| Gastric cancer is the second most common type of malignant tumors in China of which the mortality rate ranks third among all cancers.The treatment effect of patients with advanced gastric cancer is often poor.One of the reasons is that somatic mutations may cause mutations and polyclonality of gene mutations in gastric cancer tissues,resulting in long-term interaction with the microenvironment(including the body's immune system)to evolve into complex biological characteristics.The emergence of mutation and high-throughput transformation technology has enabled studies of cancer mutation landscape,and cancer genomics research has achieved significant breakthroughs.However,the characteristics of the whole exome mutation map and molecular functional mechanism of gastric cancer are not completely clear.Therefore,studying the characteristics of gene mutations in gastric cancer and identifying their underlying biological functions can provide a theoretical basis,and form the basis for translational medicine for gastric cancer precision medicine,which will have important clinical significance and social value.In this study,a total of 46 tissues from patients with gastric cancer were collected from the Jiangnan University Affiliated Hospital(Wuxi,China).Whole Exome Sequencing(WES)and bioinformatics analysis were performed on cancer tissues and adjacent tissues,respectively.Mutation maps were created and molecular mutation characteristics in the gastric cancer were identified,and further analyzed and verified in WES data set from the International Cancer Genome Consortium(ICGC)database.Data were combined with patient information and prognostic follow-up data to determine relevant clinical treatment,identify candidate mutation genes and mutation sites,and verify their the biological function in vivo and in vitro experiments.The main content and results of this study are as follows:1.Construction of a gastric cancer gene mutation landscape.A total of 46 cancer samples and adjacent normal tissues with complete diagnosis,follow-up information,and intact and sufficient content were screened,and raw sequencing data was obtained in the Fastq format through genomic DNA extraction,library construction,exon capture,and sequencing.Subsequently,FastQC carry out data quality control was performed,GATK4 mutect2 somatic mutations were detected,and Funcotator was performed for functional annotation on somatic mutations.Moreover,the Maftools package was employed to perform statistical analysis,which visually displayed somatic mutations,and gastric cancer mutation map features were finally obtained.The main results included the following:(1)The top 10 high-frequency mutation genes,which were TP53(51%),TTN(49%),FLG(30%),SYNE1(30%),HMCN1(26%),ASPM(26%),DNAH5(23%),FSIP2(23%),XIRP2(21%),and MUC16(21%).The average mutation load was 6.494 ± 1.067 bp/Mbp.(2)In the classification of mutations,missense mutations accounted for 85.51%,frameshift deletion mutations accounted for 6.56%,and nonsense mutations accounted for 4.22%,frameshift insertion mutations accounted for 1.79%,and frame deletion mutations accounted for 1.48%.Furthermore,in-frame insertion mutations accounted for 0.33%,no stop code mutations accounted for 0.09%,and regarding mutation type,single nucleotide mutations accounted for 89.83%,insertion mutations accounted for 2.12%,deletion mutations accounted for 8.07%;in single In terms of nucleotide mutation types,C>T mutations accounted for 62.30%,C>A mutations accounted for 15.86%,T>C mutations accounted for 13.62%,T>G mutations accounted for 5.32%,C>G mutations accounted for 6.84% %,T>A mutations accounted for 4.07%;conversion mutations accounted for 82.82%,transversion mutations accounted for 17.17%.(3)Genes with significant enrichment mutations in pharmacogenomics included TP53,APOB,FAT3,HMCN1,MUC16,and OBSCN;(4)Mutation pathways that showed significant enrichment included RTK-RAS,PI3 K,TGF-Beta,WNT,and TP53(P <0.01);(5)The TP53 mutation is a driver gene mutation(MutSigCV algorithm and oncodriveCLUST algorithm).Taken together,these results revealed the characteristics of gastric cancer somatic mutations at the genomic molecular level,suggesting that mutations in TP53,MUC16,and HMCN1 may affect the therapeutic drug effect,and that mutations in RTK-RAS,PI3 K,TGF-Beta,WNT,and TP53 pathway may drive the development of gastric cancer.2.Through cross-validation and further screening of gastric cancer gene mutation maps in different databases,it was found that the TP53 R337 C mutation is a potential marker for poor prognosis,and a potentially harmful mutation site.To further explore the characteristics of gastric cancer gene mutation maps,in this study,we analyzed the exon sequencing datasets of gastric cancer in the United States(443 cases),Japan(585 cases),and China(Beijing Cancer Hospital,123 cases)using the ICGC cancer sequencing database.Mutation maps were drawn,and Kaplan-Meier survival analysis,and COX and LASSO regression analysis were employed based on clinicopathological and follow-up information.In addition,loci related to prognosis were selected and verified in the data set of the affiliated hospital of Jiangnan University(Jiangnan,China).The results showed that in the Japanese data set,the TP53 mutation frequency was 56%(ranked first in high frequency),39% in the US data set(ranked second in high frequency),and 39% in China(Beijing Cancer Hospital)(high frequency ranking)the first).Univariate and multivariate COX regression analysis showed that the 10 TP53 mutation sites related to prognosis were: SNP7675101,SNP7675209,SNP7674202,SNP7675178,SNP7674954,SNP7670700,SNP7673772,SNP7675161,SNP7675203,and SNP7675229.In the data set of the Affiliated Hospital of Jiangnan University,it was found that the TP53 mutation was related to chemotherapy resistance.Patients with gastric cancer carrying a TP53 SNP7670700(R337C)site mutation not only had a significantly shorter overall survival,but also had a significantly shortened progression-free survival.The above-mentioned results suggest that the TP53 R337 C mutation is a potentially poor prognostic marker and a potentially harmful mutation site that is worth further studying to identify its biological function.3.Through experimental verification,it was found that the TP53 R337 C mutation promoted chemotherapy resistance of gastric cancer.To explore the relationship between the TP53 R337 C mutation and drug resistance,in this study,AGS and SGC7901 gastric cancer cells were transfected with the TP53 R337 C mutant plasmid and TP53 wild-type plasmid,respectively,and the 5-fluorouracil(5-FU)levels of gastric cancer cells was measured by a semi-inhibitory concentration(IC50)cytotoxicity test to assess its sensitivity to 5-FU.The results showed that overexpression of R337 C mutant TP53 significantly increased the IC50 of AGS and SGC7901 cells when compared with overexpression of wild-type TP53,thereby suggesting that the TP53 R337 C mutation may promote 5-FU resistance of gastric cancer cells.Further use of stable overexpression of R337 C mutant and wild-type TP53 gastric cancer AGS cells in a subcutaneous tumor formation model in nude mice that underwent 5-FU treatment,showed that the tumor volume of nude mice bearing the TP53 R337 C mutation was significantly increased compared with the wild-type group,which further confirmed that the TP53 R337 C mutation may promote 5-FU resistance.Furthermore,in AGS and SGC7901 cells overexpressing TP53 R337 C,silencing the expression of R337 C mutant TP53 significantly enhanced the sensitivity of gastric cancer cells to 5-FU,thereby suggesting that interfering with the expression of TP53 R337 C may reverse 5-FU resistance.Taken together,the above-mentioned results suggest that TP53 R337 C may be a potential drug resistance marker for chemotherapy treatment in patients with gastric cancer.Thus,targeting the TP53 R337 C mutation provides a novel idea for reversing drug resistance and has potential clinical value.In this study,WES technology was used to draw a mutation map of gastric cancer genes in Wuxi(Jiangsu,China),and the mutation characteristics were analyzed.The mutation maps of gastric cancer in the United States,China(Beijing Cancer Hospital),and Japan were compared.TP53 high-frequency mutations,combined with clinical analysis,showed 10 potentially harmful mutation sites of the TP53 gene,and in vitro and in vivo experiments confirmed that the TP53 R337 C mutation can promote 5-FU resistance to gastric cancer cells.Thus,the first proposed TP53 R337 C mutation is a potential drug resistance marker and therapeutic target that provides a theoretical basis for the use of gastric cancer and the discovery of novel drug targets. |
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