Studies On Inhibition Of Hep G2 Proliferation And Migration In Hepatocellular Carcinoma Cells By Targeting FOXO4 With MiR-5589-5p And MiR-5589-3P

Objective:To find miRNAs(micro RNAs)that target FOXO4.Explore the possible roles and mechanisms of this miRNAs in HCC(Hepatocellular carcinoma).Provides potential gene targets and new ideas for the treatment of HCC.method:This study used the bioinformatics methods to screen miRNAs targeting FOXO4.Four miRNAs target gene prediction sites(Target Scan,miRDB,Pic Tar,miRanda)were used to predict all miRNAs targeting FOXO4;The expression data of miRNAs in the TCGA database(the Cancer Genome Atlas)were downloaded and calculated to find differentially expressed miRNAs;The two sets of miRNAs targeting FOXO4 and differentially expressed miRNAs were calculated by intersection calculation.After searching the literature,miRNAs that were not reported in HCC were selected for further experiments.The HCC tissues and paracancerous tissue samples were collected from patients with primary HCC at the Affiliated Tumor Hospital of Guangxi Medical University.At the same time,the patient’s general condition,past history,and clinical pathological features were collected.RT-PCR was used to detect the expression of miRNAs and FOXO4 in tissue samples.The relationship between miRNAs expression and clinicopathological features was analyzed.The correlation between miRNAs and FOXO4 was calculated.The lipofactamine2000 liposome transfection reagent was used to transfect miRNA mimics into hepatoma cells Hep G2 to induce high expression of miRNAs.A negative control group was set up to detect the changes of the corresponding miRNAs and FOXO4 expression and analyze the relationship between them.Cell proliferation experiments and cell scratch experiments were performed to calculate respectively the OD value and cell migration area,and the effect of miRNAs on the proliferation and migration of hepatocellular carcinoma cells was investigated.result:1.A total of 56 patients with liver cancer were included in the study,including 50 males and 6 females;the age ranged from 16 to 84 years and the median age was 51 years old;BCLC stage A was 28 cases,and B stage was 8 cases,C was 20 cases.2.The results of gene detection showed that the expression levels of miR-5589-5p(t=-5.908,P<0.01)and Mi R-5589-3p(t=-8.267,P<0.01)in HCC tissues were significantly lower than those adjacent tissues.organization.After analysis of the relationship between miRNAs and clinicopathological features,miR-5589-5p was significantly different in the distribution of alpha-fetoprotein(AFP)in cancer tissue of male patients with liver cancer older than 50 years(P=0.033).HBs Ag,tumor size,ES differentiation(Edmondson-Steiner),MVI(Microvascular invasion),capsule invasion,extrahepatic metastasis,BCLC stage,and portal vein tumor embolus were not significantly different(P>0.05);There were differences in the distribution of portal vein tumor thrombi(χ~2=5.543,P=0.019)and BCLC stages(χ~2=4.978,P=0.026)in the different expression levels of Mi R-5589-3p;but no significant difference in the distribution of HBs Ag,AFP,tumor size,ES differentiation,MVI,capsule invasion,and extrahepatic metastasis(P>0.05).In HCC tissues,the expression of miR-5589-5p and miR-5589-3p were not significantly associated with FOXO4,but there was a certain negative correlation between the two miRNAs and FOXO4 in adjacent tissues.3.In the cell proliferation experiment,the proliferation of hepatoma cells at the miR-5589-5p mimic transfection group was significantly inhibited(t=-2.367,P=0.039).When miR-5589-3p mimic transfected hepatoma cells,the inhibition was not significant(t=-1.356,P=0.205).The cell scratch test results showed that miR-5589-3p transfected cell migration area is smaller than the control group,indicating that the group may be inhibited cells,and miR-5589-5p transfected cell migration area and control group cell migration area similar.Conclusions:1.Mi R-5589-5p and miR-5589-3p show low expression in HCC tissue.2.FOXO4 may be suppressed as a target gene of Mi R-5589-5p and miR-5589-3p.Mi R-5589-5p can significantly inhibit the proliferation of Hep G2 cells,miR-5589-3p can inhibit the migration of Hep G2 cells to a certain extent;both may play an important role as potential tumor suppressor genes HCC.