WT1反义核酸诱导卵巢癌细胞凋亡的实验研究

Effect of WT1 antisense mRNA on the induction of apoptosis in ovarian carcinoma SKOV3 cells

关键词:  WT1基因  上皮性卵巢癌  反义核酸

Key words:  WT1 gene  ovarian epithelial carcinoma  antisense oligonucleotide

摘要:

［目的］研究WT1反义寡核苷酸转染对卵巢癌SKOV3 细胞增殖和凋亡的影响。［方法］ 实验分4组：正常对照组（加入转染液）、反义组（脂质体+反义寡合苷酸转染）、正义组（脂质体+正义寡合苷酸转染）和脂质体组（只加入脂质体）。分组转染卵巢癌SKOV₃ 细胞株后，流式细胞仪检测细胞凋亡和细胞周期，MTT法检测细胞的增殖活性，RT-PCR检测WT1 mRNA表达水平，Western blot检测其蛋白表达水平。［结果］脂质体介导的WT1反义寡核苷酸转染明显抑制卵巢癌SKOV₃ 细胞增殖，抑制率为49.48%，G₀-G₁ 期细胞分布率增加达（79.10±2.79）%，凋亡细胞群明显增多，凋亡率为（13.18±2.00）%，同时转染后细胞WT1 mRNA及蛋白表达水平下降，与对照组及其他各组相比较差异均有显著性意义（P<0.05）。 ［结论］WT1反义核酸能抑制卵巢癌细胞增殖，诱导细胞凋亡。WT1反义核酸用于卵巢癌的基因治疗具有一定的潜在意义。

Abstract:

［Objective］To study the effect of WT1 antisense oligodeoxynucleotides(ASODN) transfection on the cell proliferation and apoptosis of SKOV₃ cells.［Methods］There were 4 groups in our study: normal control group, WT1 ASODN group, WT1 SODN group and Lipofectamine group. The cell apoptosis was observed by flow cytometry. The effect of WT1 ASODN on cell proliferation was assayed by MTT method. The expression level of WT1 mRNA and protein were detected by RT-PCR and Western blot.［Results］The growth of the ovarian cancer cell lines SKOV₃ transfected by WT1 ASODN became significantly slow and its activity reduced, with the inhibition rate was 49.48%. WT1 antisense phosphorothioate oligonucleotides not only inhibited the proliferation, arrest cell cycle at G0-G1 checkpoint and induce the apoptosis in SKOV₃ ovarian carcinoma cells but also downregulated WT1 mRNA and protein expression that contribute to the apoptosis(P<0.05).［Conclusions］WT1 antisense phosphorothioate oligonucleotides could not only inhibite the proliferation but also induce the apoptosis in SKOV₃ ovarin carcinoma cell line. Antisense oligonucleotides of WT1 may serve as a potential antitumor strategy in the gene therapy of ovarian carcinoma