The Mechanism Of MiR-216b-5p Targeting TRIM44 Inhibiting PI3K/AKT/mTOR Pathway In Regulating The Biological Function Of Hepatoma Cells

Part 1 Expression of miR-216b-5p and its predicted target gene TRIM44 in hepatocellular carcinoma tissues and cells Purpose and significanceThe expression level of miR-216b-5p in hepatocellular carcinoma tissues and cells was studied,and predicted the target genes that miR-216b-5p may regulate.The expression of target genes in hepatocellular carcinoma tissues and cells and the correlation between them were further analyzed.MethodsThe carcinoma tissues and adjacent liver tissues of 30 newly diagnosed HCC patients were collected.The immortal liver cells(THLE-2)and hepatoma cells(Huh7,Hep G2,Hep3 B,MHCC97H,MHCC97 L,LM3)were cultured in vitro,and the expression level of miR-216b-5p in hepatocellular carcinoma tissues and cells was detected by fluorescence quantitative PCR.The downstream related target genes of miR-216b-5p were predicted and analyzed through the websites,and the expression of target gene was detected by fluorescence quantitative PCR or western blot in hepatocellular carcinoma tissues and cells to further analyze the correlation between miR-216b-5p and the target gene.Results 1.The expression level of miR-216b-5p in hepatocellular carcinoma tissues was 0.51 times higher than that in adjacent liver tissues,which was significantly lower than that in adjacent liver tissues(p=0.001).The expression level of miR-216b-5p in hepatoma cells were significantly lower than this in immortal liver cells(all p<0.05),and the expression levels in Hep G2,Hep3 B,Huh7,MHCC97 H,MHCC97L and LM3 cells were 0.71,0.65,0.57,0.44,0.76 and 0.26 times of those in immortal liver cells,respectively.2.The downstream target gene of miR-216b-5p was predicted to be TRIM44 by the bioinformatics websites.3.The expression level of TRIM44 mRNA in hepatocellular carcinoma tissues was 1.40 times that in adjacent liver tissues,which was significantly higher than that in adjacent liver tissues(p=0.004).The expression levels of TRIM44 mRNA in hepatoma cells were significantly higher than this in immortal liver cells(all p<0.05),and the expression levels in Huh7,Hep G2,Hep3 B,MHCC97H,MHCC97 L and LM3 cells were 1.99,1.63,1.83,2.18,1.48 and 2.72 times of those in immortal liver cells,respectively.4.The expression level of TRIM44 protein in hepatocellular carcinoma tissues was 1.22 times higher than that in adjacent liver tissues,and the difference was statistically significant(p=0.033).The expression levels of TRIM44 protein in hepatoma cells were significantly increased compared with those in immortal liver cells(all p<0.05),and the expression levels of TRIM44 protein in Huh7,Hep G2,Hep3 B,MHCC97H,MHCC97 L and LM3 cells were 1.80,1.59,1.67,2.08,1.54 and 2.18 times of those in immortal liver cells,respectively.5.The mRNA expression levels of miR-216b-5p and TRIM44 in hepatocellular carcinoma tissues were negatively correlated,r=-0.463,p<0.001.ConclusionBy detecting the expression levels of miR-216b-5p and TRIM44 in hepatocellular carcinoma tissues and cells,it was found that miR-216b-5p was an inhibitor of hepatocellular carcinoma,while the target gene TRIM44 was an oncopromoter of hepatocellular carcinoma,and the two were negatively correlated,which was consistent with the bioinformatics prediction results.Part 2 Effects of miR-216b-5p on biological function of hepatoma cells Purpose and significanceThe effect of miR-216b-5p on the biological function of hepatoma cells was analyzed in vitro using hepatoma Huh7 and Hep G2 cells.MethodsHepatoma Huh7 and Hep G2 cells were infected with miR-216b-5p mimic and inhibitor,and cell models with high and low expression of miR-216b-5p were constructed in vitro,respectively.CCK8 assay,Transwell invasion assay,cell scratch assay and colony formation assay were used to detect the effect of miR-216b-5p on the biological function of hepatoma cells,respectively.Results1.The relative expression level of miR-216b-5p in hepatoma Huh7 cells was significantly increased in mimic group,which was 754.66 times that of mimicNC(p=0.003).The relative expression level of miR-216b-5p in the inhibitor group was significantly decreased,0.10 times that of the inhibitor-NC group(p<0.001).The relative expression level of miR-216b-5p in mimic group was significantly increased in hepatoma Hep G2 cells,which was 257.60 times that in mimic-NC group(p=0.001).The relative expression level of miR-216b-5p in the inhibitor group was significantly decreased,0.28 times that in the inhibitor-NC group(p=0.001).2.CCK-8 assay showed that after transfected with miR-216b-5p mimic for24 h,the proliferation ability of hepatoma Huh7 and Hep G2 cells was significantly reduced over time compared with that of mimic-NC group.After transfection with miR-216b-5p inhibitor for 24 h,the proliferation ability of hepatoma Huh7 and Hep G2 cells was significantly enhanced over time compared with the inhibitorNC group.3.Transwell invasion assay showed that the invasion ability of hepatoma Huh7 and Hep G2 cells transfected with miR-216b-5p mimic was significantly lower than that of the mimic-NC group(all p<0.001).The invasion ability of hepatoma Huh7 and Hep G2 cells transfected with miR-216b-5p inhibitor was significantly higher than that of the inhibitor-NC group(all p<0.001).4.Cell scratch experiment showed that the scratch healing rate of hepatoma Huh7 and Hep G2 cells transfected with miR-216b-5p mimic was slower than that of the mimic-NC group(all p<0.05).The scratch healing ability of hepatoma Huh7 and Hep G2 cells transfected with miR-216b-5p inhibitor was faster than that of the inhibitor-NC group(all p<0.05).5.Colony formation assay showed that the number of hepatoma Huh7 and Hep G2 cells transfected with miR-216b-5p mimic was significantly reduced compared with the mimic-NC group(all p<0.01).The number of clone formation of hepatoma Huh7 and Hep G2 cells transfected with miR-216b-5p inhibitor was significantly increased compared with the inhibitor-NC group(all p<0.01).ConclusionAfter transfection with mimic and inhibitor,it was confirmed that miR-216b-5p inhibited the biological functions of hepatoma cells,such as proliferation,invasion,migration and clone formation,and the role of miR-216b-5p in the occurrence and development of hepatocellular carcinoma was clarified.Part 3 The mechanism of miR-216b-5p targeting TRIM44 inhibiting PI3K/ AKT /mTOR pathway in regulating the biological function of hepatoma cells Purpose and significanceThe interaction and regulatory relationship between miR-216b-5p and TRIM44 were further analyzed,and the relevant molecular mechanisms were clarified.MethodsThe interaction between TRIM44 and miR-216b-5p was verified by double luciferase report assay.miR-216b-5p mimic and overexpression-TRIM44(OETRIM44)were transfected into hepatoma Huh7 and Hep G2 cells.The reverse effect of OE-TRIM44 on the inhibition of biological function of miR-216b-5p mimic in hepatoma cells were detected by CCK8 assay,Transwell invasion assay,cell scar assay and colony formation assay,respectively.The possible mechanism was explored based on literature reports,and verified by western blot.Results1.The relative luciferase ratio of hepatoma Huh7 and Hep G2 cells transfected with miR-216b-5p mimic and wild-type(WT)plasmid of TRIM44 gene was significantly lower than that of other groups(p<0.001).2.After transfection of hepatoma Huh7 and Hep G2 cells with miR-216b-5p,the expression level of TRIM44 mRNA in the mimic-NC group was significantly decreased compared with that in the mimic-NC group(all p<0.01).Compared with inhibitor-NC group,the expression of TRIM44 mRNA in inhibitor group was significantly increased(all p<0.05).3.After transfected with miR-216b-5p into hepatoma Huh7 and Hep G2 cells,the expression of TRIM44 protein in the mimic-NC group was significantly lower than that in the mimic-NC group(all p<0.05).Compared with inhibitor-NC group,the expression level of TRIM44 protein in inhibitor group was significantly increased(all p<0.05).4.After transfection of hepatoma Huh7 and Hep G2 cells with TRIM44-NC,mRNA expression levels of TRIM44 in OE-TRIM44 group were significantly increased compared with TRIM44-NC group(all p<0.05).Compared with TRIM44-NC group,the expression level of TRIM44 protein in OE-TRIM44 group was significantly increased(all p<0.05).5.CCK8 results further showed that the proliferation ability of hepatoma Huh7 and Hep G2 cells in mimic+TRIM44-NC group was not significantly changed compared with that in mimic group;While with the passage of time,the proliferation ability of cells in vitro in mimic +OE-TRIM44 group was higher than that of mimic +TRIM44-NC group.6.The results of Transwell invasion experiment further showed that in hepatoma Hu H7 and Hep G2 cells,compared with mimic group,the invasion ability of cells in mimic+TRIM44-NC group was not significantly changed,while the invasion ability of cells in mimic+OE-TRIM44 group was significantly increased compared with mimic+TRIM44-NC group(all p<0.05).7.The results of cell scratch test further showed that in hepatoma Huh7 and Hep G2 cells,compared with mimic group,the scratch healing ability of mimic+TRIM44-NC group had no significant difference,but the scratch healing ability of mimic+OE-TRIM44 group was significantly improved compared with mimic+TRIM44-NC group(all p<0.05).8.The results of colony formation assay further showed that in hepatoma Huh7 and Hep G2 cells,compared with mimic group,the in vitro colony formation ability of cells in mimic+TRIM44-NC group had no significant change,but the colony formation ability of cells in mimic+OE-TRIM44 group was significantly increased compared with mimic+TRIM44-NC group(all p<0.05).9.Western blot test results further showed that there were no significant differences in the protein levels of PI3 K,AKT,mTOR and P70S6 K among all groups in hepatoma Huh7 and Hep G2 cells;Compared with mimic-NC group,the protein levels of p-p-PI3 K,p-AKT,p-mTOR and p-P70S6 K in mimic group were significantly reduced(all p<0.01);The protein levels of p-PI3 K,p-AKT,p-mTOR and p-P70S6 K in mimic+TRIM44-NC group were not significantly changed compared with those in mimic+TRIM44-NC group,but the protein levels of pPI3 K,p-AKT,p-mTOR and p-P70S6 K in mimic+OE-TRIM44 group were significantly increased compared with those in mimic+TRIM44-NC group(all p<0.05).ConclusionCell recovery experiments confirmed that overexpression of TRIM44 could reverse the inhibitory effect of miR-216b-5p on the biological function of hepatoma cells,and it was found that its regulation might be accomplished through PI3K/ AKT /mTOR pathway.