Pancreas β Cell Transcriptome Sequencing And The Effects Of Gsta1,Gsta2 And Nrp2 Genes On Cell Function

Objective:In this study,we constructed the pancreatic cancer mediated by palmitic acid and glucose β Cell type(Min6 cell)type two diabetes mellitus(T2DM)injury model,transcriptome sequencing analysis of m RNA in model group and normal control group,screening out differentially related genes related to cell function,studying the effects of related differential genes on Min6 cell proliferation,migration,apoptosis and other functions,and studying the molecular mechanism mediated by them,and exploring the molecular mechanism of T2 DM pathogenesis.Methods:1.Construct the high glucose and high-fat model of MIN6 cells,detect the cell proliferation ability by CCK8 experiment,detect the migration ability of MIN6 cells by scratch experiment,detect the apoptosis level of MIN6 cells by flow cytometry,detect the insulin secretion ability of MIN6 cells by ELISA and detect the levels of Bcl-2,Bak,caspase 3 and other apoptotic proteins by Western blot to verify whether the model is successfully constructed and its impact on cell function.2.The m RNA transcriptome of palmitic acid + glucose group and control group were sequenced for the second generation,and the gene expression,differential gene function and gene sequence were analyzed.According to the analysis results and relevant research progress,the differential target genes were preliminarily screened,and the expression levels of RNA and protein of the differential target genes were verified by QRT PCR and Western blot.3.construct the overexpression lentivirus vector of the target gene,transfect MIN6 cells,detect the transfection effect by fluorescence microscope,verify the transfection and expression of the target gene by QRT PCR and Western blot,detect the effect of the target gene on the viability and survival rate of MIN6 cells by CCK8,detect the effect of the target gene on the apoptosis rate of MIN6 cells by flow cytometry,and detect the migration ability of MIN6 cells by scratch test.4.The expression level of related proteins regulated by the target gene was verified by Western blot.Results:1.After adding a certain concentration of palmitic acid and glucose,we successfully constructed the T2 DM model of high-fat and high glucose MIN6 cells.Compared with the control group,the survival rate of cells in the model group decreased significantly,the apoptosis rate increased significantly,the proliferation and migration ability decreased significantly,the insulin secretion ability decreased significantly,the level of apoptotic protein Bcl-2 decreased significantly,the level of Bak increased significantly,and the level of caspase 3 increased significantly.2.Using second-generation sequencing technology,we detected the transcriptome expression levels of model cells and control cells.The analysis found 222 differential genes,including 127 genes with increased expression and 95 genes with decreased expression.Through the analysis of differential genes go and KEGG,and the m RNA and protein levels of genes with large differential expression were verified.Combined with the research progress of related genes,we selected gsta1,gsta2 and Nrp2 as the follow-up research object.3.We successfully constructed the overexpression vectors of gsta1,gsta2 and Nrp2 and transfected them into MIN6 cells.Overexpression of gsta1 and Nrp2 significantly decreased cell viability.After 48 hours of high glucose and high fat culture,overexpression of Nrp2 could significantly reduce the survival rate of cells and significantly increase the apoptosis rate;Overexpression of gsta1 and gsta2 could significantly increase the apoptosis rate and reduce the cell survival rate,but not significantly.Overexpression of gsta1,gsta2 and Nrp2 had no significant effect on cell migration.4.Under normal culture conditions,the protein level of PARP1 increased,the protein level of Bcl-2 decreased,the protein level of Bak increased,the protein level of caspase 3 increased,and the apoptosis pathway was activated;After high glucose and high-fat culture,PARP1 was highly expressed in Nrp2 group and empty body group,and Nrp2 group was higher than empty body group,and the apoptotic protein pathway was activated.5.Compared with the empty body group,the expression level of Nrp2 in the overexpression group of gsta2 did not increase,indicating that gsta2 is not the upstream regulator of Nrp2.Conclusions:The T2 DM model of high glucose and high fat MIN6 cells mediated by palmitic acid and glucose inhibits cell survival and proliferation,promotes apoptosis,inhibits the secretion of insulin and regulates the expression level of apoptotic protein;Through a series of experimental techniques such as second-generation sequencing,we screened three target genes: gsta1,gsta2 and Nrp2.A series of cell function experiments confirmed that gsta1 and Nrp2 could significantly reduce cell viability;Gsta1,gsta2 and Nrp2 can reduce the cell survival rate and increase the apoptosis rate.In addition,Nrp2 can regulate the expression level of PARP1 and then activate apoptosis related pathways.In general,we found gsta1,gsta2 and Nrp2 genes in MIN6 cell T2 DM model,explored their effects on cell function,and improved our understanding of the pathogenesis of T2 DM.