Objective: To investigate the effects of small interfering RNA (siRNA) targeting YAP on the proliferation and apoptosis of human periodontal ligament stem cells (hPDLSCs).
Methods: Synthesized sequences of siRNA were transfected into hPDLSCs by Lipofectamine™ 2000. The expression of YAP was identified by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Proliferation activity was detected by using cell counting kit-8 (CCK-8). Changes in the cell cycle and apoptosis rate were detected by using flow cytometry. Results were analyzed by using SPSS 19.0, and P < 0.05 was considered statistically significant.
Results: Expression of YAP mRNA and protein were significantly downregulated after 48 h of transfection (P < 0.001). No obvious difference was found in the expression levels of YAP protein between 48 and 72 h, thus indicating that siRNA could inhibit the expression of YAP persistently and effectively. Proliferation activity was inhibited, and apoptosis rate was increased. Cell cycle was changed as the proportion of G₁and S phases increased (P < 0.01) and G₂ phase decreased (P < 0.05).
Conclusion: Knocking down YAP gene by siRNA could inhibit proliferation activity, induce apoptosis, and change the cell cycle of hPDLSCs. Thus, YAP could regulate the proliferation and apoptosis of hPDLSCs.
目的: 本研究通过小干扰RNA（siRNA）沉默人牙周膜干细胞（hPDLSCs）YAP基因，观察YAP基因沉默后对细胞增殖凋亡的影响。
方法: 将化学合成siRNA序列以脂质体Lipofectamine™ 2000介导瞬时转染hPDLSCs，采用实时荧光定量逆转录聚合酶链反应（RT-PCR）、Western blot检测转染后YAP表达水平，细胞增殖活性检测试剂盒（CCK-8）检测siRNA在体外对hPDLSCs增殖能力的影响，流式细胞仪检测细胞周期及凋亡率的变化。采用SPSS 19.0软件对数据进行处理，设P<0.05为差异有统计学意义。
结果: 在转染48 h后hPDLSCs的YAP mRNA和蛋白水平明显降低（P<0.001），YAP蛋白表达水平在细胞被转染48、72 h后无显著差异，表明siRNA可以持续有效地抑制YAP的表达；CCK-8增殖活性实验结果显示细胞增殖活性受到抑制；沉默YAP基因后细胞早期及晚期凋亡率均增加。细胞周期检测结果显示细胞周期发生改变，G1及S期细胞比例增多（P<0.01），G2期细胞比例明显减少（P<0.05）。
结论: siRNA沉默YAP基因后hPDLSCs增殖活性降低，细胞凋亡率明显增加，细胞的周期分布发生改变；YAP基因可以调控hPDLSCs的增殖与凋亡。