ERP29通过mTOR信号通路对卵巢癌SKOV3细胞生物学行为的影响

Effect of ERP29 on biological behavior of ovarian cancer SKOV3 cells via mTOR signaling pathway

①目的通过RNA干扰技术沉默EPR29基因,探讨干扰前后mTOR通路相关蛋白的表达差异,以及沉默ERP29基因对卵巢癌SKOV3细胞增殖、侵袭及凋亡的影响,探讨ERP29通过mTOR信号通路在卵巢癌发生、发展中的作用。②方法采用Lipfectamin2000技术将靶向ERP29的小干扰RNA(siRNA-ERP29)转染SKOV3细胞并作为实验组,空白载体NC siRNA转染的SKOV3细胞作为阴性对照组,未经处理的SKOV3细胞作为空白对照组;RT-qPCR技术检测SKOV3细胞转染情况;细胞模型构建成功后采Western blot技术检测各组细胞AKT、pAKT、mTOR、pmTOR、4EBP1、p4EBP1蛋白表达情况;CCK-8实验检测干扰后各组细胞增殖能力的变化;划痕实验检测干扰后各组细胞侵袭能力的变化;流式细胞仪检测干扰后各组细胞凋亡情况。③结果实验组pAKT、pmTOR、p4EBP1蛋白表达较空白组及阴性对照组增加,差异有统计学意义(P<0.05),空白组及阴性对照组间差异无统计学意义(P>0.05)。3组细胞AKT、mTOR、4EBP1蛋白的表达差异均无统计学意义(P>0.05)。实验组较空白组及对照组相比增殖能力增强,差异有统计学意义(P<0.05)。实验组较空白组及对照组相比侵袭能力增强,差异有统计学意义(P<0.05)。实验组较空白组及对照组相比凋亡率下降,但统计学无显著性差异(P>0.05)。④结论 ERP29通过抑制mTOR通路相关蛋白的磷酸化抑制卵巢癌细胞的增殖、侵袭,但对卵巢癌细胞的凋亡无显著影响。

Objective:To silence the expression of EPR29 gene in ovarian cancer SKOV3 cells by RNA interference,explore the difference of expression of mTOR pathway related proteins before and after interference,and the effect of silencing ERP29 gene on proliferation,invasion and apoptosis of ovarian cancer SKOV3 cells,and explore the role of ERP29 in the occurrence and development of ovarian cancer through mTOR signaling pathway.Methods SKOV3 cells were transfected with small interfering RNA(siRNA-ERP29)targeting ERP29 by Lipfectamin 2000 technology and served as experimental group.SKOV3 cells transfected with blank vector NC siRNA were used as negative control group,while untreated SKOV3 cells were used as blank control group.RT-qPCR technology was used to detect the transfection of SKOV3 cells.Western blot was used to detect the expression of AKT,pAKT,mTOR,pmTOR,4EBP1 and p4EBP1 in each group after the successful construction of cell model;CCK-8 test was used to detect the changes of cell proliferation ability in each group after interference;Scratch test was used to detect the changes of cell invasion ability in each group after interference;Flow cytometry was used to detect apoptosis in each group after interference.Results The expression of pAKT,pmTOR and p4EBP1 protein in experimental group was higher than that in blank group and negative control group(P<0.05).There was no significant difference between blank group and negative control group(P>0.05).There was no significant difference in the expression of AKT,mTOR and 4EBP1 protein among the three groups(P>0.05).Compared with the blank group and the control group,the proliferation ability of the experimental group was enhanced,and the difference was statistically significant(P<0.05).The invasive ability of the experimental group was stronger than that of the blank group and the control group,and the difference was statistically significant(P<0.05).Compared with the blank group and the control group,the apoptotic rate of the experimental group decreased,but there was no significant difference(P>0.05).Conclusion ERP29 inhibits the proliferation and invasion of ovarian cancer cells by inhibiting the phosphorylation of mTOR pathway-related proteins,but has no significant effect on the apoptosis of ovarian cancer cells.