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目的研究shRNA表达质粒稳定转染,靶向沉默HER-2/neu基因对子宫内膜癌细胞株Ishikawa细胞增殖及其对孕激素敏感性的影响。方法根据HER-2/neumRNA的序列设计和合成两条shRNA.分别将其与质粒(pSilencer4.1CMV neo,Ambion)连接,并将连接好的质粒通过LipofectamineTM2000(invitrogen)转染Ishikawa细胞株。应用含有250μg·mL-1G418的DMEM培养基筛选稳定转染克隆。用RT-PCR法及Western blot分别检测稳定转染细胞HER-2/neumRNA及p185蛋白表达水平,用Annexin-V检测细胞凋亡率。用10μg.μL-1孕激素培养细胞72小时后,用MTT法分别检测转染细胞及未转染细胞的增殖率。结果转染特异性shRNA表达质粒组Ishikawa细胞HER-2/neumRNA及p185蛋白表达水平明显低于对照组细胞。细胞增殖率明显降低而细胞凋亡率明显增高(P<0.05)。用10μg.μL-1孕酮培养细胞72小时后,转染特异性shRNA的细胞组细胞增殖率明显低于未转染细胞组(P<0.05)。结论RNA干扰可以明显降低Ishikawa细胞株HER-2/neumRNA及185蛋白的表达,从而抑制细胞增殖并诱导细胞凋亡。HER-2shRNA还可以增加Ishikawa细胞株对孕激素的敏感性,与孕激素存在协同效应。
Objective To study the effects of shRNA expression plasmid stable transfection and target-directed silencing of HER-2 / neu on the proliferation and the progesterone sensitivity of Ishikawa cells. Methods Two shRNAs were designed and synthesized according to the sequence of HER-2 / neumRNA, ligated with plasmid pSilencer4.1CMV neo (Ambion), and the ligated plasmids were transfected into Ishikawa cells by LipofectamineTM2000 (invitrogen). Stably transfected clones were screened using DMEM medium containing 250 μg · mL -1 G418. The expression of HER-2 / neumRNA and p185 protein in stable transfected cells were detected by RT-PCR and Western blot respectively, and the apoptosis rate was detected by Annexin-V. After culturing cells with 10μg.μL-1 progestin for 72 hours, the proliferation rate of transfected and non-transfected cells was detected by MTT assay. Results The expression of HER-2 / neumRNA and p185 protein in Ishikawa cells transfected with shRNA-specific shRNA plasmid was significantly lower than that in control cells. Cell proliferation rate was significantly reduced and apoptosis rate was significantly increased (P <0.05). After cultured with 10μg.μL-1 progesterone for 72 hours, the proliferation rate of cells transfected with specific shRNA was significantly lower than that of untransfected cells (P <0.05). Conclusion RNAi can significantly reduce the expression of HER-2 / neumRNA and 185 protein in Ishikawa cell lines, thereby inhibiting cell proliferation and inducing apoptosis. HER-2shRNA also increases the sensitivity of Ishikawa cells to progestin and shows synergistic effect with progestin.