hybridizes with the sequence defined in (i) or (ii) under stringent conditions and is expressed to produce a functional POLR2C protein.

[0068] As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more. For example, the sequence may be the complementary sequence of the sequence defined in (i) or (ii).

[0069] The full-length nucleotide sequence of the POLR2C gene of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0070] It should be understood that the POLR2C gene of the present invention is preferably obtained from humans, and other genes obtained from other animals that are highly homologous to the human POLR2C gene (such as having a sequence identity of 50% or more, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, and more preferably 85% or more, such as 85%, 90%, 95%, 98% or even 99% or more) are also within the preferred equivalent range of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0071] The coding sequence of the POLR2C protein involved in this article can be, for example, the sequence of NM_032940.3 (SEQ ID NO:1, 828bp):

[0072] atgccgtacgccaaccagcctaccgtgcggatcacggagctcactgacgagaatgtcaagttcatcatcgagaacaccgacctggcggtggccaattcgattcggagggtcttcatcgctgaggttcccataatagccattgactgggttcagattgatgccaattcctcagttcttcatgatgaattcattgctcacaggcttggattaattcccctcattagtgatgacattgtggacaagctgcagtactctcgggactgcacatgtgaggagttctgccccgagtgctcggtggagttcaccctcgatgtgcggtgcaatgaagaccagacgcgacatgtcacgtctcgagacctcatctccaacagcccccgggtcattccggtgacatcccggaaccgagataatgaccccaatgactacgtggagcaggatgacatcctcatcgtcaagttgagaaagggccaggagctgagacttcgagcctatgccaaaaagggctttggcaaggagcatgccaagtggaaccctactgcaggggtggcttttgaatacgatccagacaatgccctgaggcacacagtgtaccccaagcccgaggaatggccaaagagtgagtactcggagctggatgaggatgagtcgcaggctccctatgaccccaacggcaagccagaaaggttttactacaatgtggagtcctgtggctctctgcgtcctgaaaccattgtcctgtcagccctctcaggattgaagaagaaactgagtgatttacaaactcaattaagccacgagatccagagtgatgtgctaaccataaattaa

[0073] The sequence of the POLR2C protein involved in this article can be, for example, the sequence of NP_116558.1 (SEQ ID NO:2, 275aa):

[0074] MPYANQPTVRITELTDENVKFIIENTDLAVANSIRRVFIAEVPIIAIDWVQIDANSSVLHDEFIAHRLGLIPLISDDIVDKLQYSRDCTCEEFCPECSVEFTLDVRCNEDQTRHVTSRDLISNSPRVIPVTSRNRDN DPNDYVEQDDILIVKLRKGQELRLRAYAKKGFGKEHAKWNPTAGVAFEYDPDNALRHTVYPKPEEWPKSEYSELDEDESQAPYDPNGKPERFYYNVESCGSLRPETIVLSALSGLKKKLSDLQTQLSHEIQSDVLTIN

[0075] In some embodiments, the polypeptides contemplated herein include polypeptides encoded by known POLR2C polypeptide encoding molecules, polypeptides encoded by encoding molecules that differ from the disclosed encoding molecule sequences due to the degeneracy of the genetic code. Therefore, the scope of the present invention includes polypeptides encoded by polynucleotides having any of the polynucleotide sequences provided herein, or polypeptides encoded by variants thereof.

[0076] The variant forms of the protein or polypeptide of the present invention include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid deletion, insertion and / or substitution, and the addition of one or more (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein or polypeptide is usually not changed. For another example, the addition of one or more amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein or polypeptide. For example, the POLR2C protein or polypeptide of the present invention may or may not include the initial methionine residue and still have the function of RNA polymerase subunit.

[0077] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by sequences that can hybridize with the POLR2C protein coding sequence under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the POLR2C protein.

[0078] Variants may include fragments of the polypeptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. The fragments of interest will generally be at least about 10 amino acids to at least about 15 amino acids in length, at least about 50 amino acids in length, and may be up to 300 amino acids or longer. The protein variants described herein are encoded by polynucleotides within the scope of the invention. The genetic code can be used to select appropriate codons to construct corresponding variants.

[0079] Inhibitors of POLR2C or its coding sequence

[0080] The present invention also relates to an "inhibitor" of POLR2C or its coding sequence. The terms "inhibitor" or "inhibitor of POLR2C or its coding sequence" are used interchangeably and refer to a substance that can reduce the level or activity of POLR2C or its coding sequence. The inhibitors of the present invention include, but are not limited to: antibodies against POLR2C, interfering RNA (siRNA) against POLR2C, antisense oligonucleotides (ASO) against POLR2C, microRNA (miRNA) against POLR2C, nucleic acid aptamers against POLR2C, decoys against POLR2C, guide RNA (gRNA) against POLR2C, POLR2C antagonists, and POLR2C blockers. In some embodiments, the inhibitors used in the present invention include, but are not limited to, antibodies, siRNA, miRNA, antisense oligonucleotides, antagonists, blockers, etc. against POLR2C or its coding sequence. The inhibitors of the present invention can reduce the level of POLR2C coding molecules or proteins, and can be further used to inhibit the proliferation, migration and / or invasion of gastric cancer cells, and become effective substances for the treatment of gastric cancer.

[0081] In some embodiments, the POLR2C inhibitor is a POLR2C-specific antibody or a functional fragment thereof. The term "antibody" is used in its broadest sense and covers fully assembled polyclonal antibodies and monoclonal antibodies, as well as antibody fragments that can bind to antigens (e.g., Fab', F'(ab)2, Fv, single-chain antibodies, diabodies, etc.). In some embodiments, the antibodies of the present application include humanized antibodies, human antibodies, bispecific antibodies, trispecific antibodies, etc.

[0082] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for a small number of possible spontaneous mutations. For example, monoclonal antibodies can be prepared by hybridoma methods or can be prepared by recombinant DNA methods. In some embodiments, antibodies or antibody fragments are isolated from antibody phage libraries. In some embodiments, high affinity (nM order of magnitude) human antibodies are produced by chain shuffling, etc. In some embodiments, recombinant antibodies against POLR2C can be produced in transgenic animals (e.g., rodents).

[0083] In some embodiments, the POLR2C inhibitor is its siRNA, such as the siRNA described in any one of SEQ ID NOs: 3 to 5. The RNAi molecule used to achieve RNAi-mediated gene expression regulation may include single-stranded or multi-stranded polymerized ribonucleotides. It may contain any number of modifications, such as modifications to the phosphate-sugar backbone, to the sugar group, or to the base to enhance stability or binding affinity. For example, the phosphodiester bond of natural RNA may be modified to contain at least one nitrogen or sulfur heteroatom. When avoiding the common emergency response produced by dsRNA in certain tissues, the modifications in the RNA structure may be deliberately adjusted so that specific genes are inhibited. Similarly, the bases may also be modified to block the activity of adenosine deaminase. RNA may be produced enzymatically or by partial / complete organic synthesis. Any modified ribonucleotide may be incorporated into RNA by in vitro enzymatic or organic synthesis. The RNAi molecule may be a dsRNA molecule.

[0084] Typically, the siRNA is at least about 10 to about 15 nucleotides long. In some applications, the RNA is less than about 20, 21, 22, 23, 24, or 25 nucleotides in length. In other cases, the RNA is at least about 50, 100, 150, or 200 nucleotides in length. In certain other applications, the RNA may be longer, for example, at least about 300, 400, 500, or 600 polynucleotides.

[0085] In some embodiments, the POLR2C inhibitor is an antisense oligonucleotide thereof. The phrase "POLR2C antisense oligonucleotide" refers to an oligonucleotide containing a nucleic acid sequence that interacts with a complementary nucleic acid sequence involved in POLR2C expression through base pairs, and the expression of POLR2C is downregulated through the interaction. Preferably, the specific nucleic acid sequence involved in POLR2C expression is a genomic DNA molecule or mRNA molecule encoding POLR2C. The genomic DNA molecule may contain a regulatory region of the POLR2C gene, and / or a coding sequence for a mature POLR2C protein. In some embodiments, the antisense oligonucleotide may include modifications, such as oligonucleotides including modified backbones and / or non-natural bases and / or sugar modifications. In some embodiments, the antisense oligonucleotide comprises a sequence of about 8 to about 100 nucleotides, more preferably the antisense oligonucleotide comprises about 15 to about 30 nucleotides or about 18 to about 26 nucleotides. Antisense oligonucleotides may also contain various modifications, for example, modifications that confer resistance to nucleolytic degradation on the antisense oligonucleotides, such as modified internucleoside linkages, modified nucleobases and / or sugar groups, and the like.

[0086] The test for inhibition of target gene expression can be performed by observing or detecting the absence or observable decrease in the level of protein encoded by the target gene (which can be detected by, for example, specific antibodies or other methods known to those skilled in the art); and / or observing or detecting the mRNA product of the target gene (which can be detected by, for example, hybridization studies); and / or observing or detecting a phenotype associated with gene expression. In the context of drug treatment, the test for inhibition of target gene expression can be performed by observing changes in the disease state of the subject (e.g., alleviation, disappearance of symptoms, changes in disease state, etc.). According to certain embodiments, the change in disease state can be a reduction in tumor growth, a reduction in tumor volume, a reduction in cell activity, a reduction in cell invasiveness, etc. Preferably, the inhibition is specific, that is, the inhibition of the target gene does not significantly affect other genes of the cell.

[0087] Host cells

[0088] When the POLR2C inhibitor comprises or is an oligonucleotide or polypeptide product, the present invention also relates to a vector for producing the POLR2C inhibitor, a host cell produced by genetic engineering using the vector, and a transgenic animal that highly expresses the POLR2C inhibitor or its components obtained by genetic engineering.

[0089] The coding sequence of the present invention can be used to express or produce recombinant POLR2C inhibitors by conventional recombinant DNA technology (Science, 1984; 224: 1431). In the present invention, the term "vector" and "recombinant expression vector" are used interchangeably to refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. In short, as long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0090] Methods well known to those skilled in the art can be used to construct expression vectors containing POLR2C inhibitor coding sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. The present invention preferably uses pcDNA3.1 vectors, pIRES2-EGFP vectors, and AdMaxTM expression systems. In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0091] The vector containing the above-mentioned appropriate DNA sequence and the appropriate promoter or control sequence can be used to transform the appropriate host cell to enable it to express the protein or polypeptide. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell. Representative examples include: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, etc. In the present invention, Escherichia coli bacterial cells and mouse dendritic cells are preferably used as host cells.

[0092] The recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0093] Drug, pharmaceutical composition or kit

[0094] The present invention also provides a medicament, a pharmaceutical composition or a kit, which contains an effective amount of the POLR2C inhibitor of the present invention and a pharmaceutically or immunologically acceptable carrier. As used herein, the terms "active substance" or "active substance of the present invention" are used interchangeably and refer to the POLR2C inhibitor.

[0095] In a preferred embodiment, the pharmaceutical composition can be used to prevent or treat diseases and / or symptoms associated with overexpression or overactivation of POLR2C, such as gastric cancer.

[0096] In some embodiments, gastric cancer is selected from: early gastric cancer; or advanced gastric cancer, such as polyp type, localized ulcer type, infiltrating ulcer type or diffuse infiltrating gastric cancer. In some embodiments, gastric cancer is selected from: adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, carcinoid, including their metastases. In some embodiments, gastric cancer is gastric adenocarcinoma or its metastases. In some embodiments, gastric cancer is selected from: well-differentiated, moderately differentiated, poorly differentiated or undifferentiated gastric cancer. In some embodiments, gastric cancer is selected from: untreated gastric cancer, or gastric cancer that has been treated. In some embodiments, the product of the present application delays or prevents or treats the progression of gastric cancer (e.g., infiltration and / or spread).

[0097] As used herein, the term "comprising" or "including" includes "including", "consisting essentially of", and "consisting of". As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that can produce a function or activity in humans and / or animals and can be accepted by humans and / or animals.

[0098] The terms "individual", "subject", "host" and "patient" are used interchangeably and refer to any mammalian subject, especially a human, for whom diagnosis, treatment or therapy is desired. Other subjects may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc.

[0099] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not necessary active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0100] The pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffer substances, etc. may also be present in these carriers. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, the pH is about 6-8.

[0101] The active substance in the composition of the present invention accounts for 0.001-99.9wt% of the total weight of the composition, preferably 1-95wt%, more preferably 5-90wt%, and more preferably 10-80wt%, and the remainder is pharmaceutically acceptable carriers and other additives.

[0102] As used herein, the term "unit dosage form" means that for the convenience of taking, the composition of the present invention is prepared into a dosage form required for a single dose, including but not limited to various solid doses (such as tablets), liquid doses, capsules, and sustained-release preparations.

[0103] In another preferred embodiment of the present invention, the composition is in unit dosage form or multiple dosage form. In another preferred embodiment of the present invention, 1 to 6 doses of the composition of the present invention are administered every day, every two days, every three days, every week, every two weeks, or every month, preferably 1 to 3 doses, for example, 1 dose is administered.

[0104] It should be understood that the effective dose of the active substance such as the POLR2C inhibitor used may vary depending on the severity of the subject to be administered or treated. The specific situation is determined according to the individual situation of the subject (e.g., the subject's weight, age, physical condition, and the desired effect), which is within the scope of judgment of a skilled physician.

[0105] The composition of the present invention may be in solid form (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (such as oral solution) or other suitable forms. The administration routes can be: (1) direct naked DNA or protein injection; (2) connecting the cDNA, mRNA and protein of POLR2C inhibitors to transferrin / poly-L-lysine complexes to enhance their biological effects; (3) forming complexes between cDNA, mRNA and proteins and positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) encapsulating cDNA, mRNA and proteins in liposomes and mediating their entry into cells, which is beneficial for the smooth entry of large molecules and protects them from hydrolysis by various extracellular enzymes; (5) combining cDNA, mRNA and proteins with cholesterol to increase their cytoplasmic retention time by 10 times; (6) transporting cDNA, mRNA and proteins with immunoliposomes to enable their specific transport to target tissues and target cells; (7) in vitro transfection of cDNA, mRNA and proteins into transfer cells (such as fibroblasts) can also better load POLR2C-related drugs into target cells; (8) electroporation, that is, introducing cDNA, mRNA and proteins into target cells with the help of electric current.

[0106] In addition, the composition of the present invention may also contain other active substances for improving and treating cancer. Alternatively, according to the present invention, treatment may also include combination therapy. "Combination therapy" as used herein refers to treating a patient who needs a drug in combination with the inhibitor of the present invention or administering another drug to treat the disease. Combination therapy can be a sequential therapy, that is, treating the patient with one or more drugs first, and then administering another, or administering two or more drugs simultaneously. Some other active substances that can be used in the composition or examples of drugs used in combination therapy include: chemotherapeutic agents (cisplatin, doxorubicin, danurubicin, tamoxifen, paclitaxel, methotrexate, etc.), aromatase inhibitors, angiogenesis inhibitors, specific and non-specific immunomodulators, biological response modifiers (BRMs), colony stimulating factors (CSFs), interferons, interleukins, autologous tumor cell vaccines, hormones, etc. Preferably, the drugs or other preparations administered in combination do not interfere with the therapeutic activity of the inhibitor of the present invention.

[0107] Example

[0108] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make appropriate modifications and changes to the present invention, and these modifications and changes are within the scope of the present invention.

[0109] The experimental methods for which the specific conditions are not specified in the following examples can be conventional methods in the art, for example, refer to "Molecular Cloning Laboratory Manual" (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or follow the conditions recommended by the supplier. The DNA sequencing method is a conventional method in the art, and sequencing can also be provided by commercial companies.

[0110] Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0111] Example 1: Expression of POLR2C in gastric cancer tissue

[0112] 1. Experimental Materials

[0113] Gastric cancer tissue chip: purchased from Shanghai Xinchao Biotechnology, array number: HStmA150CS02 (150 points);

[0114] POLR2C primary antibody: purchased from abcam, #ab182150;

[0115] Horseradish peroxidase-labeled secondary antibody: purchased from Dako, #SM802.

[0116] (II) Experimental methods

[0117] The gastric cancer tissue chip was baked at 56°C overnight, and then the chip was dewaxed and hydrated. The POLR2C antigen in the slice was renatured in antigen retrieval solution (pH 9.0); the slide was incubated with rabbit anti-POLR2C (1:10000, monoclonal antibody, abcam) at 4°C overnight. After washing with phosphate buffer, the secondary antibody was incubated at room temperature for 30 minutes. Finally, the slice was stained with diaminobenzidine (DAB), counterstained with hematoxylin, and sealed with neutral gum, and then photographed and observed.

[0118] The immunohistochemical results of POLR2C were scored by staining intensity (no staining, 0; light yellow, 1; yellow, 2; brown, 3) and percentage of positive cells (≤24%, 1; 25-49%, 2; 50-74%, 3; ≥75%, 4).

[0119] (III) Experimental results

[0120] The expression of POLR2C in gastric cancer tissues and adjacent tissues was analyzed by immunohistochemical staining. Figure 2 shown.

[0121] The results showed that the expression of POLR2C in gastric cancer tissues was significantly higher than that in adjacent adjacent tissues. The above results indicate that POLR2C is highly expressed in gastric cancer tissues.

[0122] Example 2: Expression of POLR2C in Gastric Cancer Cell Lines

[0123] 1. Experimental Materials

[0124] Gastric cancer cell BGC823 (human poorly differentiated gastric adenocarcinoma cells, purchased from Shanghai Fuheng Biotechnology, cat# FH0262)

[0125] Gastric cancer cell HGC27 (human undifferentiated gastric cancer cells, purchased from Shanghai Fuheng Biotechnology, cat# FH0271)

[0126] Gastric cancer cell MGC803 (primary poorly differentiated mucinous gastric adenocarcinoma cell line established in China, purchased from Shanghai Fuheng Biotechnology, cat# FH0269)

[0127] Gastric cancer cell AGS (human gastric adenocarcinoma cells, ATCC source, purchased from Shanghai Fuheng Biotechnology, cat# FH0261)

[0128] Gastric cancer cell line SGC7901 (human gastric adenocarcinoma lymph node metastasis cell line, established in China, purchased from Shanghai Fuheng Biotechnology, cat# FH0264)

[0129] Gastric cancer cell MKN45 (poorly differentiated gastric adenocarcinoma liver metastasis cells, purchased from Shanghai Fuheng Biotechnology, cat# FH0267);

[0130] Human gastric epithelial cells GES1 (purchased from Shanghai Fuheng Biotechnology, cat# FH0273):

[0131] qPCR detection reagents were purchased from Roche;

[0132] Reverse transcription reagents were purchased from TOYOBO;

[0133] POLR2C antibody was purchased from abcam, #ab182150 (same as the primary antibody in Example 1).

[0134] (II) Experimental methods

[0135] q-PCR and Western blot were used to detect the mRNA and protein expression of POLR2C in six gastric cancer cells (BGC823, HGC27, MGC803, AGS, SGC7901, MKN45). In short, gastric cancer cells were lysed by Trizol and their RNA was extracted, reverse transcribed into cDNA, and qPCR was used to detect the expression of POLR2C. At the same time, the cells were collected and the protein was extracted by SDS lysis, and the expression of POLR2C was detected by Western blot. The mRNA and protein expression of POLR2C in the six gastric cancer cells detected by the above q-PCR and Western blot methods were compared with their expression levels in the normal gastric mucosal epithelial cell GES1 control to analyze the expression differences.

[0136] (III) Experimental results

[0137] q-PCR and Western blot results are as follows Figure 3 The results showed that POLR2C was expressed in normal gastric mucosal cells and various gastric cancer cell lines.

[0138] Example 3: Construction of POLR2C overexpressing stable cell line

[0139] 1. Experimental Materials

[0140] Plasmid pcDNA3.1 / POLR2C-over: Using AGS cell cDNA as a template, PCR amplification (high-fidelity enzyme purchased from Nanjing Novozymes Biotech Co., Ltd., cat#P515) POLR2C, homologous recombination (homologous recombination enzyme purchased from Nanjing Novozymes Biotech Co., Ltd., cat#C112-02) into the pcDNA3.1 vector (double-digested by EcoRI and BamHI), transformed into DH5α competent cells (purchased from Nanjing Novozymes Biotech Co., Ltd., cat#C502), positive clones were sent for sequencing (sequencing by Genewise Biotech Co., Ltd.), and clones with correct sequencing results were amplified.

[0141] jetPEI plasmid transfection reagent was purchased from Polyplus;

[0142] G418 was purchased from Shenggong Biotechnology Co., Ltd.

[0143] (II) Experimental methods

[0144] AGS gastric cancer cells were plated in 6-well plates, 2 × 10 5cells / well, and transfected with 2 μg plasmid (pcDNA3.1 / POLR2C-over) on the second day. After 48 hours, the screening concentration medium (containing G418, 800 μg / ml) was added. After about 14 days of screening, resistant clones appeared. The 96-well monoclonal dilution method was used to screen stable cell lines. After the cells were collected, the protein was extracted by SDS lysis method, and the expression of POLR2C was detected by Western blot.

[0145] (III) Experimental results

[0146] The experimental results are as follows Figure 4 shown.

[0147] The results showed that the above method successfully obtained the AGS gastric cancer cell line stably overexpressing POLR2C. The AGS#5 gastric cancer cell line stably overexpressing POLR2C was selected for subsequent experiments.

[0148] Example 4: POLR2C expression and efficiency detection after transient siRNA interference

[0149] 1. Experimental Materials

[0150] The three siRNAs specific to the POLR2C gene and the unrelated negative control siRNA were synthesized by Shanghai Genema Gene:

[0151] siRNA1 sequence (SEQ ID NO:3) :

[0152] GCUCACAGGCUUGGAUUAAUU;

[0153] siRNA2 sequence (SEQ ID NO:4) :

[0154] CCUCGAUGUGCGGUGCAAUUU;

[0155] siRNA3 sequence (SEQ ID NO:5) :

[0156] GCUGAGACUUCGAGCCUAUUU;

[0157] Negative control siRNA (SEQ ID NO: 6) :

[0158] UUCUCCGAACGUGUCACGUUU.

[0159] siRNA transfection reagent: purchased from Polyplus;

[0160] q-PCR detection reagents: purchased from Roche;

[0161] Reverse transcription reagent: purchased from TOYOBO.

[0162] (II) Experimental methods

[0163] After MGC803 cells were transfected with POLR2C siRNA for 48 hours, the cells were collected and lysed with Trizol, RNA was extracted, reverse transcribed into cDNA, and the interference efficiency of POLR2C siRNA was identified by q-PCR.

[0164] MGC803 cells were transfected with POLR2C siRNA for 72 hours, and the protein was extracted by SDS lysis method. The expression of POLR2C was detected by Western blot.

[0165] (III) Experimental results

[0166] The experimental results are as follows Figure 5 shown.

[0167] The results showed that all three POLR2C siRNAs had good interference effects, and POLR2C siRNA-3 will be used for subsequent experiments.

[0168] Example 5: Effect of POLR2C on the proliferation of gastric cancer cells in vitro

[0169] 1. Experimental Materials

[0170] CCK8 reagent was purchased from Dakoway Biotechnology Co., Ltd.

[0171] (II) Experimental methods

[0172] MGC803 cells interfered with by siRNA-3 and AGS#5 cells overexpressing POLR2C were plated in 96-well plates, and 2-5×10 3 Cells / well, 100μl culture medium, routine culture (set 3-5 duplicate wells, add culture medium blank control group), add 10μl CCK8 working solution to each well, routine culture for 1-4h, use microplate reader to measure the light absorption value of each well at 450nm wavelength, and adjust to zero with blank control. The light absorption value of each experimental group is detected every day, with light absorption value as the vertical axis and each action time as the horizontal axis. The experiment is terminated on the 7th day, and the growth curve of each experimental group is drawn.

[0173] (III) Experimental results

[0174] The CCK8 experiment observed the effect of POLR2C expression changes on the proliferation ability of gastric cancer cells in vitro for 7 consecutive days. Figure 6 shown.

[0175] The results showed that overexpression of POLR2C in gastric cancer cells significantly promoted cell proliferation, while after knocking down POLR2C expression in gastric cancer cells, cell proliferation was significantly lower than that in the control group.

[0176] The above results show that overexpression of POLR2C in gastric cancer cells can significantly promote cell proliferation, while reduced expression can inhibit cell proliferation. Therefore, inhibiting POLR2C expression can inhibit the proliferation of gastric cancer cells, thereby achieving the effect of gastric cancer treatment.

[0177] Example 6: Effect of POLR2C on the invasion of gastric cancer cells

[0178] 1. Experimental Materials

[0179] Matrigel and Transwell chambers: purchased from Corning;

[0180] Rapid Wright-Giemsa stain: purchased from Nanjing Jiancheng Technology Co., Ltd.

[0181] (II) Experimental methods

[0182] Matrigel was dissolved at 4°C overnight, and diluted with serum-free medium on ice (1:5-1:10). 100 μl of the diluted Matrigel was added to the upper chamber of a 24-well Transwell chamber (8 μm). After incubation in an incubator for 1-5 hours, the gel was washed with serum-free medium to fully hydrate it. Cells from each group in the logarithmic growth phase were routinely digested with trypsin and resuspended in serum-free medium. The cell density of AGS#5 cells overexpressing POLR2C was adjusted to 2×10 5 / ml, and the density of MGC803 cells knocking down POLR2C expression by siRNA-3 was adjusted to 5×10 5 / ml. Set up three replicate wells, take 200μl of cell suspension and add it to the upper chamber of the Transwell chamber covered with Matrigel, and add 600μl of culture medium containing 10% FBS to the lower chamber, and culture for 24h. Take out the Transwell chamber, remove the culture medium in the upper chamber, wipe off the cells on the inner membrane surface of the chamber with a cotton swab, stain with Giemsa stain at room temperature for 3min, stop staining for 8min, wash 3 times with PBS, and air dry naturally at room temperature. Randomly select several fields of view under a microscope, take pictures at 200× and count the number of cells that have penetrated the membrane.

[0183] (III) Experimental results

[0184] The experimental results are as follows Figure 7 and Figure 8 shown.

[0185] The results showed that the invasive ability of gastric cancer cells was significantly increased after overexpression of POLR2C. Figure 7 ); When POLR2C expression was knocked down in gastric cancer cells, the invasion ability of cells was significantly reduced ( Figure 8 ).

[0186] The above results indicate that inhibiting POLR2C expression can inhibit the invasion of gastric cancer cells, thereby achieving the effect of inhibiting the development of gastric cancer.

[0187] Example 7: Effect of POLR2C on the Migration of Gastric Cancer Cells

[0188] 1. Experimental Materials

[0189] Rapid Wright-Giemsa stain: purchased from Nanjing Jiancheng Technology Co., Ltd.

[0190] (II) Experimental methods

[0191] MGC803 cells with POLR2C knockdown by siRNA-3 were plated in 24-well plates, and 2 × 10 5 cells / well, 1 ml of culture medium, when the confluence is >90%, replace with serum-free culture medium, scratch vertically with 200 μl pipette tip after 24 hours, wash 3 times with PBS, add serum-free culture medium, culture conventionally, terminate after 24 hours, stain, take pictures, and measure the scratch distance or healing area.

[0192] (III) Experimental results

[0193] The results are as follows Fig. 9 shown.

[0194] The results showed that when POLR2C expression was knocked down in gastric cancer cells, the migration ability of cells was significantly decreased.

[0195] The above results indicate that inhibiting the expression of POLR2C can inhibit the migration of gastric cancer cells, thereby achieving the effect of inhibiting the development of gastric cancer.

[0196] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0197] Attachment: Sequence information

[0198]

[0199] Note: According to the WIPO Sequence requirements, "U" in siRNA in the ST26 sequence list is recorded as "T".

[0200] Therefore, the sequences of the siRNAs of SEQ ID NOs: 3 to 6 provided herein are consistent with the sequences in the computer readable sequence listing.

Claims

1. Use of an inhibitor of RNA polymerase II subunit C (POLR2C) or a vector, cell or organism for producing the inhibitor in the preparation of a product for inhibiting gastric cancer cell proliferation, metastasis and / or invasion.

2. A product for inhibiting the proliferation, metastasis and / or invasion of gastric cancer cells, comprising an inhibitor of RNA polymerase II subunit C (POLR2C) or a vector, cell or organism for producing the inhibitor.

3. The use according to claim 1 or the product according to claim 2, wherein: The inhibitor is one or more selected from the following group: a substance that knocks down or knocks out POLR2C gene expression, a substance that reduces the level of POLR2C nucleic acid and / or polypeptide, and a substance that weakens the function of POLR2C nucleic acid and / or polypeptide.

4. The use according to claim 1 or the product according to claim 2, wherein: The inhibitor is one or more selected from the following group: an antibody against POLR2C, an interfering RNA (siRNA) against POLR2C, an antisense oligonucleotide (ASO) against POLR2C, a microRNA (miRNA) against POLR2C, a nucleic acid aptamer against POLR2C, a decoy against POLR2C, a guide RNA (gRNA) against POLR2C, a POLR2C antagonist, and a POLR2C blocker; For example, among them, The inhibitor is selected from the group consisting of: siRNA of SEQ ID NO:3, siRNA of SEQ ID NO:4, and siRNA of SEQ ID NO:

5.

5. The use according to claim 1 or the product according to claim 2, wherein: The POLR2C is a polypeptide molecule having an amino acid sequence as shown in SEQ ID NO: 2, or a homologous sequence thereof; and / or The coding molecule of POLR2C has the nucleotide sequence shown in SEQ ID NO: 1, or a homologous sequence thereof.

6. The use according to claim 1 or the product according to claim 2, wherein: The product is in a form suitable for a route of administration selected from the group consisting of: enteral administration, parenteral administration, such as intravenous, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal (such as iontophoresis); and / or The product is in a form suitable for an administration route selected from the group consisting of: direct injection of liposome-encapsulated DNA, gold-coated DNA gene gun bombardment, replication-defective bacteria carrying plasmid DNA, and replication-defective adenovirus carrying target DNA; and / or The product is administered to mammals, such as primates, rodents, livestock, pets, etc., preferably humans, rats, mice, dogs, horses, cows, rabbits or monkeys.

7. The use according to claim 1 or the product according to claim 2, wherein: The product is selected from: medicine, health care product, test kit, medical device, or a combination thereof.

8. The use according to claim 1 or the product according to claim 2, wherein: The product is used for preventing and / or treating gastric cancer.

9. The use according to claim 1 or the product according to claim 2, wherein: The product also comprises a pharmaceutically acceptable carrier.

10. The use according to claim 1 or the product according to claim 2, wherein: The product is combined with one or more other tumor immunotherapy drugs or therapies, such as tumor vaccines, adoptive cellular immunotherapy, T-cell targeting immunomodulatory drugs, immune checkpoint inhibitors (ICIs), such as PD1 / PDL1 inhibitors (such as PD1 antibodies, pembrolizumab).

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