PGRMC1抑制对子宫内膜癌细胞化疗敏感性影响的研究

Inhibited progesterone receptor membrane component-1 by shRNA plasmid to increased drug sensitivity of endometrial carcinoma cell

目的:观察抑制孕激素膜受体1(PGRMC1)对子宫内膜癌细胞化疗敏感性的影响。方法:设计合成以PGRMC1为靶标的shRNA1、2质粒,对照质粒shRNA3,转染入瘤细胞。转染1周后,检测细胞绿色荧光蛋白(GFP)阳性率,RT-PCR检测转染shRNA后PGRMC1基因mRNA水平变化,蛋白质印迹法检测蛋白表达变化。CCK8法检测转染后第2代的瘤细胞对化疗药物ADM、5-FU及DDP的敏感性,流式细胞仪检测AnnexinⅤ标记的转染组与对照组细胞加化疗后细胞凋亡率的差异;双氢二氯荧光染色阳性率判定细胞内活性氧簇(ROS)水平的差异。结果:shRNA可在细胞稳定表达>1周,并可传代。shRNA1、2可显著抑制PGRMC1基因mRNA及蛋白表达,提高子宫内膜癌细胞化疗敏感性。相同化疗压力下,PGRMC1抑制的子宫内膜细胞凋亡率及细胞内ROS水平明显高于对照组。结论:抑制PGRMC1基因表达能够增加子宫内膜癌细胞对化疗的敏感性。

OBJECTIVE：To determine whether chemo-sensitivity of endometrial cancer cell line Ishikawa will be in- creased through the silence of progesterone receptor membrane component-1 （PGRMCi） gene by shRNA plasmid. METH- OD： Design and construct the shRNA1 and 2 plasmid targeting PGRMC1 ,and shRNA3 was control. One week later,GFP positive ratio was detected, the mRNA and protein of PGRMC1 by FCM, RT-PCR and Western blot method. The drug sensitivity for ADM,5-FU and DDP were detected by CCK8 method. Annexin V staining was used to identify apoptotic cells by FCM. Meanwhile the intracellular ROS level was detected by FCM too. RESULTS： shRNA could duplicate and passage stably, shRNA1 and 2 inhibited the expression of PGRMC1 both on mRNA and protent level significantly. After inhibiton of PGRMC1 ,the drug sensitivity of Ishikawa increased. In the same drug concentration, the apoptosis ratio and ROS level of shRNA1 and 2 groups were more higher than that of the shRNA3 and control group. CONCLUSION： PGRMC1 play important role in regulating drug resistance of endometrial cancer.