Research On MiR-1246 Targets Kindlin-2 To Regulate The Autophagy,Migration And Invasion Of Cervical Cancer Cells And Establishment Of Autophagy-related Prognosis Model

Part ? miRNA-1246 targets Kindlin-2 to regulate the migration andinvasion of cervical cancer cellsObjective: Screen and confirm the functional target genes for miR-1246 to regulate cervical cancer cells migration and invasion.Methods: Bioinformatics database was used to predict the potential target genes of miR-1246,and combined with literature and gene expression levels,target genes were selected;Western blot was used to detect the effect of miR-1246 on target gene protein expression;Dual-luciferase reporter assay verification of the specific binding site of miR-1246 to the target gene;miR-1246 and target gene in clinical specimens were detected by RT-qPCR and IHC,and the correlation between the expression of the two was analyzed;Rescue experiments verified the necessity of target genes for miR-1246 to regulate migration and invasion processes.Results: Kindlin-2 was selected as the target gene;RT-qPCR and immunohistochemistry results showed that miR-1246 and Kindlin-2 were negatively correlated in cervical cancer tissues;Western blot results showed that miR-1246 can down-regulate the expression of Kindlin-2;Dual-luciferase reporter assay confirmed Kindlin-2 3`UTR and miR-1246 binding site;Rescue experiments confirmed that Kindlin-2 could reverse miR-1246 induced cell migration and invasion.Conclusion: Kindlin-2 is the target gene of miR-1246.miR-1246 downregulates the expression of Kindlin-2 by binding to the site on the 3`UTR of Kindlin-2,which enhances the migration and invasion of cervical cancer cells.Part ? Kindlin-2 suppresses cervical cancer cell migration and invasion through AKT/mTOR-mediated autophagy inductionObjective: To explore the regulation and specific mechanism of Kindlin-2on cervical cancer cell autophagy,migration and invasion,analyzed the expression of Kindlin-2 in cervical cancer,and its relationship with clinicopathological factors and prognosis,and explore the biological effect and clinical value of Kindlin-2 in cervical cancer.Methods: The expression of Kindlin-2 in cervical cancer was analyzed by bioinformatics database and clinical specimen.In two cervical cancer cell lines,Kindlin-2 was up-regulated and down-regulated respectively.Autophagy related proteins were tested by Western blot,autophagy flux was detected by confocal microscopy,and autophagy ultrastructure was observed by transmission electron microscopy to explore the effect of Kindlin-2 on autophagy.The changes of proteins in the downstream pathway were detected by Western blot,and the necessity of the pathway in the process of Kindlin-2 regulating autophagy was explored in the rescue experiment.The wound healing assay and the transwell assay explored the influence of Kindlin-2 on cell migration and invasion,and the rescue experiment explored the necessity of autophagy in Kindlin-2regulating cell migration and invasion.RT-qPCR was used to detect the expression level of Kindlin-2 in cervical cancer tissues,and to analyze its correlation with clinicopathological factors.The relationship between kindlin-2expression and prognosis was investigated in combination with a public database.Results: the m RNA and protein levels of Kindlin-2 were down-regulated in cervical cancer.Kindlin-2 increased the expression level of autophagy-related protein LC3-? and decreased the expression level of p62;Autophagy flux detection results showed that Kindlin-2 increased the number of yellow and red spots.Transmission electron microscopy observed that Kindlin-2 increased the number of autophagosomes and autophagolysosome.Western blot demonstrated that Kindlin-2 reduced the expression levels of p-AKT and p-mTOR,and the rescue experiment showed that the regulation of Kindlin-2 on autophagy depended on the AKT/mTOR pathway.The wound healing assay and transwell assay showed that Kindlin-2 inhibited cell migration and invasion,and the rescue experiments showed that autophagy was necessary for this process.Low expression of Kindlin-2 is associated with deeper interstitial infiltration and higher rates of lymph node metastasis,as well as with shorter DFS.Conclusion: Kindlin-2 inhibits the migration and invasion of cervical cancer cells through autophagy mediated by AKT/mTOR pathway,and the low expression of Kindlin-2 indicates poor prognosis of cervical cancer.Part ? Establish a prognostic model of cervical cancer based on autophagy-related genesObjective: The high throughput gene expression data was analyzed by bioinformatics to establish a prognostic model for cervical cancer based on the expression profiles of autophagy-related genes(ATGs).Methods: Gene expression profiles and clinical data of cervical cancer cases were obtained fromThe Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)databases,and the list of autophagy-related genes(ATGs)was obtained from the human autophagy database(HADb).TCGA cases were randomly divided into the training set and the test set,and univariate Cox regression analysis was carried out in the training set to preliminarily screen out the ATGs related to the prognosis of patients,and then the number of genes was further reduced through the least absolute shrinkage and selection operator(LASSO)regression.Prognostic models were constructed based on the coefficients of multivariate Cox regression and gene expression,and the risk score of each case was calculated,and patients were divided into high-and low-risk groups according to the risk score.The Kaplan-Meier survival analysis and receiver operating characteristic curve were used to evaluate the predictive efficacy of the prognostic model.Multivariate analysis and stratified analysis assess the independent prognostic value of prognostic models.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were used to analyze the enrichment of ATGs and related genes in the model.Gene set enrichment analysis(GSEA)explored the gene enrichment pathway of high-risk patients.Reveal the possible mechanism of action of this prognostic model.Results: Univariate Cox analysis showed that 33 ATGs were related to the prognosis of patients in the training set,and then 8 genes(VEGFA,TM9SF1,NAMPT,KLHL2,HSPA8,HSP90AB1,HGS,ATG4D)were selected by LASSO regression method to construct a prognostic model.This model can divide cervical cancer patients in TCGA and GEO data sets into high-and low-risk groups with significantly different prognosis.The ROC curve shows the area under the curve of 1-year,3-year and 5-year survival rates are 0.746,0.750 and 0.791,respectively.Multivariate analysis and stratified analysis further confirmed the independent prognostic value of the model.In addition,GO,KEGG and GSEA analysis revealed several basic signaling pathways and biological processes related to the prognostic model.Conclusion: We constructed and verified a new prognostic model based on8 ATGs,which can be used as an independent prognostic factor for cervical cancer patients.