MiR-22-3p And MiR-29a-3p Synergistically Inhibit The Activation Of LX-2 Cells By Targeting AKT3

Objectives To investigate the effects of miR-22-3p and miR-29a-3p on the activation of human hepatic stellate cells LX-2 by targeting serine/threonine kinase 3(AKT3),to provide a new avenue for the combined application of miR-22-3p and miR-29a-3p in the study and treatment of hepatic fibrosis(HF).Methods 1 H&E,Masson’s trichrome staining,and Sirius Red staining were used to observe the pathological changes of the liver tissue in the sham-operated group(Sham)and the HF group with common bile duct ligation(BDL).2 miRNAs microarray was used to analyze the differentially expressed miRNAs in liver tissue of rats in Sham group and BDL group.3 Kyoto Encyclopedia of Genes and Genomes(KEGG)database,Gene Ontology(GO)and ECR Browser were used to analyze the signal pathways,GO function and conservation involved by miR-22-3p and miR-29a-3p.4 q RT-PCR was performed to detect the expression levels of miR-22-3p and miR-29a-3p in BDL rat liver tissue and activated LX-2 cells.5 CCK-8,β-galactosidase staining,double immunofluorescence,Transwell,wound healing,colony formation,q RT-PCR and Western Blot assays were performed to detect the effects of miR-22-3p and miR-29a-3p mimics/inhibitor on proliferation,senescence,migration,colony formation ability and the expression of α-smooth muscle actin(α-SMA),collagen type I α1 chain(COL1A1),p16 and p21 in LX-2cells.6 q RT-PCR was used to detect the molecular mechanisms of the interaction of miR-22-3p and miR-29a-3p.7 Bioinformatics was used to predict the common target genes of miR-22-3p and miR-29a-3p.KEGG and GO were used to analyze the screened target genes.RNAhybird and String were used to analyze the binding free energy and protein interactions.8 Dual luciferase reporter assay was used to determine the targeting relationship between miR-22-3p/miR-29a-3p and target gene AKT3.9 q RT-PCR,Western Blot,double immunofluorescence and immunohistochemistry were used to detect the expression of AKT3 in BDL rat liver tissue and activated LX-2 cells.10 CCK-8,β-galactosidase staining,double immunofluorescence,Transwell,wound healing,colony formation assay,q RT-PCR and Western Blot assay were performed to detect the effect of si-AKT3/pc DNA3.1-AKT3 on proliferation,senescence,migration,colony formation ability and expression of α-SMA,COL1A1,p16 and p21 in LX-2 cells.11 CCK-8,Transwell,and q RT-PCR assays were performed to detect whether AKT3 could reverse the inhibitory effects of miR-22-3p and miR-29a-3p on proliferation,migration,m RNA expression of α-SMA and COL1A1 in LX-2 cells.Results 1 The levels of deposition of collagen,and the number of collagen fibers were increased in BDL rat liver tissue,as detected by H&E,Sirius Red staining and Masson’s trichrome staining,respectively,compared to those in the sham rat liver tissue.The HF model of BDL rats was successfully induced.2 The levels of miR-22-3p and miR-29a-3p were downregulated in the liver tissues of BDL group rats,as detected by miRNAs microarray.3 miR-22-3p and miR-29a-3p were related to HF-related signal pathways,biological processes,and were species conserved,as analyzed by KEGG,GO and ECR Browser.4 The expression levels of miR-22-3p and miR-29a-3p were significantly downregulated in the liver tissue of BDL rats and activated LX-2 cells,the differences were statistically significant,compared with the control group,respectively(P<0.05).5The proliferation,migration,colony-forming ability,and α-SMA and COL1A1 expression of LX-2 cells were synergistically inhibited,and the cellular senescence and the expression of p16 and p21 were synergistically induced by miR-22-3p and miR-29a-3p mimics.However,the opposite results were obtained in LX-2 cells transfected with miR-22-3p and miR-29a-3p inhibitors.The above results showed that the differences were statistically significant,compared with the control group,respectively(P<0.05).6 miR-22-3p or miR-29a-3p expression in LX-2 was dramatically enhanced or decreased by the transfection of the another miRNA mimics or inhibitors,the differences were statistically significant,compared with the control group,respectively(P<0.05).7 24 common candidate target genes of miR-22-3p and miR-29a-3p were predicted,as shown by bioinformatics analysis.The HF-related signaling pathways and biological processes were enriched in these target genes,as analyzed by KEGG and GO.AKT3 was screened as a common candidate target gene of miR-22-3p and miR-29a-3p.AKT3 could bind with miR-22-3p and miR-29a-3p,as analyzed by RNAhybrid.8 The luciferase activity was significantly reduced in LX-2cells co-transfected with both the wild-type reporter vector and miR-22-3p/miR-29a-3p mimics,compared with the control group,the difference was statistical significance(P<0.05).The luciferase activity was no significant difference in LX-2 cells co-transfected with both the mutant reporter vector and miR-22-3p/miR-29a-3p mimics,compared with the control group(P>0.05),as shown by the results of the dual luciferase reporter assays.9AKT3 was highly expressed in liver tissue of BDL rats and activated LX-2 cells,the differences were statistically significant,compared with the control group,respectively(P<0.05).10 The proliferation,migration,colony-forming ability,and α-SMA and COL1A1 expression of LX-2 cells were inhibited,and the cellular senescence and the expression of p16 and p21 were induced by si-AKT3.However,the opposite results were obtained in LX-2 cells transfected with pc DNA3.1-AKT3.The above results showed that the differences were statistically significant,compared with the control group,respectively(P<0.05).11 The synergistic inhibitory effects of miR-22-3p and miR-29a-3p mimics on LX-2 cell proliferation,migration ability,α-SMA and COL1A1 m RNA expressions were rescued by pc DNA3.1-AKT3.The above results showed that the differences were statistically significant,compared with the control group,respectively(P<0.05).Conclusions 1 miR-22-3p and miR-29a-3p synergistically inhibited the activation of LX-2 cells.2 miR-22-3p and miR-29a-3p co-targeted AKT3 and regulated its expression.3AKT3 promoted the activation of LX-2 cells.4 AKT3 overexpression reversed the synergistic inhibitory effects of miR-22-3p and miR-29a-3p on proliferation in LX-2 cells.In conclusion,miR-22-3p and miR-29a-3p might synergistically inhibit the activation of LX-2 cells by targeting AKT3.Figure 38;Table 7;Reference 124.