| To study the effect of microRNA on hepatocytes proliferation and liver regeneration,high-throughput sequencing method was used to detect and analyze samples from rat regeneration liver tissue, the results showed that the expression of 129 microRNAs(miRNAs) including mi R-182 were significantly increased. The qRT-PCR method was used to confirm expression change of LR( liver regeneration)-related miRNAs. The result showed that 11 miRNAs(miR-21,-21*,-125 a,-142,-143,-181 c,-182,-183,-191,-199 a,-429) had similar expression trends with high-throughput sequencing results.Combining literature with the function analysis of those predictive target genes, previously miR-182 was selected to study its function in vitro. The rat hepatocytes BRL-3A were transfected by mimics and inhibitor of miR-182, cell proliferation respectively was promoted and inhibited significantly compared with control. MiR-182 was indicated directly bound with the 3’UTR of caspase3 mRNA through the detection result of dual-luciferase reporter, which suggested caspase3 is one of the target genes of miR-182.Caspase3 is a protein that executes apoptosis, so we speculated that mi R-182 accelerates rat hepatocyte BRL-3A proliferation by inhibiting expression of caspase3.In this study, mi R-182 over-expression plasmid and interference plasmid were constructed to further investigate the role of miR-182 in rat LR, over-expressed plasmid C1 and interference plasmid SC1 of miRNA-182 were respectively transfected into rat livers by tail vein hydrodynamics-based transfection(HDT). We first detected the expression dynamics of plasmid fluorescent protein, then the expression of the miR-182, we successfully established the most appropriate transgenic model. By detecting liver coefficient we found that the liver coefficient of C1 group was significantly higher than the control at 72 h and 120 h after PH, but there was no obvious change at 168 h. In the SC1 group, the liver coefficient was significantly lower than the control at 168 h. The results showed that the over-expression of miR-182 promoted liver regeneration, and rat LR could not be completed as scheduled when interfered the expression of miR-182.The qRT-PCR and WB analyzing effect of miR-182 on 9 genes/proteins related to cell proliferation 、apoptosis and survival showed that the m RNA changes of caspase3、TNFRS1A、SMAD7 and MAPK9 were up-regulated, but WNT4 was down-regulated at 72h; caspase3、TNFRS1A、TGFBR1、WNT4、MAP3K12、SMAD7、MAPK9、MAP2K1 were down-regulated at 120h; BCL2、TNFRS1A、TGFBR1、WNT4 wereup-regulated, but caspase3、SMAD7、MAPK9 were down-regulated at 168 h. The protein showed WNT4、MAPK9、SMAD7、MAP3K12、MAP2K1 were up-regulated at 72h; BCL2、WNT4、MAPK9、SMAD7、MAP3K12、MAP2K1 were up-regulated at 120h; WNT4 was up-regulated, but caspase3、TGFBR1、TNFRSF1A were down-regulated at 168 h. These results implied that PH 72-120 h mi R-182 promote rat LR by up-regulated proliferation-related genes, regeneration stopped at PH 168 h mainly relying on the inhibition of proliferation-related genes and promotion of apoptosis-related genes to suppress the excessive proliferation of LR.Comparing with the proteomics results in "Protein expression changes of hepatocytes in rat liver regeneration predicted their proliferation and apoptosis activity"(to be published), we found that the expression of target genes MAPK9, SMAD7, MAP3K12, MAP2K1, BCL2 and WNT4 in MAPK, SMAD,WNT4 pathways was promoted, but the expression of target genes caspase3, TNFRS1 A and TGFBR1 in caspase3 pathway was inhibited by mi R-182. Conclusion:mi R-182 regulated the expression of 9 targeted genes including caspase3 and affected their pathways activity to promote the rat hepatocyte proliferation and liver regeneration. |
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