| Background and objectiveGastric cancer is one of the most common malignant tumors in the world.Recurrence and metastasis of gastric cancer is the main cause of failure of gastric cancer treatment.Peritoneal metastasis is the most common site of recurrence and metastasis.Once peritoneal metastasis occurs,the prognosis is very poor,which is the main cause of death in gastric cancer.The rate of false negatives in peritoneal metastasis was high in conventional imaging diagnosis.And the sensitivity and positive predictive value of commonly used tumor markers for diagnosis of peritoneal metastasis were poor.Diagnostic laparoscopy combined with intraperitoneal cancer detection is an invasive procedure.Patients with a history of abdominal pelvic surgery or abdominal adhesions are unable to undergo laparoscopy,which limits their clinical applications.Moreover,there is no clear predictor of peritoneal metastasis for postoperative gastric cancer.It is agreed that “risk factors for peritoneal metastasis include late TNM stage(T3/T4 and N positive),extranodal lymph node infiltration,Borrmann III,IV and Lauren diffuse patients.".However,the study found that only 25% of patients with T3/T4 and N+ developed peritoneal metastasis.Therefore,it is difficult to accurately predict whether peritoneal metastasis occurs in gastric cancer patients following curative resection by virtue of the currently known high-risk factors.Medicine has entered the era of molecular subtyping.Even if the histology is the same,the genetic characteristics of the tumor may be completely different,leading to differences in treatment and prognosis.Gastric cancer is also a highly heterogeneous tumor,and the TCGA divides gastric cancer into four molecular subtypes by high-throughput sequencing.However,the molecular typing is not based on the characteristics of the Chinese population(there were only about 20% East Asian population in the TCGA gastric cancer database),and this genotyping does not focus on patients with peritoneal metastasis.Therefore,this project intends to analyze the gastric cancer samples of specific populations through target sequence capture and sequencing(focusing on the postoperative peritoneal metastasis population),combined with bioinformatics analysis and clinical survival data to screen out the key genes predicting peritoneal metastasis and construct corresponding Lentivirus-mediated silencing and overexpression of stable cell lines,to explore the impact of key genes of peritoneal metastasis on the phenotype of gastric cancer cells through in vitro and in vivo experiments.We hoped that this transformational study will provide new biomarkers for gastric cancer and provide new ideas for individualized treatment.Methods1.Analysized 73 patients with newly diagnosed gastric cancer tissue FFPE samples,in which 63 patients with stage I-III were divided into peritoneal metastasis group(PM)and non-peritoneal group(non-PM)according to whether or not postoperative peritoneal metastasis occurred by whole-exome sequencing technique retrospectively.And we searched for characteristic gene mutations in postoperative PM patients by analyzing clinical pathology combined with survival data,to screen out differential genes between the two groups,and to construct a predictive model of mutated gene for peritoneal metastasis in gastric cancer patients following curative resection.2.By combined the results of FFPE samples by WES of?-? stage and 10 peripheral blood ctDNA samples of advanced gastric patients with peritoneal metastasis patients by 508 gene sequencing,We screened out the most relevant gene CDC27 for gastric cancer postoperative peritoneal metastasis by bioinformatics analysis and clinical data comparison,which lays the foundation for follow-up Research.3.IHC was used to detect the expression of CDC27 in human gastric cancer tissues and to analyze the correlation between CDC27 expression and clinical pathology and survival data.Western blot and RT-qPCR were used to detect the expression of CDC27 in multiple gastric cancer cell lines and construct lentiviral-mediated shRNA-CDC27 stably transfected cell line and lentiviral-mediated overexpressing CDC27 stably transfected cell lines.4.CCK-8 method,colony formation assay,Transwell invasion assay,scratch assay and flow cytometry PI single staining method were used to detect the changes of proliferation,colony formation,invasion and migration ability and cell cycle distribution of gastric cancer cell of CDC27 silencing and overexpression.And the effect of CDC27 silencing on the tumorigenic ability of MGC-803 gastric cancer cell line was observed by subcutaneous tumor formation in nude mice.Results1.After exome sequencing of FFPE samples from patients with stage I-III gastric cancer,the rate of coincidence of mutations in surgical specimens between patients with postoperative peritoneal metastasis(PM)and those without peritoneal metastasis(non-PM)was 19.7.%.The number of mutant gene and TMB were significantly higher in PM patients than those of non-PM patients.There was a significant positive correlation between TMB and the number of mutant genes;the risk of peritoneal metastasis was significantly higher in patients with high TMB than in the low TMB group.2.There are 49 differential genes between PM and non-PM patients,and 8 high-frequency differential genes are screened out.The risk of peritoneal metastasis in patients with 8 high-frequency differential genes is significantly higher than that of non-carrying patients;Univariate and multivariate regression model analysis of the factors confirmed that the 8 high-frequency differential gene was an independent risk factor for postoperative peritoneal metastasis in gastric cancer.The peritoneal metastasis prediction model based on high-frequency differential gene and pathological risk factors(T3-4)was superior to the simple pathological prediction model.3.Targeted capture of 508 genes sequencing of peripheral blood ctDNA in 10 advanced gastric cancer patients found that the rate of coincidence of genetic variation between patients with advanced peritoneal metastasis and non-peritoneal metastases was very low.The heat map showed that patients with advanced peritoneal metastasis had characteristic genetic mutations.4.Sequencing of FFPE samples from patients with postoperative gastric cancer and ctDNA samples from advanced gastric patients using bioinformatics combined with clinical survival data showed that CDC27 mutations were associated with postoperative peritoneal metastasis,and patients with CDC27 mutations had a significant higher risk of postoperative peritoneal metastasis,and CDC27 mutations are associated with DFS and OS shortening.Further mutation site analysis revealed that the CDC27 mutation is located in the Apc3 region,the TPR region and the phosphorylation region,and were new mutation sites not included in the TCGA database.5.IHC found that CDC27 is highly expressed in gastric cancer tissues,and high CDC27 expression is positively correlated with lymph node metastasis and tumor invasion depth.Patients with high CDC27 expression have shorter DFS and an increased risk of postoperative peritoneal metastasis.6.Western blot and RT-qPCR showed that CDC27 had the highest expression in MGC-803 and the lowest expression in AGS relatively.Based on this,we successfully constructed MGC-803_shCDC27 cell which stably silencing CDC27 and AGS_CDC27 cell which stably overexpressing CDC27.In vitro experiments showed that silencing CDC27 inhibited the proliferation,clonal formation,invasion and migration of gastric cancer cells MGC-803.Cell cycle analysis showed G2/M phase arrest and S phase decreased.Overexpression of CDC27 enhanced the proliferation,colony formation,invasion and migration ability of gastric cancer cells AGS.Cell cycle analysis showed that G2/M phase decreased and S phase increased.In vivo experiments showed that silencing CDC27 reduced the subcutaneous tumorigenic ability of gastric cancer cell line MGC-803 in nude mice.Conclusion1.Using the whole exome sequencing technology to retrospectively analyze the gene mutation of FFPE samples in stage I-III gastric cancer,we found that patients with PM after surgery have carried characteristic genetic mutations in the surgical samples,The characteristic genetic variations included increased Ti ratio,Ti/Tv,number of mutant genes,TMB significantly,and carrying 8 high-frequency differential genes.2.The risk of PM in patients carried 8 high-frequency differential genes was significantly higher than that in non-carriers,and it was an independent risk factor for postoperative PM in gastric cancer.The peritoneal metastasis prediction model based on high-frequency differential genes and pathological risk factors(T3-4)was significantly better than the simple pathological prediction model.3.Bioinformatics analysis and clinical prognosis analysis showed that CDC27 mutation was closely related to postoperative PM in gastric cancer.Patients with CDC27 mutation had significantly increased risk of postoperative PM and significantly shortened DFS and OS.The mutation sites found in this study were new mutation sites that not included in the TCGA database.4.CDC27 is highly expressed in gastric cancer tissues,and high expression of CDC27 is associated with shorter DFS and an increased risk of postoperative PM.5.In vitro and in vivo experiments confirmed that silencing CDC27 inhibited the malignant phenotype of gastric cancer cell line MGC-803,but overexpression of CDC27 enhanced the malignant phenotype of gastric cancer cell AGS.It maybe related to G2/M arrest and S phase reduction.6.CDC27 mutation and high expression are both poor prognostic factors for gastric cancer,and CDC27 mutation is closely related to gastric cancer peritoneal metastasis.CDC27 may be a new biomarker and potential therapeutic target for gastric cancer. |
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