Gastric cancer prognosis marker and application thereof

Abstract

The invention provides a gastric cancer prognosis marker and application thereof, and belongs to the technical field of biology. According to the invention, survival analysis is carried out to explore the prognosis correlation of m6A'write 'RBM15 in gastric cancer. The result proves that the RBM15 is a prognostic marker related to a better clinical result and can be used for judging the prognosis condition of a gastric cancer patient. The invention verifies the relationship among RBM15, ECT2 and EMT signal pathway, and the result proves that RBM15 mediates m6A methylation of ECT2 through IGF2BP3, affects the stability of ECT2 RNA, affects the expression of ECT2, and regulates EMT signal pathway at the same time. The invention further discloses a mechanism of the RBM15 for influencing proliferation, invasion and migration of gastric cancer cells and sensitivity to 5-fluorouracil chemotherapy, and a new therapeutic target is provided for gastric cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gastric cancer prognosis marker and application thereof. Background Art

[0002] Gastric cancer (GC) is one of the most common digestive tract tumors in the world, with more than 1 million new cases each year. Globally, it is the third leading cause of cancer-related mortality. The clinical stage at the time of GC diagnosis is a key determinant of patient prognosis and survival. Currently available biomarkers for early detection of GC, such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), CA72-4, CA125, etc., lack sufficient sensitivity and specificity, and new biomarkers are urgently needed to improve the early detection and prognosis of GC.

[0003] M6A is involved in almost all aspects of RNA metabolism, including translation, degradation, input, output, and folding. The key components of the m6A methyltransferase complex include METTL3, METTL14, WTAP, METTL16, VIRMA, and RBM15. Among them, RBM15 can promote the progression of laryngeal squamous cell carcinoma, and its knockdown has been shown to inhibit the development of colorectal cancer. However, the expression and mechanism of action of RBM15 in gastric cancer are still unclear.

[0004] Epithelial cell transformation sequence 2 (ECT2) is a guanine nucleotide exchange factor (GEF) for RhoGTPases, including RhoA, Rac1, and Cdc42

[23] . ECT2 is associated with malignant transformation, tumorigenesis, and metastasis. Current research on ECT2 focuses on its role in other tumors. For example, ECT2 promotes the malignant phenotype by activating the AKT / mTOR pathway and contributes to cisplatin resistance in cervical cancer. ECT2 also drives the progression of lung adenocarcinoma by affecting extracellular matrix dynamics and focal adhesion signals. Its carcinogenic effects are influenced by its subcellular localization. However, the role of ECT2 in gastric cancer remains largely unexplored. Summary of the invention

[0005] The purpose of the present invention is to provide a gastric cancer prognosis marker and application thereof. The gastric cancer marker is RBM15, which can be used to predict the prognosis of gastric cancer and can also be used as a therapeutic target for treating gastric cancer.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a gastric cancer prognosis marker, wherein the gastric cancer prognosis marker is RBM15.

[0007] The present invention also provides an application of a gastric cancer prognostic marker RBM15 in predicting the prognosis of gastric cancer.

[0008] Furthermore, high expression of the marker RBM15 is judged as a good prognosis, and low expression of the marker RBM15 is judged as a poor prognosis.

[0009] The present invention also provides a use of the gastric cancer prognosis marker RBM15 as a therapeutic target in the preparation of a gastric cancer therapeutic drug.

[0010] Furthermore, the gastric cancer treatment drug is a drug that promotes high expression of RBM15.

[0011] Furthermore, the drug for promoting high expression of RBM15 includes an RBM15 activator or an RBM15 overexpression recombinant lentivirus.

[0012] Furthermore, the RBM15 overexpression drug can regulate the EMT signaling pathway by mediating the m6A methylation modification of ECT2 through IGF2BP3.

[0013] Furthermore, the RBM15 overexpression drug can inhibit the proliferation, migration and invasion of gastric cancer cells.

[0014] Furthermore, the RBM15 overexpression drug can enhance the drug sensitivity of gastric cancer cells.

[0015] The beneficial effects of the present invention compared with the prior art are: (1) This study performed survival analysis to explore the prognostic relevance of m6A "writer" RBM15 in gastric cancer. The results showed that RBM15 is a prognostic marker associated with better clinical outcomes and can be used to determine the prognosis of gastric cancer patients.

[0016] (2) The present invention verifies the relationship between RBM15, ECT2 and the EMT signaling pathway. The results show that RBM15 mediates ECT2 m6A methylation through IGF2BP3, affecting the stability of ECT2 RNA, thereby affecting the expression of ECT2 and regulating the EMT signaling pathway. The present invention also reveals the mechanism by which RBM15 affects gastric cancer cell proliferation, invasion, migration and sensitivity to 5-fluorouracil chemotherapy, providing a new therapeutic target for gastric cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1-1 This is the survival analysis of RBM15 in human gastric cancer in Example 1 of the present invention, wherein A is a meta-analysis of 23 m6A regulatory molecules, highlighting the enriched pathways and prognosis; B is the prognostic significance of 23 m6A regulatory molecules; C is the RFS forest map of 23 m6A regulatory molecules in the TCGA database; D is the OS forest map of 23 m6A regulatory molecules in the TCGA database, and E is the OS forest map of 23 m6A regulatory molecules in the ACRG database; F is the RFS forest map of 23 m6A regulatory molecules in the ACRG database; G is the survival analysis of 84 gastric cancer patients based on RBM15 expression; Figure 1-2 This is the survival analysis of RBM15 in human gastric cancer in Example 1 of the present invention, wherein H is the Kaplan-Meier curve showing the PFS and OS stratified by high and low RBM15 expression in the TCGA database; I is a comprehensive analysis of the relationship between RBM15 expression and prognosis PFS; PPS and OS are studied in GC patients in GC chip data sets GSE14210, GSE15459, GSE22377, GSE29272, GSE51105 and GSE62254; J is the RBM15 expression of different subtypes of CIN, EBV, GS and MSI in the TCGA database; K is the RBM15 expression of different subtypes of CIN, EBV, GS and MSI in the TCGA database; L is the RBM15 expression of different subtypes of EMT, MSI, MSS / TP53-, MSS / TP53+ in the ACRG database; M represents the expression of RBM15 in different subtypes of EMT, MSI, MSS / TP53-, and MSS / TP53+ in the ACRG database; Figure 2 This is an analysis of 23 m6A-related genes in cancer in Example 1 of the present invention, wherein A is 23 m6A-related genes in 115 samples out of 437 samples that have mutated, B is a co-occurrence analysis of 23 m6A-related genes; C is a correlation analysis of 23 m6A-related genes in the TCGA database; D is a correlation analysis of 23 m6A-related genes in the ACRG database; Figure 3-1: The effect of knockdown or overexpression of RBM15 on gastric cancer cells in Example 1 of the present invention; A is a quantitative PCR combined with Western-blot analysis to confirm the knockdown of RBM15 in AGS cells or the forced expression of RBM15 in MKN-45 cells; B is a cell proliferation experiment (colony formation and cell counting kit-8) result diagram of RBM15-scr, shRBM15-1, shRBM15-2 RBM15 knockdown and MKN-45 vector control or RBM15 overexpression cells in AGS cells; C is a line graph of cell proliferation experiment (colony formation and cell counting kit-8) of RBM15-scr, shRBM15-1, shRBM15-2 RBM15 knockdown and MKN-45 vector control or RBM15 overexpression cells in AGS cells; D is a cell migration and invasion test result diagram of RBM15-scr, shRBM15-1, shRBM15-2 (RBM15 knockdown) AGS cells; E is a graph showing the results of cell migration and invasion assays of AGS cells with RBM15-scr, shRBM15-1, and shRBM15-2 (RBM15 knockdown); F is a graph showing the results of cell migration and invasion assays of MKN-45 cells under the action of vector (vector control) or RBM15 overexpression; G is a graph showing the results of cell migration and invasion assays of MKN-45 cells under the action of vector (vector control) or RBM15 (RBM15 overexpression); H is a bar graph showing cell migration and invasion assays of AGS cells with RBM15-scr, shRBM15-1, and shRBM15-2 (RBM15 knockdown); I is a bar graph showing cell migration and invasion assays of MKN-45 cells under the action of vector (vector control) or RBM15 (RBM15 overexpression); Figure 3-2: The effect of knockdown or overexpression of RBM15 on gastric cancer cells in Example 1 of the present invention; J is a graph showing the results of a cell migration experiment of AGS cells with RBM15-scr, shRBM15-1, and shRBM15-2 (RBM15 knockdown); K is a graph showing the results of a cell migration experiment of AGS cells with RBM15-scr, shRBM15-1, and shRBM15-2 (RBM15 knockdown); L is a graph showing the results of a cell migration experiment of MKN-45 cells under a vector (vector control) or RBM15 (RBM15 overexpression); M is a graph showing the results of a cell migration experiment of MKN-45 cells under a vector (vector control) or RBM15 (RBM15 overexpression); N is a graph showing the lymphatic vessel formation ability of HLEC cells co-cultured with AGS cells with RBM15-scr, shRBM15-1, and shRBM15-2RBM15 knockdown; O is a graph showing the results of a cell migration experiment of HLEC cells co-cultured with AGS cells with RBM15-scr, shRBM15-1, and shRBM15-2 RBM15 knockdown is the lymphatic vessel formation ability of HLEC cells co-cultured with AGS cells; P is the result of lymphatic vessel formation ability of HLEC cells co-cultured with MKN-45 cells and Vector (vector control) or RBM15 (RBM15 overexpression); Q is the lymphatic vessel formation ability of HLEC cells co-cultured with MKN-45 cells and Vector (vector control) or RBM15 (RBM15 overexpression); R is the migration diagram of HLEC co-cultured with AGS cells by transwell method; S is the migration result of HLEC co-cultured with AGS cells by transwell method; T is the result of HLEC co-cultured with MKN-45 cells, vector (vector control) or RBM15 (RBM15 overexpression) migration diagram; U is the migration results of HLEC co-cultured with MKN-45 cells, vector (Vector control) or RBM15 (RBM15 overexpression); Figure 4-1The EMT pathway regulated by RBM15 in Example 1 of the present invention; A is based on the KEGG pathway data set in the ACRG database; B is based on the REACTOME pathway data set in the ACRG database; C is based on the HALLMARK pathway data set in the ACRG database; D is the correlation between RBM15 and emt-related genes analyzed using Spearman; E is the correlation between RBM15 and Vim analyzed by Spearman; F is a flow chat diagram of RNA sequences; G is the KEGG analysis of RNA-seq of shRBM15 gene AGS cells; H is the expression levels of CDH1, CDH2, CTNNB1 and Vimentin in AGS cells with knockdown of RBM15 gene by Western-blot analysis; Figure 4-2 The EMT pathway regulated by RBM15 in Example 1 of the present invention; wherein, I is the expression levels of CDH1, CDH2, CTNNB1 and Vimentin in AGS cells knocked down by RBM15 gene by quantitative PCR q-PCR analysis; J is the immunofluorescence assay of E-cadherin in AGS cells by shRBM15; K is the immunofluorescence assay of N-cadherin in AGS cells by shRBM15; L is the immunofluorescence assay of Vimentin in AGS cells by shRBM15; Figure 5-1 The results of the analysis in Example 1 of the present invention, wherein A is the correlation analysis between ARBM15 and CDH1; B is the correlation analysis between RBM15 and CDH2; C is the correlation analysis between RBM15 and TWIST2; D is the correlation analysis between RBM15 and ZEB2; E is the correlation analysis between RBM15 and SNAI2; F is the expression levels of CDH1, CDH2, CTNNB1 and vimentin in MKN-45 cells overexpressing RBM15 detected by Western blot analysis; G is the expression levels of CDH1, CDH2, CTNNB1 and vimentin in MKN-45 cells overexpressing RBM15 detected by quantitative PCR (q-PCR); H is the immunofluorescence analysis of E-cadherin by RBM15 overexpressed in MKN-45 cells; I is the immunofluorescence analysis of N-cadherin by RBM15 overexpressed in MKN-45 cells; J is the immunofluorescence analysis of Vimentin by RBM15 overexpressed in MKN-45 cells; Figure 5-2 The analysis results in Example 1 of the present invention, wherein K is the volcano map of RBM15 differential genes in the TCGA database; L is the volcano map of RBM15 differential genes in the ACRG database; M is the volcano map of RBM15 differential genes in RNA-seq; Figure 6-1 The EMT signaling pathway mediated by RBM15 through the regulated ECT2 in Example 1 of the present invention; A is a Venn diagram of RBM15 differentially expressed genes in RNA-seq, TCGA and ACRG databases; B is a western blot detection of AGS cells after knocking out RBM15; C is a q-PCR detection of AGS cells after knocking out RBM15; D is a q-PCR and western blot detection of AGS cells after treatment; E is a q-PCR and western blot detection of MKN-45 cells after treatment; F and G are survival analysis of ECT2 in ACRG and TCGA databases; H is a joint survival analysis of RBM15 and ECT2 in the ACRG cohort; Figure 6-2 is the EMT signaling pathway mediated by RBM15 through the regulated ECT2 in Example 1 of the present invention; I is the joint survival analysis of RBM15 and ECT2 in the TCGA cohort; J is the correlation between ECT2 and RBM15 in ACRG patients; K is the correlation analysis of RBM15 in the ACRG database; L is the enrichment analysis based on ECT2 expression in the KEGG database; M is the enrichment analysis based on ECT2 expression in the HALLMARK database; N is the enrichment analysis based on ECT2 expression in the REACTOME database; Figure 7-1 The figures are the animal experiments and tissue chips in Example 1 of the present invention; A is a mouse model and a mouse tumor picture; the upper part is the vector group, and RBM15 is the RBM15 group overexpressing RBM15; B is the volume of mouse tumors after injection of MKN-45 cells; C is the weight of mouse tumors after injection of MKN-45 cells; D is the expression levels of RBM15, ECT2, CDH1, CDH2, CTNNB1 and Vimentin in mouse tumor tissues detected by Vimentin (q-PCR); E is the expression levels of RBM15, ECT2, CDH1, CDH2, CTNNB1 and Vimentin in mouse tumor tissues detected by CTNNB1; Figure 7-2 1 is the animal experiment and tissue chip in Example 1 of the present invention; F is the expression level of RBM15, ECT2, CDH1, CDH2, CTNNB1 and Vimentin in mouse tumor tissues detected by immunohistochemistry; Figure 7-3 The animal experiment and tissue chip in Example 1 of the present invention; G is a representative image of high or low expression of RBM15, ECT2, E-cadherin, β-catenin, and vimentin in 87 GC tumor tissues; each scale bar represents 20 μm; Figure 8-1In Example 1 of the present invention, RBM15 inhibits GC cell proliferation and migration by promoting ECT2 expression; A is a photo of cell proliferation experiment of AGS and MKN-45 cells after colony formation and cell counting kit-8 CCK-8 relative treatment; B is the result of cell proliferation experiment of AGS and MKN-45 cells after colony formation and cell counting kit-8 CCK-8 relative treatment; C is the cell migration of AGS cells after relative treatment; D is the cell invasion test of AGS cells after relative treatment; Figure 8-2 RBM15 inhibits GC cell proliferation and migration by promoting ECT2 expression in Example 1 of the present invention; E is the cell migration of AGS cells after relative treatment; F is the cell invasion test of AGS cells after relative treatment; G is the expression level of CDH1, CDH2, CTNNB1, Vimentin and ECT2 in AGS and MKN-45 cells after relative treatment; H is the expression level of CDH1, CDH2, CTNNB1, Vimentin and ECT2 in AGS and MKN-45 cells after relative treatment; Figure 9-1 : The methylation modification of IGF2BP3 dependent on ECT2 regulated by RBM15 in Example 1 of the present invention; A is the total methylation level detection of AGS cells; B is the total methylation level detection of MKN-45 cells; C is the distribution of ECT2 prediction scores; D is the agarose gel electrophoresis of ECT2 extracted by MeRIP in AGS cells; E is the agarose gel electrophoresis of ECT2 extracted by MeRIP in MKN-45 cells; F is the luciferase activity of 293T cells co-transfected with ECT2-WT or ECT2-Mut and vector or RBM15; G is the RNA pull-down experiment confirming that ECT2 binds to IGF2BP3 in AGS and MKN-45; H is the enrichment of IGF2BP3 binding to ECT2 m6A modification sites shown by RIP-qPCR in AGS cells; Figure 9-2: RBM15 regulates ECT2-dependent methylation modification of IGF2BP3 in Example 1 of the present invention; I is MKN-45 cell RIP-qPCR showing enrichment of IGF2BP3 binding to ECT2 m6A modification sites; J is AGS cell RIP-qPCR showing enrichment of IGF2BP3 binding to ECT2 m6A modification sites; K is MKN-45 cell RIP-qPCR showing enrichment of IGF2BP3 binding to ECT2 m6A modification sites; L is the luciferase activity of 293T cells co-transfected with ECT2-WT or ECT2-Mut and vector or IGF2BP3; M is AGS cell RNA stability analysis showing that different transfection groups have different effects on ECT2 mRNA half-life; N is MKN-45 cell RNA stability analysis showing that different transfection groups have different effects on ECT2 mRNA half-life; Figure 10-1 The results in Example 1 of the present invention, wherein A is a forest plot of OS Overall Survival and RFS Relapse - FreeSurvival of 23 m6A regulatory molecules for GC patients after receiving XP chemotherapy in the ACRG database and 5-FU chemotherapy in the TCGA database; B is a survival analysis of OS and RFS of XP chemotherapy in the ACRG database; Figure 10-2 The results in Example 1 of the present invention, wherein C is the GEO data set for 5-fluorouracil application, and the relationship between RBM15 and the prognosis of OS, FPS, and PPS in GC patients is comprehensively analyzed; D is the IC50 of 5-FU in MKN-45 cells after CCK-8 detection; E is the IC50 of 5-FU in AGS cells after CCK-8 detection; Figure 10-3 The results in Example 1 of the present invention, wherein F is a schematic diagram of GC organoids; G is western blot detection of the expression of RBM15 and ECT2 in organoids; H is CCK-8 method detection of cell viability of organoids after 5-FU treatment CTRL: control, 5-FU: 5-fluorouracil 20 μM; I is a tumor organoid drug sensitivity test detecting the organoid diameter size of the RBM15 high group and the RBM15 low group at different time points after drug treatment CTRL: control, 5-FU: 5-fluorouracil 20 μM; J is the flow chart of this study. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Example 1 Example 1 of the present invention verifies the effect of BM15 as a potential therapeutic target for GC treatment, and the specific steps are as follows: (1) Tissue specimens and patient data: The first group consisted of 84 gastric cancer tissue samples obtained from tissue microarrays provided by Shanghai Xinchao Biotechnology Co., Ltd. (Shanghai, China) between January 2011 and December 2012. The second group included 29 gastric cancer tissue samples from patients who underwent gastrectomy at Shandong Provincial Hospital (Shandong, China) between January 2019 and December 2022. Human materials were obtained with the informed consent of the patients, and the study was approved by the Clinical Research Ethics Committee (National Natural Science Foundation of China: NO.2021-529). Prior to the present invention, all patients did not receive neoadjuvant chemotherapy. (2) Collection and preprocessing of publicly available datasets: Gene expression profile data and clinical annotations of gastric cancer samples were retrospectively collected from publicly accessible datasets of NCBI-GEO (https: / / www.ncbi.nlm.nih.gov / geo / ) and TCGA (https: / / cancergenome.nih.gov / ). The datasets include GSE14210, GSE15459, GSE22377, GSE29272, GSE51105, and GSE62254 (ACRG) datasets. TCGA RNA sequencing data are in FPKM format and downloaded from the UCSC Xena database (https: / / gdc.xenahubs.net / download / TCGA-STAD.htseq_fpkm.tsv.gz).

[0025] (3) Analysis of RBM15 and gastric cancer prognosis: metscape and gene set enrichment analysis (GSEA) The present invention screened a group of 23 m6A modification-related genes (Chong, W., et al., m(6)A regulator-based methylation modification patterns characterized by distinct tumor microenvironment immune profiles in colon cancer. ). The Metascape tool was then used for protein-protein interaction enrichment analysis. In addition, the present invention used the GSEA algorithm and the R software package "clusterProfiler" to examine the changes in biological processes in different RBM15 expression subgroups. Well-defined biological features came from Hallmark, Reactome, and KEGG gene sets, which were downloaded from the MSigDB database v7.1 (Chen, H., et al., Molecular characterization and clinical relevance of metabolic signature subtypes in gastric cancer).

[0026] Metascape analysis of 23 m6A regulatory molecules revealed that these factors are mainly involved in pathways related to mRNA modification, transport, destabilization, and progression ( Figure 1-1 In order to clarify the interaction and prognostic significance of these m6A regulatory factors in gastric cancer, the present invention conducted correlation and prognostic analysis ( Figure 1-1 Notably, patients with high RBM15 expression showed significantly better prognosis in both ACRG and TCGA cohorts.

[0027] Mutation spectrum analysis showed that the mutation frequency of RBM15 in GC was 3%, ranking in the top three among m6A molecules ( Figure 2 Further analysis of the co-mutation rates of 23 m6A regulatory factors revealed that there were significant differences in the co-mutation rates of RBM15, ELAVL1, IGF2BP2, YTHDC2, and YTHDF1 / 2 ( Figure 2 Using data from the TCGA and ACRG databases for correlation analysis, we found that RBM15 is closely related to several other m6A regulatory factors, indicating that it plays a key role in the m6A regulatory network ( Figure 2C in 1-1 and D in 1-1). Previous studies have identified several molecular subtypes of GC based on transcriptomic and genomic analyses. The present invention further performed tissue microarray analysis on 84 gastric cancer patients and found that the subgroup of patients with high RBM15 expression had a better prognosis. In addition, by integrating microarray data from multiple GEO databases (including GSE14210, GSE15459, GSE22377, GSE29272, GSE51105, GSE62254), the present invention further verified that high expression of RBM15 was associated with long-term survival of gastric cancer patients ( Figure 1-2 The present invention also explored the relationship between RBM15 expression and previously identified molecular subtypes ( Figure 1-2 In the TCGA cohort, RBM15 was highly expressed in EBV and MSI subtypes, both of which were associated with a good prognosis ( Figure 1-2 Similarly, in the ACRG cohort, RBM15 expression was elevated in both MSI and MSS / TP53+ subtypes ( Figure 1-2 L and M).

[0028] (2) RBM15 inhibits the proliferation, migration and invasion of GC cells: In order to explore the functional role of RBM15 in gastric cancer cells, the present invention conducted a series of experiments: A. Cell culture and transfection AGS cells and MKN-45 cells were purchased from the American Type Culture Collection (ATCC). AGS and MKN-45 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (PAN), penicillin (10U / mL, Thermo Fisher) and streptomycin (10U / mL, Thermo Fisher). The cells were cultured in a saturated humidity incubator at 37°C and 5% CO2. During the experiment, AGS and MKN-45 cells were cultured at 2×10 5 Cells were seeded at a density of 10 cells / well in a 6-well plate and incubated overnight. Lentiviral vectors for knockdown or overexpression of RBM15 and ECT2, as well as their respective blank control vectors, were prepared from Genechem (Shanghai). Transfection was performed according to the instructions of the transfection reagent. After transfection, cells were selected with puromycin (0.5 μg / ml, MedChemExpress) for 7 days. siRNA targeting IGF2BP3 was purchased from Keyybio (Shandong, China).

[0029] B. CCK-8 assay: One day before the experiment, cells were plated at 5×10 3The density of cells / well was seeded in a 96-well plate. Cell proliferation assay was evaluated using Cell Counting Kit-8 (DojinDo, Japan) according to the manufacturer's instructions. Absorbance was measured at 0h, 24h, 48h, 72h and 96h to evaluate cell viability and proliferation.

[0030] C. Clone formation experiment: cells were cultured at 2×10 3 The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured at 37°C for 7 days, with the culture medium replaced every 4 days. After the incubation period, colonies containing more than 50 cells were observed and counted under an optical microscope.

[0031] D. Transwell assay: For cell invasion and migration analysis, 200 μL of cell suspension in serum-free medium (migration: 3×10 5 / mL, invasion: 5×10 5 / mL) were seeded onto transwell inserts (Corning). For invasion assays, inserts were pre-coated with Matrigel, whereas for migration assays, they were not coated with Matrigel. The lower chamber was filled with 600 μl of complete medium. After incubation, cells on transwell inserts were fixed with 4% paraformaldehyde in PBS for 30 min. Subsequently, they were stained with crystal violet (Sigma-Aldrich) for 30 min.

[0032] E. Wound healing assay: Cells were seeded in 6-well plates and cultured until the confluence exceeded 95%. The monolayer was then scraped along the central axis with a sterile plastic tip and washed with PBS to remove loose cells. The culture medium was replaced with low serum medium (1%). Cell motility was quantified by measuring the distance between the cell edges in three randomly selected microscopic fields (×100 magnification) at each time point (0h, 24h, 48h).

[0033] In the clone formation experiment ( Figure 3-1 B) and Cell Counting Kit-8 (CCK-8) assay ( Figure 3-1 In C), knockdown of RBM15 in AGS cells resulted in a significant increase in cell proliferation, whereas overexpression of RBM15 in MKN-45 cells resulted in a significant decrease in cell proliferation.

[0034] In addition, Transwell assay ( Figure 3-1 D, E, H) and wound healing assay ( Figure 3-2 (J and 3-2K) showed that RBM15 knockdown significantly enhanced cell migration and invasion, while RBM15 overexpression inhibited these behaviors ( Figure 3-1 F, G, I and Figure 3-2Co-culture experiments with HLECs further revealed that knockdown of RBM15 increased the lymphangiogenesis capacity of HLECs ( Figure 3-2 N and O), while overexpression of RBM15 attenuated lymphangiogenesis ( Figure 3-2 In addition, knockdown of RBM15 promoted the migration ability of HLECs ( Figure 3-2 R and S), while overexpression of RBM15 inhibited the migration ability of HLECs ( Figure 3-2 Middle T, U).

[0035] Taken together, these findings underscore the role of RBM15 as a tumor suppressor in gastric cancer and highlight its important function in regulating gastric cancer cell proliferation, migration, and invasion in vitro.

[0036] (3) RBM15 regulates the EMT pathway To investigate the biological significance of RBM15 dysregulation, we performed gene set enrichment analysis (GSEA) to compare subgroups of RBM15 high and low expression in the ACRG database. GSEA used KEGG, REACTOME, and HALLMARK pathway datasets and found that RBM15 is involved in the regulation of m6A methylation-related functions, including RNA processing and splicing. In addition, RBM15 is also involved in the regulation of EMT, cell adhesion, and TGF-β signaling pathways ( Figure 4-1 Molecular subtype analysis further confirmed that RBM15 expression was downregulated in the EMT subtype ( Figure 1-2 L and M).

[0037] Correlation analysis showed that RBM15 was significantly negatively correlated with stromal markers such as CDH2 (N-cadherin), VIM, ZEB2, TWIST, and SNAI2 in GC, and significantly positively correlated with epithelial markers such as CDH1 (E-cadherin). Figure 4-1 D, E and Figure 5-1 A, B, C, D, E).

[0038] To further elucidate its mechanism, the present invention performed RNA-seq on GC cells with RBM15 knockdown ( Figure 4-1 Middle F). GSEA results reinforce the involvement of RBM15 In pathways related to focal adhesion, ECM receptor interaction, etc. ( Figure 4-1 qPCR assays showed that knockdown of RBM15 resulted in decreased CDH1 RNA levels and increased CDH2, CTNNB1, and Vimentin levels ( Figure 4-1 In contrast, RBM15 overexpression had the opposite effect ( Figure 4-1Western blot and immunofluorescence (IF) assays confirmed these findings, showing similar trends ( Figure 4-2 I, J, K, L and Figure 5-1 In conclusion, these results suggest that RBM15 may function by regulating the EMT signaling pathway.

[0039] (4) RBM15 mediates the EMT signaling pathway through regulated ECT2 A. Real-time quantitative PCR (qPCR) Total RNA was extracted from cells using Trizol reagent (Vazyme, China). RNA was then reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vazyme, China) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on an Applied Biosystems QuantStudio1 real-time PCR system (Applied Biosystems, ThermoFish) using ChamQUniversal SYBR qPCR Master Mix (Vazyme, China) to detect mRNA expression levels. The primer sequences used for qRT-PCR are shown in Table S1. GAPDH was used as an internal reference gene for mRNA standardization. The relative expression of mRNA was calculated using the 2-ΔΔCt method, and the higher the 2-ΔΔCt value, the higher the gene expression.

[0040] B. Western blot analysis and antibody detection Briefly, total cell lysate was prepared using cell lysis buffer and then denatured by boiling. Protein concentration was determined using a BCA protein assay kit (Solarbio). Equal amounts of protein were separated by SDS-PAGE at 80 V for 2.5 h and then transfected onto a PVDF membrane for 1.5 h. After transfer, the membrane was washed three times with 1% TBST for 5 min each time.

[0041] Primary antibodies RBM15 (Proteintech, 10587-1-AP), ECT2 (Santa Cruz, sc-514750), E-cadherin (Proteintech, 60335-1-Ig), N-cadherin (Proteintech, 66119-1-ig), Vimentin (Proteintech, 60330-1-Ig), β-catenin (Proteintech, 66379-1-Ig), IGF2BP3 (Proteintech, 14642-1-AP), and β-actin (Proteintech, 20536-1-AP) were incubated with the membrane overnight at 4°C. The membrane was then incubated with secondary antibodies (Proteintech, SA00001 and SA00002) according to standard protocols.

[0042] C. In vivo experiments Balb / c male nude mice, 4-5 weeks old, weighing 20-25 g, were purchased from Vital River Laboratories (Beijing, China). Ten mice per group were used to evaluate the effects of RBM15 overexpression and its respective control vectors in tumor growth studies. Each mouse was subcutaneously injected with 100 μl of tumor cells (5 × 10 6 ). Tumor volume was measured every 2 days. After 4 weeks, tumors were harvested, volume and weight were measured, and photos were taken. This study was approved by the Clinical Research Ethics Committee (NSFC: NO.2021-529).

[0043] C. Transwell assay For cell invasion and migration analysis, cells were suspended in 200 μL serum-free medium (migration: 3 × 10 5 / mL, invasion: 5×10 5 / mL) were seeded onto transwell inserts (Corning). For invasion assays, inserts were pre-coated with Matrigel, whereas for migration assays, they were not coated with Matrigel. The lower chamber was filled with 600 μL of complete medium. After incubation, cells on transwell inserts were fixed with 4% paraformaldehyde in PBS for 30 min. Subsequently, they were stained with crystal violet (Sigma-Aldrich) for 30 min.

[0044] In order to identify the potential regulatory targets of RBM15 in GC, the present invention combined the data from TCGA and ACRG databases to jointly analyze the differentially expressed genes (DEGs) in RBM15 knockdown cells.

[0045] This analysis identified 604 rbm15-related DEGs, including ECT2, ARHGAP5, HIPK2, TJP2, COL17A1, and MATR3 ( Figure 6-1 Middle A and Figure 5-2 K, L, M in the middle). qPCR and western blot analysis confirmed that ECT2 is a downstream target molecule of RBM15 ( Figure 6-1 Knockdown or overexpression of RBM15 alters the RNA and protein levels of ECT2 ( Figure 6-1 Survival analysis showed that GC patients with high ECT2 expression had improved prognosis in both ACRG and TCGA databases ( Figure 6-1 By integrating RBM15 and ECT2 expression subgroups, the present invention divided GC samples into four categories: RBM15-low / ECT2-low, RBM15-low / ECT2-high, and RBM15-high / ECT2-high. It was found that the RBM15-low / ECT2-low subgroup had the worst prognosis ( Figure 6-1 H in 6-2 and I in 6-2). Correlation analysis showed that ECT2 was significantly positively correlated with RBM15 ( Figure 6-2 Middle J), ​​and was significantly negatively correlated with emt-related molecules CDH2, VIM, ZEB2, TWIST, and SNAI2 ( Figure 6-2 Middle K). Enrichment analysis based on differential expression of ECT2 also highlighted its role in inhibiting the EMT process ( Figure 6-2 These findings suggest that, like RBM15, ECT2 plays a crucial role in the negative regulation of the EMT process.

[0046] To further evaluate the effect of RBM15 on GC cell progression in vivo, we injected MKN-45 cells overexpressing RBM15 subcutaneously into mice (n=20). As expected, the tumor volume and weight of the RBM15 overexpression group were reduced compared with the vector control group ( Figure 7-1 A, B, and C in the middle. Subsequent analysis of tumor protein and RNA levels showed that the expression of CDH1 and ECT2 was increased, while the expression of CDH2, CTNNB1 (β-catenin), and vimentin was decreased in the RBM15 overexpression group (D and E in 7-1). Immunohistochemical analysis further confirmed these findings, showing stronger staining of RBM15, ECT2, and E-cadherin and weaker staining of N-cadherin, β-catenin, and vimentin in the RBM15 overexpression group compared with the vehicle group (F in 7-2).

[0047] The present invention also explored the clinical relevance of RBM15, ECT2, E-cadherin, β-catenin and vimentin in GC by tissue microarray analysis (n=29) ( Figure 7-3 (G) Immunohistochemical analysis showed that the expressions of ECT2 and E-cadherin were significantly increased, while the expressions of β-catenin and vimentin were significantly decreased in RBM15-high tumor tissues.

[0048] Although the key role of RBM15 in the development of GC by the EMT signaling pathway has been established, further studies are needed to determine whether these effects are mediated by ECT2. To explore this, the present invention transfected AGS cells with shRBM15 and / or overexpressed ECT2, while MKN-45 cells were transfected with RBM15 overexpression and / or shECT2. These modified cells were then subjected to various functional assays. Figure 8-1 As shown in A and B, colony formation assay and CCK-8 assay showed that ECT2 overexpression restored the proliferation ability of GC cells caused by RBM15 knockdown. In contrast, shECT2 reversed the reduction of cell proliferation caused by RBM15 overexpression.

[0049] Transwell experiments further showed that ECT2 overexpression restored the migration and invasion abilities of GC cells that were reduced by RBM15 overexpression, while shECT2 restored the migration and invasion abilities of RBM15 overexpressing cells ( Figure 8-1 C, D, Figure 8-2 Middle E, Figure 8-2 In addition, ECT2 overexpression counteracted the upregulation of N-cadherin and vimentin and the downregulation of E-cadherin induced by RBM15 knockdown. Conversely, ECT2 knockdown reversed the inhibitory effect of RBM15 overexpression on the EMT process ( Figure 8-2 (G, H).

[0050] (5) RBM15 mediates ECT2 methylation and activates IGF2BP3 A. MeRIP Experiment RNA extraction was performed as described previously, and 2 μg of total RNA was used for MeRIP-PCR (P-9018-24, EpigenTek) according to the manufacturer’s protocol. 1% agarose gel was prepared and electrophoresed at 100 V for 1 h to detect methylated RNA levels.

[0051] B. Luciferase reporter gene assay 293T cells were seeded in 24-well plates and transfected with luciferase reporter gene or Renilla reporter gene, RBM15 overexpression plasmid or control vector. The absorbance was measured within 48 hours after transfection.

[0052] C. RIP-qPCR and RNA pull-down experiments RIP-qPCR: cells were lysed with tris-triton buffer and incubated with protein A / G magnetic beads pre-coupled with IGF2BP3 antibody. After overnight incubation, the beads were washed and RNA bound to the beads was detected by qPCR.

[0053] RNA pull-down: Biotinylated ECT2 transcripts were incubated with streptavidin magnetic beads. Cell lysate prepared with tris-triton buffer was then incubated with magnetic beads for 6 h. IGF2BP3 protein levels were detected by Western blot.

[0054] In order to study the molecular mechanism of RBM15 regulating m6A modification in GC, the present invention used EpiQuik m6A RNA methylation quantification kit to quantify the m6A methylation level in GC cells with RBM15 knockdown or overexpression. The results showed that after RBM15 inhibition, the m6A methylation level was significantly reduced, and after RBM15 overexpression, the methylation level was increased ( Figure 9-1 Further analysis using SRAMP methylation prediction sites identified potential m6A sites ( Figure 9-1 Middle C). MeRIP analysis showed that RBM15 downregulation reduced the mA modification of ECT2 mRNA, while RBM15 overexpression enhanced this modification ( Figure 9-1 D and E). Based on SRAMP analysis, the present invention mutated the AAACT sequence at 2909 bp of the 3'UTR to AACCT, and performed luciferase assay. The results showed that RBM15 overexpression significantly increased the relative luciferase activity of WT-ECT2, while the mut-ECT2 group was resistant to the effect of RBM15 overexpression ( Figure 9-1 Middle F).

[0055] The regulatory role of "m6A readers" in m6A-modified transcripts is crucial. The present invention first detected the enrichment of IGF2BP3 binding to ECT2 mRNA through RNA pull-down assay ( Figure 9-1 Middle G). RIP-qPCR assay confirmed that IGF2BP3 binds to ECT2 mRNA in AGS and MKN-45 cells ( Figure 9-1 Middle H, Figure 9-2In addition, RIP-qPCR showed that RBM15 knockdown attenuated the direct interaction between IGF2BP3 and ECT2 mRNA in GC cells, while RBM15 overexpression enhanced this interaction ( Figure 9-2 (J, K).

[0056] Luciferase assays with AGCCA 3'UTR 2887bp AGACA sequence mutations further demonstrated that WT-ECT2 luciferase activity increased significantly with IGF2BP3 overexpression, whereas the mut-ECT2 group was resistant to the effects of IGF2BP3 ( Figure 9-2 In addition, actinomycin treatment showed that the stability of ECT2 was decreased in the shRBM15 group compared with the control group, while the stability of ECT2 was increased in the RBM15 overexpression group compared with the vector control ( Figure 9-2 (in N).

[0057] Taken together, these findings once again confirm that RBM15-mediated modification of ECT2 by mA enhances ECT2 stability through IGF2BP3.

[0058] (6) RBM15 enhances the sensitivity of gastric cancer cells to 5-fluorouracil Gastric cancer tissue of GC patients after gastrectomy, GC tissue is about 0.5 cm 3 , rinsed three times in DPBS containing antibiotics, and then cut into 1 mm 3 The fragments were incubated in 10-15 ml digestion solution at 37 °C for 30 minutes to 1 hour. The resulting suspension was filtered through a purification mesh and centrifuged at 2000 G for 5 minutes at 4 °C. The cell pellet was resuspended in red blood cell lysis buffer and centrifuged again to collect the cells. The cell pellet was then mixed with Matrigel (BD Biosciences, California, USA) 1:1 and seeded into 24-well plates. After digestion of organoids, 50 organoid fragments (containing 250 cells) were mixed with 10 ul Matrigel and seeded into each well of a 96-well plate. The PDOs were allowed to grow to an average diameter of 50 μM and treated with 10 μM 5-Fu (selleckchem, USA) dissolved in DMSO. PDO images were taken every 3 days. After 9 days, cell viability was measured using the CellTiter-Lumi™ Luminescent 3D Cell Viability Assay Kit (Beyotime, Shanghai, China). Briefly, organoids were mixed with 100 μL of CellTiter-Glo® 3D reagent, shaken for 5 min, and incubated at room temperature for 30 min. The average diameter was measured using a fluorescence absorbance spectrophotometer and bright-field images were analyzed using Image J.

[0059] To further investigate the role of RBM15 in regulating drug sensitivity, the present invention analyzed the sequencing data of GC patients treated with 5-fluorouracil in the ACRG and TCGA cohorts. The ACRG database includes 91 GC patients treated with the XP regimen (5-fluorouracil and cisplatin), while the TCGA cohort includes 68 patients who received 5-fluorouracil chemotherapy. Cox regression analysis of 23 m6A regulatory molecules showed that GC patients with high expression of RBM15 had improved prognosis after 5-fluorouracil chemotherapy, suggesting that 5-fluorouracil chemotherapy can benefit patients more ( Figure 10-1 Middle A). Kaplan-Meier survival analysis further showed that GC patients with high RBM15 expression had longer OS and RFS when receiving XP chemotherapy ( Figure 10-1 An independent GEO dataset confirmed this finding, showing that patients with high RBM15 expression could benefit from 5-fluorouracil treatment ( Figure 10-2 In vitro CCK-8 assay showed that RBM15 overexpression significantly increased the sensitivity of GC cells to 5-fluorouracil compared with the vector control ( Figure 10-2 In contrast, RBM15 knockdown reduced the sensitivity of GC cells to 5-fluorouracil treatment ( Figure 10-2 Middle E).

[0060] The present invention proposes to establish gastric cancer organoids from patients ( Figure 10-3 F). These organoids reflect the complex dynamics of chemotherapy response. It may provide a valuable tool for improving personalized treatment and predicting individual patient responses to chemotherapy. In organoid culture, the present invention divides samples into RBM15-high group and RBM15-low group according to the expression of RBM15. The RBM15-high group includes #1, #2 and #3, while the RBM15-low group includes #4, #5, and #6. Western blot analysis showed that the expression levels of RBM15 and ECT2 in the RBM15-high expression group were higher than those in the RBM15-low expression group ( Figure 10-3 Middle G). CCK-8 test found that the RBM15-high group was more sensitive to 5-fluorouracil than the RBM15-low group ( Figure 10-3 In addition, the drug sensitivity test of tumor organoids showed that after 5-fluorouracil treatment, the organoid diameter of the RBM15-high group was smaller than that of the RBM15-low group ( Figure 10-3 Together, these findings suggest that RBM15 enhances the sensitivity of GC cells to 5-FU.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gastric cancer prognosis marker, characterized in that: The gastric cancer prognostic marker is RBM15.

2. An application of the gastric cancer prognostic marker RBM15 according to claim 1 in predicting the prognosis of gastric cancer.

3. The use of the gastric cancer prognostic marker according to claim 2 in predicting the prognosis of gastric cancer, characterized in that: A high expression of the marker RBM15 is judged as a good prognosis, and a low expression of the marker RBM15 is judged as a poor prognosis.

4. A use of the gastric cancer prognosis marker RBM15 according to claim 1 as a therapeutic target in the preparation of a gastric cancer therapeutic drug.

5. The use of the gastric cancer prognosis marker RBM15 as a therapeutic target in the preparation of a gastric cancer therapeutic drug according to claim 4, characterized in that: The gastric cancer treatment drug is a drug that promotes high expression of RBM15.

6. The use of the gastric cancer prognosis marker RBM15 as a therapeutic target in the preparation of a gastric cancer therapeutic drug according to claim 5, characterized in that: The drug for promoting high expression of RBM15 includes an RBM15 activator or an RBM15 overexpression recombinant lentivirus.

7. The use of the gastric cancer prognosis marker RBM15 as a therapeutic target in the preparation of a gastric cancer therapeutic drug according to claim 5, characterized in that: The RBM15 high expression drug can mediate the m6A methylation modification of ECT2 through IGF2BP3, affect the stability of ECT2, and regulate the EMT signaling pathway.

8. The use of the gastric cancer prognosis marker RBM15 as a therapeutic target in the preparation of a gastric cancer therapeutic drug according to claim 5, characterized in that: The RBM15 high-expression drug can inhibit the proliferation, migration and invasion of gastric cancer cells.

9. The use of the gastric cancer prognosis marker RBM15 as a therapeutic target in the preparation of a gastric cancer therapeutic drug according to claim 5, characterized in that: The RBM15 high-expression drug can enhance the drug sensitivity of gastric cancer cells.

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