Effect Of MiR-199a/b-3p Targeting DJ-1 Gene On HCC Via Ras/PI3K/AKT Axis

Objective : To investigate the regulatory relationship and mechanism of miR-199a/b-3p targeting DJ-1 gene in HCC development.Methods : The expression,pathological correlation and molecular interaction of DJ-1 gene were analyzed using bioinformatics databases(TCGA and HPA).Upstream miR-199a/b-3p was screened using miRWalk and Target Scan databases.Real-time quantitative technique(RT-qPCR)was used to verify the expression of miR-199a/b-3p.Dual luciferase reporter assay and protein imprinting technique(West bloting)were used to verify the targeting relationship between miR-199a/b-3p and DJ-1 gene and the molecular mechanism.Cell proliferation,scratch,Transwell and apoptosis were used to detect the cell function after the regulation of DJ-1 gene.SPSS26.0 statistical technology was used to analyze the data results.Results :1.Compared with the matched and unmatched samples in the TCGA database,the expression of DJ-1 gene in HCC tissues was higher than that in Normal tissues(P<0.001).Secondly,HPA database and HIC staining results of clinical samples showed that the expression of DJ-1 gene in HCC tissues was significantly higher than that in paracancer tissues and normal liver tissues.It was also closely correlated with clinical stage T(T1T2 vs T3T4),pathological grade(ⅠⅡvs ⅢⅣ),portal vein invasion and survival rate of HCC patients(P<0.05).In addition,DJ-1 gene had certain predictive value for HCC clinical diagnosis(area under ROC curve was 79.6%,sensitivity was 84%,specificity was71.7%).2.The bioinformatics results predicted that miR-199a/b-3p may regulate miRNA upstream of DJ-1 gene,and the molecular correlation in TCGA database showed that Spearman correlation coefficient r=-0.113,P = 0.031,indicating that the molecular relationship was negative targeting.The expression of miR-199a-3p was up-regulated in HepG2 hepatocellular carcinoma cell line and normal liver cells LO2 by RT-PCR assay(P<0.05).The results of double luciferase gene assay confirmed the existence of targeted interaction between miR-199a-3p and DJ-1 gene.interaction between miR-199a-3p and DJ-1 gene.r=-0.113,P = 0.031,AUC=0.882,sensitivity =0.90,specificity =0.821,CI(0.849-0.915),and its molecular relationship was negative targeting.The ROC curve results were better than those of other miRNAs,and the results reported by double luciferase gene detection again showed that there was a negative targeting interaction between miR-199a-3p and DJ-1 gene.3.Cell function of HepG2 cells transfected with miR-199a-3p mimics,inhibitor and OE-DJ-1 was detected by CCK8,cell scratch,Transwell migration and flow cytometry.The results showed that proliferation,migration and invasion of HepG2 cells were enhanced after DJ-1 gene overexpression.The apoptosis process of HepG2 cells was weakened,the difference was statistically significant,and the cell function of HepG2 cells was opposite after the intervention of miR-199a-3pmimics(P<0.005).4.Protein imprinting technology(West bloting)was used to detect oncogene Ras protein and PI3K/AKT pathway key molecule AKT after transfection of miR-199a-3p mimics,inhibitor and OE-DJ-1 HepG2 cells.The results showed that miR-199a-3p mimic can inhibit the expression of oncogene Ras and the activation of AKT signal protein molecules by inhibiting the expression of DJ-1 gene.Conclusion : 1.DJ-1 gene is highly expressed in HCC tissues and is significantly correlated with TNM stage,pathological grade,portal vein invasion and survival of HCC patients,which has predictive value for HCC clinical diagnosis,promotes proliferation,migration and invasion of HepG2 cells,and inhibits cell apoptosis2.miR-199a/b-3p is low expressed in HCC,which has high predictive value for HCC clinical diagnosis,and can inhibit proliferation,migration and invasion of HepG2 cells and promote cell apoptosis3.miR-199a/b-3p inhibits the proliferation,proliferation,migration and invasion of HepG2 cells and promotes apoptosis of HepG2 cells by negatively targeting DJ-1 gene to inhibit the activation of Ras and PI3k/AKT signaling pathway.4.Overexpressed DJ-1 gene partially reversed the inhibition of miR-199a/b-3p.