| PurposeGastric cancer(GC)is highly heterogeneous.Drug resistance is an important factor for recurrence and metastasis of GC after surgery,and peritoneal metastasis is one of the most commen type of GC metastasis,C1ORF112,as a protein of unknown function,has not been reported to promote drug resistance of gastric cancer.At present,there is a lack of multi-omics models to predict the prognosis of gastric cancer,and the abnormal gene events of peritoneal metastasis need to be clarified.With the extensive development of deep sequencing technology and the generation of multi-omics data,the molecular typing and metastasis mechanism of gastric cancer based on gene sequencing has been widely researched.This study used a variety of molecular biology and bioinformatics methods to explore the role of C1ORF112 in 5-FU resistance of gastric cancer,integrate multi-omics data to predict the prognosis and molecular typing of gastric cancer,and analyze the exome sequencing of gastric adenocarcinoma with peritoneal metastasis,which will help to uncover the mechanism of C1ORF112induced 5-FU resistance in GC.A more comprehensive understanding of the heterogeneity of gastric cancer and the characteristics of peritoneal metastasis can provide strategic support for clinical individualized treatment.Materials and methods:1.Use Protein Atlas、GENT and TCGA databases to analyze the expression of C1ORF112 in gastric cancer and normal tissues;2.Q-PCR and WB assays were used to detect the expression of C1ORF112 in frozen tissues of gastric cancer;3.Use the Kaplan-Meier Plotter database to analyze the relationship of C1ORF112 expression and the prognosis of GC patients treated with 5-FU chemotherapy;4.Construction of C1ORF112 overexpression and knockdown lentivirus stable transfected cell lines;5.Transwell and wound healing assays were used to detect the migration ability of gastric cancer cells along with change of C1ORF112 expression during 5-FU chemotherapy;6.Flow cytometry、CCK8 and EdU reagent were adopted to test the effect of C1ORF112 on the proliferation,apoptosis,and cell cycle of gastric cancer cells treated with 5-FU;7.Detection of C1ORF112 participates in DNA damage response by using IF assay;8.Construction of 5-FU resistant cell lines,use WB to detecte the expression changes of C1ORF112、FIGNL1 and DNA homologous recombinase after drug resistance;9.IF was used to analyze the impact of C1ORF112 on the formation of RAD51 nuclear focus;WB was used to uncover that wether C1ORF112 affected the expression of RAD51 protein in gastric cancer cells treated with 5-FU;10.Analysis of the interaction of C1ORF112 and FIGNL1 using STRING and TCGA databases,and IF was used to detect the co-localization of these two proteins by transfection of exogenous tag protein;11.Co-IP assay was used to analyze the interaction of C1ORF112、FIGNL1 and RAD51;12.Screen out data containing mRNA、LncRNA、microRNA and DNA methylation in the TCGA GC data set,establish a random forest model,and analyze the survival subtypes of GC;13.Analyze the functional characteristics of different types of GC;14.Perform WES on gastric adenocarcinoma with peritoneal metastasis;15.Sanger sequencing verifies WES sequencing results and analyzes somatic mutations.Results:1.C1ORF112 is highly expressed in gastric cancer tissue,and its expression level is negatively correlated with the prognosis of gastric cancer patients treated with 5-FU;2.Over expression of C1ORF112 increases the migration of gastric cancer cells treated with 5-FU;3.C1ORF112 enhances the tolerance of gastric cancer cells to 5-FU.Flow cytometry analysis found that C1ORF112 reduced apoptosis of gastric cancer cells treated with 5-FU and promoted cell cycle arrest.C1ORF112 protein promoted the proliferationof GC cells treated with 5-FU;4.We performed IF assay to found that C1ORF112 reduced the expression of γH2AX and participated in DNA damage response;5.C1ORF112 promotes homologous recombination repair.Construction of 5-FU drug-resistant cells,WB assay indicated that C1ORF112 and DNA homologous recombination repair related proteins were up-regulated in drugresistant celllines;after knocking down C1ORF112、RAD51 formed many nuclear foci in cells treated with 5-FU;6.Co-IP and IF assays were employed to verify the interaction of C1ORF112、RAD51 and FIGNL1;7.Three types of survival subtypes were identified from the TCGA GC data set.There were differences in gene enrichment pathway,TMB,characteristics of immune cell infiltration and correlation with Lauren typing among these 3 subtypes;8.Peritoneal metastasis 59%of somatic mutations in gastric adenocarcinoma are consistent in the primary foci and peritoneal nodules,9 genes(ERBB4、ZNF721、NT5E、PDE10A、CA1、NUMB、NBN、ZFYVE16 and NCAM1)only mutated in peritoneal metastasis may be potential driver genes.Conclusion:1.C1ORF112 is highly expressed in gastric cancer,combined with FIGNL1 to promote homologous recombination repair,and enhance 5-FU resistance of gastric cancer cells;2.Through deep learning and integration of multi-omics data,a prediction model is established to perform molecular typing on gastric cancer;3.There is a specific distribution of somatic mutations in gastric cancer peritoneal metastasis. |
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