背景：最近研究发现，长链非编码RNA可调控干细胞的增殖与分化，但关于长链非编码RNA NR_033474在干细胞增殖及细胞周期调控中的作用，目前尚不清楚。目的：通过在C3H10T1/2间质干细胞中过表达NR_033474，观察NR_033474对C3H10T1/2细胞增殖和细胞周期的影响，进一步分析NR_033474影响间质干细胞C3H10T1/2增殖的可能作用机制。方法：通过慢病毒载体在C3H10T1/2细胞中过表达NR_033474，应用Real-time PCR检测其表达效率，以转染空载体慢病毒的C3H10T1/2细胞为对照，利用细胞计数法绘制生长曲线，观察过表达NR_033474后C3H10T1/2细胞的生长变化；PI染色流式细胞仪检测细胞周期改变；通过Western蛋白印迹法检测过表达NR_033474后，细胞周期相关蛋白(P53、Cyclin B1、CDK1、Cyclin D1)的表达。结果与结论：①与对照组比较，感染NR_033474慢病毒后的C3H10T1/2间质干细胞NR_033474表达上调约100倍(P<0.01)；②与对照组比较，过表达NR_033474后的C3H10T1/2细胞增殖能力明显降低(P<0.05)；③与对照组比较，过表达NR_033474后的C3H10T1/2间质干细胞阻滞于G2/M期；④与对照组比较，过表达NR_033474后的C3H10T1/2间质干细胞Cyclin B1和CDK1蛋白表达下调，P53、Cyclin D1蛋白水平无明显变化；⑤结果表明，NR_033474可能通过下调 Cyclin B1及CDK1蛋白表达水平，抑制C3H10T1/2间质干细胞的增殖生长能力，使细胞阻滞于G2/M期。%BACKGROUND:Recent studies have found that long non-coding RNAs (lncRNAs) can regulate stem cel proliferation and differentiation. But it is unclear that how lncRNA NR_033474 regulate stem cel proliferation and cel cycle. OBJECTIVE:To investigate the effect of lncRNA NR_033474 on the proliferation and cel cycle regulation in C3H10T1/2 mesenchymal stem cel s after the NR_033474 overexpressed by lentivirus, and to study the possible regulation mechanism of NR_033474 on mesenchymal stem cel s. METHODS:LncRNA NR_033474 was cloned into a lentivirus vector. Lentivirus particles were infected into C3H10T1/2 cel s to upregulate the expression of NR_033474. The NR_033474 expression level was detected by real-time PCR. Compared with the empty lentivirus vector, the proliferation of C3H10T1/2 cel s which overexpressed NR_033474 was detected by cel counting assay and cel cycle was detected using flow cytometry. The expression of cel cycle-associated proteins such as CDK1, Cyclin B1, Cyclin D1 and P53 were detected by western blot assay. RESULTS AND CONCLUSION:Compared with the control group, lncRNA NR_033474 in C3H10T1/2 cel s which overexpressed NR_033474 was increased by about 100 times (P<0.01), and the proliferation of C3H10T1/2 cel s was significantly inhibited after NR_033474 overexpression by lentivirus (P<0.05). In addition, flow cytometry showed that C3H10T1/2 cel s overexpressing NR_033474 were arrested in G2/M phase compared to the control group. Western blot showed that the expression levels of CDK1 and Cyclin B1 were downregulated, while there were no changes in Cyclin D1 and P53 expression. To conclude, these findings suggest that the NR_033474 overexpression significantly inhibits the cel growth of C3H10T1/2 cel s, at least in part, through induction of cel cycle arrest at G2/M phase.
展开▼
