The Study Of Gastric Cancer Molecular Typing And The Relationship Between AKR1C2 And 5-FU/DDP Sensitivity

Gastric cancer(GC)is a genomically heterogeneous disease,is the second-mostcommon cancer and the second-leading cause of cancer deaths in digestive system cancers.At present,Lauran classification is the most widely used classification in gastric cancer.With the development of gene sequencing technology,new typing forms,such as molecular typing,have been proposed.And analysis of its molecular and clinical characteristics demonstrates an urgent requirement for a new classification system to complement and perfect the current one to promot its clinical application.In addition,the treatment strategy of gastric cancer is single,the target drugs are few,and the curative effect of drugs is poor,so the cure rate of gastric cancer has always very low.Therefore,this paper focuses on the above two issues to carry out the research.In the present study,differential gene expression analysis and intersection analysis were used to screen typing molecules based on the RNA-seq data from the four current subtypes(CIN,EBV,GS and MSI)and the normal and tumor samples in each subtype in two GC data sets(2014 and 2018)from TCGA.Finally,74 unique genes that were differentially expressed not only between each subtype but also between normal and tumor samples were identified.Moreover,the 74 genes have a substantially lower percentage of genomic alterations,involve in multiple biological processes and the methylation status of them is opposite to the expression status.And few of them found to have interactive relationships.We used the method of unsupervised hierarchical clustering reclassify the GC samples into three subtypes from the TCGA cohort and two subtypes from the ACRG cohort on the basis of the expression data of the 74 genes.Kaplan–Meier survival analysis revealed that there was a substantial difference in overall survival and disease free survival among our three TCGA-derived subtypes and two ACRG-derived subtypes within that cohorts(median overall survival(months),subtype A: 19.94,subtype B: 72.17,subtype C: 30.88 and subtype A: 42.933,subtype B: NA;median disease free survival(months),subtype A: 19.05,subtype B: 45.20,subtype C: 42.28 and subtype A: 30.00,subtype B: NA).Then,the multivariate Cox regression analysis was used to analyze the relationship between clinical characteristics and survival outcome in each subtype of the TCGA and ACRG cohorts.We found that subtype B was remained significantly associate with a better survival outcome in the TCGA cohort and ACRG cohort(Hazard Ratio,0.566 [95% CI: 0.379-0.846],P = 0.005 and Hazard Ratio,0.706 [0.507-0.981],P = 0.038).And found that older age,diffuse type,higher stage and no response to treatment were also major risk factors for survival.Meanwhile,we found the subtype A occurred at a significantly younger age,included diffuse-type tumors,high TNM staging,and poor differentiation.The B subtype included patients who had intestinal-type tumors occurring at older age,low TNM staging,and had a well differentiation.The C subtype also had intestinal-type tumors and had a well differentiation.Then,the clinical and molecular characteristics of the 74 genes typing system were compared with TCGA and ACRG typing systems.It was found that the GS and EMT subtypes with the worst prognosis in other typing systems were mostly distributed in subtype A,and GS subtype was mainly diffuse-type,poorly differentiated and highly staging tumors,while MSI subtype with better prognosis was mainly distributed in subtype B,and was intestinal-type,highly differentiated and poorly staging tumors.In the comparison of molecular characteristics in each subtype,the gene mutation rates of subtype A and subtype GS were the lowest,while those of subtype B and subtype MSI were both high,and the gene expression trends and methylation status of the corresponding subtypes in each typing system were similar.It can be concluded that the subtypes in 74 genes typing system not only have the clinical characteristics of other typing systems' subtypes,but also have similar molecular characteristics to them,which indicates that we have established a molecular typing system with clinical significance.Subsequently,the expression of 74 genes in 30 fresh tissue samples of gastric cancer was detected by qRT-PCR,and the 30 samples were typed based on unsupervised hierarchical clustering.It was found that the 30 samples were divided into two subtypes,subtype A and subtype B.And then,we analyzed the clinical information of these two subtypes,found the clinical characteristics of subtypes A and B were consistent with those of the subtypes typed on RNA-seq and chip platform.Therefore,it showed that the 74 genes typing system can not only use RNA-seq and chip platform data for subtyping,but also use qRT-PCR platform data for subtyping.Furthermore,through Cox risk regression analysis of different drug response groups in each subtype showed that drug non-response was a high risk factor,and the differential expression analysis in 5-FU and DDP response group and non-response group revealed that AKR1C2 gene was highly expressed in non-response group.Subsequently,in the interference and overexpression experiments of three gastric cancer cell lines,it was found that the cells in siAKR1C2 group were more sensitive to 5-FU and DDP,and the number of apoptotic cells increased significantly.At the same time,it was found that the cells in pDONR223-AKR1C2 group were not sensitive to 5-FU and DDP,and the number of apoptotic cells decreased significantly.Therefore,the relationship between AKR1C2 and sensitivity of 5-FU and DDP was preliminarily elaborated in this part,which could provide a basis for the research and development of new drugs and clinical treatment of gastric cancer.In summary,this study not only provides a complement for the molecular typing system of gastric cancer,but also provides a basis for drug development and treatment of gastric cancer.