| BackgroundThe liver is an organ with full regenerative ability and this function is helpful for alleviating liver dysfunction caused by chemical or traumatic injury.miRNA participates in a series of physiological and pathological processes in the body,such as cell differentiation,body metabolism,immune response,inflammation and tumor development.Similarly,some miRNAs also play an important role in the regulation of liver regeneration.The precursor of miR-149 can produce two active chains,including miR-149-5p(miR-149)and miR-149-3p(miR-149*),which play different biological functions respectively.In this study,we will explore the role and mechanism of miR-149 in liver regeneration by constructing the experimental model of partial hepatectomy(PHx)in mice.ObjectiveTo explore the effects of miR-149 and potential target genes on liver regeneration,and to elucidate the role and mechanism of miR-149 in liver regeneration.Methods1.To study the difference in regenerative capacity between miR-149*-/-mice and normal WT mice(1)The WT and miR-149*-/-mice at 8 weeks age were given 2/3 PHx and sacrificed at 0 h,40 h and 5 d after surgery.The curve of liver regeneration rate was drawn,and liver tissues were stained with Ki-67 by immunohistochemical technology,the mRNA levels of genes related to liver cell proliferation,including Cyclin D1,Cyclin E1,Cyclin A2,p21 and p27,were measured by RT-PCR.and Western blot was used to detect the protein expression of p-Akt,thus to compare the differences in liver regeneration.(2)The WT and miR-149*-/-mice at 48 weeks age were given the same operation and sacrifice to detect changes in liver regeneration capacity,and to explore the differences in liver regeneration capacity compared with the mice at 8 weeks age.2.To explore the effects of miR-149 and miR-149*on liver regeneration(1)NC agomir,miR-149 agomir and miR-149*agomir were intravenously injected into different groups of WT mice at 8 weeks age,2/3 of PHx were performed 24 hours later and all mice were sacrificed at 0 h or 36 h after the surgery(2)The curve of liver regeneration rate was drawn,and liver tissues were stained with Ki-67 by immunohistochemical technology,the mRNA levels of genes related to liver cell proliferation,including Cvclin D1,Cyclin E1,Cyclin A2 were measured by RT-PCR.and Western blot was used to detect the protein expression of p-Akt,thus to compare the differences in liver regeneration.3.To screen out the potential target gene of iniR-149The potential target genes of miR-149 were predicted by TargetScan,Pictar,miRDB and other websites,the luciferase plasmids containing binding sites were constructed and verified in human hepatic stellate cells LX2 cells by double luciferase test.Through,the accuracy of miR-149 binding to the target was verified by comparing the activity of luciferase wild-type and mutant plasmids4.To explore the stage in which miR-149 exerts the role in liver regenerationThe WT mice at 8 weeks age were subjected to 2/3 PHx and sacrificed at 0 h,1 h,4 h,6 h and 12 h after surgery.The miR-149 and PHLPP2 mRNA levels were measured by RT-PCR,and Western blot was used to detect the expression level of PHLPP2 protein.5.To study the role of target genes during liver regeneration(1)To detect the mRNA level of PHLPP2 gene in miR-149*-/-mice and mice with the overexpression of miR-149.(2)The plasmid with the overexpressed PHLPP2 gene was constructed and intravenously injected into WT mice at 8 weeks age by using fluid dynamics method.All mice were performed with 2/3 PHx 24 hours later and sacrificed at 0 h,40 h and 5 days after the operation.(3)The curve of liver regeneration rate was drawn,and liver tissues were stained with Ki-67 by immunohistochemical technology,the mRNA levels of genes related to cellular proliferation and apoptosis,including Cyclin D1,Cyclin E1,Cyclin A2,FOXO3 and BAD,were measured by RT-PCR,and Western blot was used to detect the protein expression of p-Akt,thus to compare the differences in liver regeneration in control group and PHLPP2 overexpression group.Results1.Differences in liver regeneration ability between WT mice and miR-149*-/-mice at different ages(1)The liver regeneration rate of miR-149*-/-mice was significant lower than that of WT mice(2)After Ki-67 staining,it was found that miR-149*-/mice had weaker liver cell division ability.(3)The expression levels of pro-proliferation-related genes in the liver tissues of miR-149*-/-mice increased,while the expression levels of anti-proliferation-related genes decreased.(4)The phosphorylated Akt protein in miR-149*-/-mice was decreased,indicated the liver regeneration ability of miR-149*-/-mice at different ages was weaker than that of WT mice2.The effect of miR-149 and miR-149*on liver regeneration(1)36 hours after the PHx,the regeneration rate of miR-149 group was significantly higher than that of the NC group,while that of the miR-149*group was not significantly different(2)The mRNA levels of pro-proliferation genes in miR-149 group were significantly higher than those in NC group,while those in miR-149*group had no significant difference(3)After immunohistochemical staining,it was found that the number of Ki-67 positive cells in miR-149 group was significantly higher than that in NC group(4)The phosphorylated Akt protein level in miR-149 group was higher than that in NC group,indicated the promoting role of miR-149 in liver regeneration.However,miR-149*had no such function in liver regeneration3.miR-149 regulates phosphorylation of Akt protein during liver regeneration(1)It was found that the 3’UTR in mRNA of PHLPP2 gene contains a potential binding site with the seed region of miR-149(2)Luciferase experiment results show that miR-149 could reduce the dual luciferase activity of PHLPP2 recombinant plasmid,suggesting that PHLLP2 was the direct target gene of miR-149(3)Overexpression of miR-149 could significantly reduce the mRNA and protein levels of the PHLPP2 gene in LX2 cells4.The effect of miR-149 and target gene PHLPP2 on liver regeneration in the early stage of liver regeneration(1)The mRNA and protein of PHLPP2 gene were negatively correlated with the expression level of miR-149.(2)In the early stage of liver regeneration,the increased expression of miR-149 could inhibit the expression of PHLPP2,which in turn promotes the phosphorylation of downstream Akt,thereby promoting liver regeneration.(3)The upregulation of PHLPP2 expression in young and old mice miR-149*-/-indicated that the weaker regeneration ability of miR-149*-/-mice might be related to the abnormally increased level of PHLPP2 gene expression.5.The effect of PHLPP2 on liver regeneration(1)The liver regeneration rate of mice with the overexpression of PHLPP2 was significantly lower than that of pcDNA3.1 control mice.(2)Ki-67 staining showed that mice with the overexpression of PHLPP2 had weaker liver cell division ability.(3)Overexpression of PHLPP2 could inhibit the expression of proliferation-related genes,while upregulate the expression level of proliferation-related genes.(4)The level of phosphorylated Akt protein in mice with the overexpression of PHLPP2 was reduced,indicated that PHLPP2,as a negative regulator of Akt phosphorylation,exerted an inhibitory effect on the liver regeneration stage,which was not conducive to the effective liver regeneration.Conclusions1.miR-149*-/-mice at different ages show poor liver regeneration ability.2.Overexpression of miR-149 can promote liver regeneration after PHx.3.miR-149 induces the activation of Akt and promotes liver regeneration after PHx by targeting PHLPP2. |
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