Identifying Therapeutic Target Genes For Gastric Cancer Using Functional Genomics Screening Based On Lentiviral ShRNA Library And Studies On The Biological Function Of DARS In Gastric Cancer

Background:Gastric cancer is one of the most common and destructive malignant tumors in the world,accounting for the 5th highest incidence of cancer worldwide,but the 3rd highest mortality.Gastric cancer is a group of biologically and genetically diverse tumor types,and there are many factors at play in its etiology,including environmental factors and genetic factors.Although endoscopy and imaging techniques have improved the detection rate of early gastric cancer related lesions,gastric cancer has extensive morphological heterogeneity.It is easy to ignore some cases only by morphology.Most gastric cancer patients are already in moderate or severe stages when they are first diagnosed.The application of biomarkers may improve the accuracy of early detection and diagnosis of gastric cancer screening,as well as the prognosis and treatment of various diseases including cancer.As the molecular mechanism of gastric cancer development continues to be further studied,so far,targeted therapies for a small number of gastric cancer patients have replaced or supplemented Trastuzumab and Ramuzumab,which are second-line drugs for advanced gastric cancer when HER-2 or VEGFR2 expression is upregulated,respectively.Pd-1 inhibitor pembrolizumab has been approved by the FDA as a third-line（or higher）treatment for recurrent,locally advanced,or metastatic gastric adenocarcinoma.Targeted therapy and immunotherapy provide clinicians with a new way to treat patients with advanced gastric cancer,but the clinical efficacy of most targeted drugs is still unsatisfactory.Therefore,personalized therapy for gastric cancer urgently needs to find genes with strong sensitivity and specificity in the occurrence and development of gastric cancer,and further explore the molecular mechanism of its role,which has important theoretical and practical significance for the clinical diagnosis and treatment of gastric cancer and provide research targets for anti-cancer drugs.Next Generation Sequencing（NGS）is a powerful technique for elucidating the pathogenesis of human cancer and identifying potential therapeutic targets.The main applications of NGS include DNA sequencing,RNA sequencing,and chromatin immunoprecipitation sequencing,depending on the input material.While the initial use of NGS focused on sequencing individual genomes or transcripts from various tissues,the recent use of NGS in cancer biology has revolutionized cancer research in several ways.In recent years,with the development of high-throughput nucleic acid synthesis methods and next-generation sequencing（NGS）platforms,functional genomic screening using large libraries of lentiviruses has become possible,aimed at discovering new cancer therapeutic targets.Objective:In this study,we will use an originally constructed shRNA lentivirus library for all human metabolic genomes to perform NGS-based functional genomics screening on human gastric cancer cell lines,aiming to screen out key genes targeting gastric cancer cells.At the same time,biological functions and downstream targets of target genes in gastric cancer cells were investigated by human gastric cancer cell line experiment.Research methods:1)NGS-based functional genomics screening was performed on human gastric cancer cell lines MKN1 and HGC-27 using a shRNA library of 6,399 shRNA for all2,096 human metabolic genes to identify shRNA phenotypes with growth inhibition and promotion,and GC-related differentially expressed genes,respectively.Candidate genes with identical phenotypic functions are obtained by Enriched pathway analysis.2)Use GEPIA and Kaplan-Meier Plotter public databases to analyze the expression differences of different candidate genes in the Aminoacyl-t RNA synthases（ARSs）family in gastric cancer tissue samples and normal gastric mucosal tissue samples adjacent to cancer.The relationship between DARS gene expression and prognosis in different gastric cancer tissue types was analyzed.The correlation between DARS gene histopathological expression and clinicopathological features of 346 patients with gastric cancer was analyzed by internal tissue microarray.DARS protein expression level was verified in a genetically engineered mouse model of diffuse gastric cancer.3)Human gastric cancer cell lines MKN1 and HGC-27 were selected,and the knockdown DARS cell model was constructed by shRNA transfection technology.The expression level of DARS after knockdown was verified and analyzed by RT-QPCR and Immunoblot.The effects of knockdown DARS on gastric cancer cells were verified by CCK-8 cell proliferation assay,clone formation assay,Edu assay,cell cycle assay,cell apoptosis assay and cell migration assay.4)We established a mouse subcutaneous tumor-bearing model,and further detected the tumorigenesis of two DARS knockdown gastric cancer cell lines in mice.HE staining and immunohistochemical analysis were performed on mouse tumors.5)RNA high-throughput sequencing was performed on gastric cancer cell lines with low DARS knockdown mediated by shRNA to analyze the molecular pathway mechanism of DARS knockdown inhibition.Immunoblots were used to verify the expression of downstream key transcription factors involved in the signaling pathway and the regulatory relationship between DARS and Ca²⁺/MAPK/CREB/ELK-1 signal transduction pathway.Results:1)By functional genomic screening of MKN1 and HGC-27 cell lines,a total of137 and 204 shRNA expressing reproducible phenotypes of growth inhibitory genes and 38 and 156 shRNA expressing reproducible phenotypes of growth promoting genes were obtained,respectively.There were 38 common growth inhibitory phenotypic genes in the two cell lines,including DARS,KARS and VARS genes in ARSs family.2)Three genes of the ARSs family were verified by GEPIA and Kaplan-Meier plotter databases.It was found that the expression levels of DARS and VARS genes in gastric cancer were significantly higher than those in normal stomach tissues,while there was no significant difference in KARS expression level.High expression of DARS and KARS genes showed poor overall survival,while the expression level of VARS was not related to the overall survival of gastric cancer patients.Histological protein expression level of DARS gene was analyzed by internal tissue microarray in 346 patients with gastric cancer.243 patients had strong protein expression level and 103 patients had relatively weak protein expression level.Clinicopathological analysis showed that the high expression of DARS protein was related to T stage and lymph node metastasis,and this difference was reflected in the intestinal and diffuse types of gastric cancer histopathological classification.In immunohistochemical analysis of genetically engineered mice,protein expression levels of DARS were higher in diffuse invasive carcinoma than in early intramucosal carcinoma.3)By RT-q PCR and Immunoblot assay,mRNA level and protein expression level of DARS knockdown were significantly decreased.Knockdown DARS gene in the two gastric cancer cell lines inhibited the proliferation and activity of gastric cancer cells,inhibited the ability of colony formation and migration of gastric cancer cells,led to G0/G1 phase arrest of gastric cancer cells,and promoted apoptosis of gastric cancer cells.4)In the mouse xenotransplantation model,the growth ability of gastric cancer cells with DARS knockdown was inhibited in mice,and the tumor volume and weight of mice with DARS knockdown were significantly decreased compared with the control group.HE staining of mouse tumor sections indicated that the mitotic capacity of gastric cancer cells was significantly reduced after DARS knockdown.5)According to the results of RNA high-throughput sequencing,there were 340down-regulated genes in MKN1 cell line,277 down-regulated genes in HGC-27 cell line,90 down-regulated genes in common between the two,and 38 up-regulated genes in common between the two.Immunoblot results suggested that DARS gene knockdown could inhibit the expression of p-ERK1/2,p-CREB and p-ELK-1,suggesting that DARS gene may affect the occurrence and development of gastric cancer by regulating Ca²⁺/MAPK/CREB/ELK-1 signaling pathway.Immunohistochemical results of mouse tumor sections indicated that the positive expression levels of DARS and p-ERK1/2 protein were significantly decreased in the DARS knockdown group.Conclusion:1)DARS,KARS and VARS are the candidate target genes for gastric cancer screened out in this study.2)The high protein expression level of DARS in clinical gastric cancer tissue samples was significantly correlated with T staging and lymph node metastasis of gastric cancer,and the prognosis was worse,especially in diffuse gastric cancer.3)DARS gene knockdown can inhibit the proliferation,colony forming ability and migration of gastric cancer cells,block the G0/G1 phase of gastric cancer cell cycle,promote apoptosis of gastric cancer cells,and inhibit the growth and mitosis of gastric cancer cells in xenograft tumors.4)DARS gene may affect the occurrence and development of gastric cancer by regulating Ca²⁺/MAPK/CREB/ELK-1 signaling pathway.