| Background and Objective:At present,the most effective treatment for a variety of benign and malignant liver diseases is partial hepatectomy(PH).However,the resection extent can be debatable: too little resection may cause tumor recurrence or treatment failure,while excessive resection may causeliverfailure.This contradiction between the effect of treatmentand the possiblility of liver failurehas severely limited the application of liver surgery.There are also a variety of limitations for the alternative treatments such as liver transplantation,which include donor shortage,immunological rejection and highcosts.In addition,the currentresearch situation for developing artificial liveris still atabasic stage,making this method far from clinical application.Therefore,the researchers tend to concentrate on the field of liver regeneration.Due to the strong regeneration potential of liver,effective stimulation for the liver regeneration may be an ideal way to solve these problems mentioned above.Scholars have been working hard to understand the molecular mechanism of the hepatocyte proliferation and to find a way to accelerate the process of liver regeneration,makingthis filed the focus of hepatobiliary researches these days.This study is the further exploration based onthe previous study,we focusesondeterming the effect of Tmub1 silencing on the phosphorylation of Akt and the proliferation and growth of hepatocytes.Materials and Methods:1.RNA interference(RNAi)technique was used to silence the expression of Tmub1 in BRL-3A cells.The interfere efficiency was detected by Western Blotting;2.Experimental groups:control group;Tmub1interference group(si RNA group);Tmub1 interferencewith 24 h treatment of 0.5μmol/LAkt phosphorylation inhibitor MK2206 group(si RNA+MK2206 group).3.The colony forming assay and the CCK-8 assay were used to detect the cell proliferation rate;4.All groups were treated by the montioned methodsduringthe logarithmic growth phase.Total RNA was extracted by Trizol method,then RT-q PCRwere conducted to detect the expression levels of Tmub1,Akt,p-Akt,cyclin D1 and c-myc mRNA.GAPDH was used as control;5.All groups were treated by the montioned methodsduringthe logarithmic growth phase.Western Blottingassays were conducted to detected the expression levels of Tmub1,Akt,p-Akt,cyclin D1 and c-myc protein;6.All groups were treated by the montioned methodsduringthe logarithmic growth phase.The cell cycle was detected by flow cytometry.Results:1.The expression of Tmub1 was significantly down regulated by RNAi;2.Compared tocontrl group and siRNA+MK2206 group,the number of colony formation of siRNA group was significantly increased(p<0.05),whilethe phosphorylation of Akt was inhibited by MK2206,and the cell proliferation rate wassignificantly inhibited(p<0.05);3.CCK-8 assay also showed thatthe proliferation ability of siRNA group was higher than that ofcontrolgroup and si RNA+MK2206 group at 1d,2d and 3d(p<0.05);4.Comparedtothe control group,the expression of cyclin D1 and c-myc mRNA genein siRNA groupwere increased(p<0.05),andthe expression of these mRNAs were significantly decreasedin si RNA+MK2206 group(p<0.05);5.Compared tothe control group,the p-Akt phosphorylation and the expression of cyclin D1 and c-myc protein wereincreased in si RNA group(p<0.05),and the phosphorylation of Akt was inhibited by MK2206,the expressionlevel of theseproteinsaresignificantly inhibitedin siRNA+MK2206 group(p<0.05);6.Compared tothe control group,the percentage of cells in the S+G2/M phasewas significantly inceased in si RNA group(p<0.05),and the percentage of cells in S+G2/M phase was decreased in siRNA+MK2206 group(p<0.05).Conclusion:1.Tmub1 silencing can increase the expression of cyclin D1 and c-myc mRNA;2.Tmub1 silencing can increase the phosphorylation level of Akt protein,and the protein expression levels of cyclin D1 and c-myc;3.Tmub1 silencingcan promote cell entry into S + G2 / M phase;4.Tmub1 silencing can promote the proliferation and growth of BRL-3A cells. |
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