TPG对HaCaT细胞增殖、凋亡、细胞周期及miR-320/RASA1表达的影响

Effects of total paeony glycosides on HaCaT cell proliferation,apoptosis,cell cycle and miR-320/RASA1 expression

目的观察赤芍总苷(TPG)对HaCaT细胞增殖、凋亡、细胞周期分布、miR-320和Ras p21蛋白活化子1(RASA1)表达的影响。方法 TPG处理HaCaT细胞后,细胞计数试剂盒(CCK-8)检测细胞活力。流式细胞术检测细胞凋亡和周期分布;实时荧光定量PCR(RT-qPCR)和蛋白质印记(Western blot)检测miR-320和RASA1表达。双荧光素酶报告实验和western blot确定miR-320和RASA1之间调控关系。检测抑制miR-320表达后HaCaT细胞增殖、凋亡和细胞周期分布情况。结果TPG可降低HaCaT细胞的活力,促进HaCaT细胞凋亡,诱导G0/G1期阻滞,抑制miR-320表达,促进RASA1表达,差异有统计学意义(P<0.05)。miR-320靶向负性调控RASA1表达。干扰miR-320可抑制HaCaT细胞增殖,诱导细胞凋亡和G0/G1期阻滞,差异有统计学意义(P<0.05)。结论TPG可抑制HaCaT细胞增殖,诱导细胞凋亡和细胞周期G0/G1期阻滞,其机制可能与下调miR-320/RASA1通路有关。

Objective To investigate the effects of total paeony glucosides(TPG) on the proliferation,apoptosis,cell cycle distribution,miR-320 and Ras p21 protein activator 1(RASA1)expression of HaCaT cells,Methods HaCaT cells were treated with TPG. Cell Counting Kit 8(CCK-8)assay was used to detect cell viability. Flow cytometry detected apoptosis and cycle distribution;real-time fluorescent quantitative PCR(RT-qPCR)and Western blot were applied to detect the expression of miR-320 and RASA1.The dual luciferase reporting experiment and western blot were used to confirm the regulatory relationship between miR-320 and RASA1. The effect of miR-320 inhibition on proliferation,apoptosis,and cell cycle distribution of HaCaT cells was also observed. Results TPG can reduce the viability of HaCaT cells,promote HaCaT cell apoptosis,induce G0/G1 phase arrest,inhibit the expression of miR-320,and promote the expression of RASA1. The difference was statistically significant(P<0.05). MiR-320 targets and negatively regulates RASA1 expression. Interfering miR-320 can inhibit the proliferation of HaCaT cells,induce apoptosis and G0/G1 phase arrest,and the difference is statistically significant(P<0.05). Conclusions TPG can inhibit the proliferation of HaCaT cells,induce apoptosis and cell cycle G0/G1 arrest. The mechanism may be related to down-regulation of miR-320/RASA1 pathway.