干扰LRP16基因表达对人卵巢癌耐药SKOV3/DDP细胞耐药性的影响及机制研究

Effects of Downregulation of LRP16 Gene Expression on the Drug Resistance of Ovarian Cancer SKOV3/DDP Cells and Its Possible Mechanisms

目的:探究干扰人白血病相关蛋白16(LRP16)基因表达对人卵巢癌耐药SKOV3/DDP细胞耐药性的影响及其相关机制。方法:采用Real-time PCR和蛋白免疫印迹(WB)检测LRP16在敏感组(SKOV3细胞)、耐药组(SKOV3/DDP细胞)、LRP16干扰组(SKOV3/DDP细胞-稳定转染LRP16 shRNA质粒)和NC组(即阴性对照组,SKOV3/DDP细胞-稳定转染阴性对照质粒)细胞中的表达情况;MTT试验检测LRP16对SKOV3/DDP细胞耐药性的影响;彗星试验检测LRP16对顺铂(DDP)诱导DNA损伤的影响;流式细胞术(FCM)试验检测细胞凋亡变化;WB试验检测PTEN、p-Akt和NF-κB蛋白表达水平。结果:LRP16干扰组细胞的耐药指数(RI)值(3.19±0.21)显著低于耐药组细胞(6.84±0.37)(P<0.05)。DDP(25μmol/L)处理24 h后,LRP16干扰组DNA损伤的细胞百分比、细胞凋亡百分比均显著低于耐药组和NC组(P均<0.05),而耐药组和NC组比较差异无统计学意义(P>0.05);LRP16干扰组细胞PTEN蛋白相对表达量高于耐药组和NC组(P均<0.05),而p-Akt和NF-κB相对表达量低于耐药组和NC组(P均<0.05)。结论:干扰LRP16基因表达可逆转卵巢癌耐药SKOV3/DDP细胞的耐药性,PTEN/Akt/NF-κB可能是其中的关键信号通路。

Objective： To investigate the effects of downregulation of LRP16 gene expression on the drug resistance of ovarian cancer SKOV3/DD cells, and explore the possible mechanisms. Methods： The expressions of LRP16 in the sensitive group（SKOV3）, resistant group（SKOV3/DDP）, LRP16 interference group（SKOV3/DDP-stably transfected with the LRP16 shRNA plasmid） and NC group（SKOV3/DDP-stably transfected with the negative-control plasmid） were detected by the real-time PCR and Western blotting（WB） assay. The influence of LRP16 expression on the drug-resistance of SKOV3/DDP was analyzed by the MTT assay. Moreover, Cisplatin（DDP）-induced DNA damage was determined by the Comet tail assay, flow cytometry（FCM） was applied to detect the cell apoptosis,and the protein levels of PTEN, p-Akt and NF-κB were detected by the WB assay. Results： The resistance index（RI） value of LRP16 interference group was（3.19±0.21）, which was much lower than（6.84±0.37） of the resistant group（P〈0.05）. At 24 h post treatment of DDP（25 μmol/L）, the percentage of DNA-damage cells and apoptotic rate of LRP16 interference group were significantly lower than those in the resistant group and the NC group（P〈0.05）, but there was no differences between the resistant group and the NC group（P〈0.05）. Meanwhile, the protein expression of PTEN in the LRP16 interference group was significantly lower than that in the resistant group and the NC group（P〈0.05）, and the protein expressions of p-Akt and NF-κB in the LRP16 interference group were significantly higher than those in the resistant group and the NC group（P〈0.05）. Conclusion： Downregulation of LRP16 gene expression was able to reverse the drug-resistance of SKOV3/DDP, and the PTEN/Akt/NF-κB may be the key pathway involved.