X染色体相关凋亡抑制蛋白的小分子干扰RNA对卵巢上皮性癌细胞生长的影响

Inhibition of ovarian cancer growth by small interfering RNA targeting X-linked inhibitor of apoptosis gene

目的探讨 X 染色体相关凋亡抑制蛋白(XIAP)的小分子干扰 RNA 对卵巢上皮性癌(卵巢癌)细胞系 SKOV3细胞生长的影响。方法设计和合成长度为64 nt 的脱氧寡核苷酸链,克隆至 pSUPER 质粒,转化大肠埃希菌 DH5α菌株,构建重组质粒 pSUPER-siXIAP,对其进行双酶切和DNA 序列分析鉴定。间接免疫荧光染色法鉴定 SKOV3细胞 XIAP 蛋白的表达。实验分为4组:分别用重组质粒 pSUPER-siXIAP(pSUPER-siXIAP 组)及阴性无关序列重组对照质粒 pSUPER-nsRNA(pSUPER-nsRNA 组)及空载体质粒 pSUPER(pSUPER 组)转染 SKOV3细胞,以未转染质粒的 SKOV3细胞为空白对照组。RT-PCR 技术检测各组细胞 XIAP mRNA 的表达,蛋白印迹法检测各组细胞XIAP 蛋白的表达,流式细胞仪分析各组细胞细胞周期的变化,四甲基偶氮唑蓝(MTT)比色法观察各组细胞生长的变化。结果本实验成功构建了 XIAP 特异性的 RNA 干扰载体 pSUPER-siXIAP 质粒。间接免疫荧光染色法检测证实 SKOV3细胞中存在 XIAP蛋白的过度表达。转染后72 h,SKOV3细胞XIAP 蛋白表达水平降低,空白对照组、pSUPER 组、pSUPER-nsRNA 组及 pSUPER-siXIAP 组的相对密度值分别为3584±124、2138±65、1973±80和110±12,各组间比较,差异有统计学意义(P=0.0334);转染后72 h,SKOV3细胞 XIAP mRNA 表达水平明显下降,空白对照组、pSUPER 组、pSUPER-nsRNA 组及 pSUPER-siXIAP 组各组的相对密度值分别为6674±274、4532±107、2322±57和1864±78,各组间比较,差异有统计学意义(P=0.0127)。流式细胞仪检测结果显示,pSUPER-siXIAP组 G_1期细胞较空白对照组、pSUPER 组明显增加(P<0.05);pSUPER-siXIAP 组 S 期细胞较空白对照组、pSUPER 组明显减少(P<0.05)。MTT 比色法检测结果显示,pSUPER-siXIAP 组细胞生长增殖被抑制,生长速度较空白对照组细胞明显减慢(P=0.0237);pSUPER-nsRNA 组和 pSUPER 组细胞生长速度较空白对照组细胞也明显减慢(P=0.0441,P=0.0472)。结论本研究成功构建了 XIAP RNA干扰载体 pSUPER-siXIAP 质粒,将其转染 SKOV3细胞后可有效降低其 XIAP mRNA 及蛋白的表达水平,并将 SKOV3细胞阻滞于 G_1期,造成转染细胞生长速度减慢,这有望为卵巢癌基因治疗提供一个新的有希望的分子靶点。

Objective To construct an RNA interference vector to down-regulate X-linked inhibitor of apoptosis （XIAP） gene and study the RNA interference effect on the cell cycle and growth of ovarian cancer. Methods Oligonucleotides of 64 base pairs for hairpin RNA targeting XIAP were designed, chemically synthesized, annealed, and cloned into the pSUPER vector. After identification by restriction digestion,the correct vectors were transiently transfected into SKOV3 cells, a human ovarian cancer cell line. The XIAP mRNA was detected by RT-PCR~ The proteins were detected by western blot and indirect staining. Flow cytometry （FCM） analysis and methyl thiazolyl tetrazolium （MTT） assay method were applied to measure cell cycle, cell growth and sensitiveness to cisplatin. Results SKOV3 cells had a high level expression of XIAP. The vector of RNA interference, which can interfere with XIAP gene was successfully constructed. After transient transfection, the expression of XIAP protein was significantly decreased in SKOV3 cells and the value of relative density was 3584 ± 124,2138 ±65,1973 ±80 and 110 ± 12, respectively （P = 0. 0334 ） . At the same time, the expression of XIAP mRNA was decreased accordingly and the value of relative density was 6674 ± 274,4532 ± 107,2322 ± 57 and 1864 ± 78 , respectively （P =0. 0127）. The FCM results showed that, the vector could increase the number of cells in G1 phase compared with parent cells and compared with the cells transfected with pSUPER （ P 〈 0. 05 ） ; the number in S phase decreased compared with parent cells and compared with the cells transfected with pSUPER （ P 〈 0. 05 ）. MTF results showed that pSUPER-siXIAP could inhibit the gro~,h and proliferation of SKOV3 cells in a time-dependent manner, and inhibit the tumor cell growth. Condusions We successfully constructed an expressing hairpin RNA against the XIAP vector. The vector can effectively silence XIAP gene, increase the number of cells in G1 phase , and slow down the growth of tumor cells. This may be a useful therapeutic strategy for XIAP over-expressing ovarian carcinomas.