Tmub1沉默提高Akt蛋白活性促进肝细胞增殖

Tmub1 silencing promotes hepatocytes proliferation by enhancing Akt activity

目的探讨Tmub1沉默对Akt磷酸化和肝细胞增殖及生长能力的影响。方法应用RNAi技术对BRL-3A细胞株Tmub1基因进行沉默,采用qRT-PCR和Western blot分别检测Tmub1、Akt、p-Akt、Cyclin D1、c-myc等基因的mRNA和蛋白表达水平;采用平板集落形成实验和CCK-8检测细胞增殖能力;流式细胞术检测细胞周期。结果与对照组相比,Tmub1基因沉默后,Cyclin D1和c-myc基因mRNA表达增强(P<0.05),p-Akt磷酸化水平以及Cyclin D1和c-myc蛋白的表达增高(P<0.05);BRL-3A细胞的增殖及生长能力增强(P<0.05);且处于细胞周期S+G2/M期的细胞比例明显增加(P<0.05)。通过MK2206抑制Akt磷酸化后,可显著抑制上述基因的mRNA及蛋白的表达水平,细胞的增殖及生长能力明显降低(P<0.05),且处于S+G2/M期的细胞比例下降(P<0.05)。结论 Tmub1沉默通过增加Akt蛋白的磷酸化及Cyclin D1、c-myc的表达水平从而促进BRL-3A细胞的增殖和生长。

Objective To determine the effect of Tmubl silencing on the phosphorylation of Akt and the proliferation and growth of hepatocytes. Methods RNA interference technique was used to knockdown Tmubl gene in rat liver cell line BRL-3A. qRT-PCR and Western blotting were used to detect the expression of Tmubl, Akt, p-Akt, Cyclin D1 and c-myc at mRNA and protein levels. Plate colony formation assay and CCK-8 assay were employed to measure the proliferation and growth of BRL-3A cells. Flow cytometry was used to determine the cell cycle. Results Compared to the control group, the mRNA levels of Cyclin DI and c-myc were up-regulated （P 〈 0.05 ）, the protein levels of p-Akt, Cyclin D1 and c-myc were increased （P 〈 0.05 ） , the proliferation and growth of BRL-3A cells were enhanced （P 〈0.05 ） , and the percentage of ceils at S ＋ GJM period was significantly increased （ P 〈 0.05 ） in the cells after Tmubl silencing. When the phosphorylation of Akt was inhibited by MK2206, the mRNA and protein levels of the genes mentioned above were significantly down-regulated, the proliferation ability of the BRL-3A cells was obviously reduced （P 〈 0. 05）, and the percentage of cells at S ＋ G2/M period was notably deceased （P 〈 0.05）. Conclusion Tmubl silencing promotes the proliferation and growth of BRL-3A cells by enhancing the phosphorylation of Akt and increasing the expression levels of Cyclin D1 and c-myc.