| 郑学忠,高 婧,陈裕浩,李青青,王恩阳,万怡轩,王 红.siRNA干扰TCAB1对人主动脉平滑肌增殖的影响及机制[J].,2019,19(6):1039-1043 |
| siRNA干扰TCAB1对人主动脉平滑肌增殖的影响及机制 |
| The Effect and Mechanism of siRNA-targeting TCAB1 on the Proliferation of HASMC |
| 投稿时间：2018-06-18 修订日期：2018-07-13 |
| DOI：10.13241/j.cnki.pmb.2019.06.008 |
| 中文关键词: 平滑肌细胞 端粒酶 卡扎尔体蛋白1 细胞增殖 细胞周期 |
| 英文关键词: Smooth muscle cells Telomerase TCAB1 Cell proliferation Cell cycle |
| 基金项目:国家自然科学基金项目（81270224） |
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| 中文摘要: |
| 摘要 目的：探究TCAB1沉默对人主动脉平滑肌细胞(HASMC)增殖的影响及可能机制。方法：采用RNAi技术设计并合成靶向沉默TCAB1基因表达的三对特异性siRNA序列(siTCAB1-331、siTCAB1-619、siTCAB-749)和一对阴性对照序列(NC)，使用lipo2000将siTCAB1、NC转染HASMC，分为3个组：干扰组（siTCAB1）、空白对照组（BC）、阴性对照组（NC），转染24小时倒置荧光显微镜观察细胞转染情况；通过RT-qPCR和Western blot从3个干扰靶点中筛选效果最好的干扰靶点。进一步转染siTCAB1-749后，MTS检测HASMC 24、48、72 h的增殖能力，48小时用RT-qPCR和Western blot检测CyclinD1表达量变化，流式细胞术检测HASMC的细胞周期变化。结果：RT-qPCR和WB结果显示siTCAB1-749为最好的干扰靶点；转染24、48、72 h后，siTCAB1-749组增殖水平明显低于NC组、BC组 (P<0.05)。流式结果显示：siTCAB1-749组处于G1期细胞比率有所增加，处于S期细胞比率减少 (P<0.05)，且siTCAB1-749组细胞周期蛋白cyclinD1表达也下降(P<0.05)。结论：沉默TCAB1能抑制HASMC的增殖，其机制可能与阻碍细胞周期蛋白cyclinD1有关。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect and potential mechanism of silencing TCAB1 on the proliferation of human aortic smooth muscle cells (HASMC). Methods: Three siRNA sequences(siTCAB1-331, siTCAB1-619, siTCAB-749) targetingTCAB1 and one negative sequence(NC) were designed, synthesized and then transfected into HASMC with Lipofectamine-TM2000. HASMC were divided three groups: interference group, blank control group, negative control group. Cell transfection was observed under fluorescence microscope. The mRNA and protein expression levels of TCAB1 were detected by RT-qPCR and Western blotting, the best target was selected from the three interfering targets. The effective siRNA (siTCAB1-749) was further transfected into HASMC with LipofectamineTM2000. The changes of cell cycle and CyclinD1 expression of HASMC cells were assessed using flow cytometry, RT-qPCR and Western blotting, respectively. The proliferation of HASMC was assessed using MTS assay after transfecting siTCAB1-749 at 24 h, 48 h,72 h. Results: RT-qPCR and Western blot indicated that the siTCAB1-749 was the best target in the three interfering targets. MTS revealed that the number of cell growth in siTCAB1-749 group was significantly lower than BC group and NCgroup at 24 h, 48 h, 72 h (P<0.05); the percentages of S phases in the cell cycle of siTCAB1-749 group was decreased and G1 phases was increased at 48 h (P < 0.05). In addition, the expression of cyclinD1 was decreased in siTCAB1-749 group(P<0.05). Conclusion: Silencing TCAB1 can inhibit the proliferation of HAVSMC, and its mechanism may be related to the inhibition of Cell cycle protein D1. |
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