Tmub1对再生肝细胞有丝分裂的影响及其机制

Effect of Tmub1 on mitosis of regenerating hepatocytes in rats

目的探讨Tmub1对再生肝细胞有丝分裂的影响及与securin的相互作用机制。方法使用40只雌性SD大鼠建立肝大部分切除术后肝再生模型,于术后0、6、12、24、48、72、96、168 h分别取再生肝组织,RT-qPCR和Western blot检测Tmub1和securin在肝再生过程中的mRNA与蛋白表达变化趋势。在大鼠正常肝细胞系BRL-3A中分别建立慢病毒稳定过表达Tmub1、干扰Tmub1和阴性对照组细胞株,用诺考达唑处理获取同步于有丝分裂期的细胞,免疫荧光观察Tmub1过表达组和阴性对照组细胞有丝分裂情况,并用免疫沉淀和Western blot检测Tmub1过表达、Tmub1干扰和阴性对照组BRL-3A细胞中securin的泛素化水平。结果 Tmub1的mRNA和蛋白水平在肝切除术后24、48 h最高,securin的mRNA水平也在48 h最高,而蛋白水平在48 h最低。Tmub1过表达可影响大鼠肝细胞有丝分裂,且securin泛素化水平显著低于阴性对照组(P<0.01);Tmub1干扰组securin泛素化水平显著高于阴性对照组(P<0.01)。结论 Tmub1可通过抑制securin的泛素化影响大鼠再生肝细胞的有丝分裂。

Objective To determine the effect of transmembrane and ubiquitin like domain containing 1（Tmub1） on the mitosis in regenerating hepatocytes and its interaction with securin. Methods Totally 40 SD female rats were randomly divided into 8 groups according to the time points after 70% partial hepatectomy（PH）. Regenerating liver samples were collected in 0,6,12,24,48,72,96 and 168 h after PH. RT-qPCR and Western blotting were conducted to measure the mRNA and protein expression patterns of Tmub1 and securin in the process of liver regeneration. BRL-3A cells with stable Tmub1 over-expression and knockdown were established respectively by corresponding lentiviral vectors. Prometaphase arrest was accomplished by nocodazole treatment,and cell mitosis was observed by immunofluorescence staining in the cells with Tmub1 over-expression and knockdown,and control cells respectively. Securin ubiquitylation levels were determined by immunoprecipitation and Western blotting in the 3 groups of cells. Results The mRNA and protein levels of Tmub1 reached the highest at 24 and 48 h after PH. Securin mRNA level also peaked at48 h after PH,but its protein level was the lowest at this time point. Compared to negative control group,Tmub1 over-expression slowed down the mitosis process and significantly lower ubiquitylation level of securin（P〈0. 01）,while Tmub1 interference improved the securin ubiquitylation level（P〈0. 01）. Conclusion Tmub1 inhibits hepatocyte mitosis by suppressing securin ubiquitylation in regenerating hepatocytes from rats after PH.