miR-147靶向MDM2调控卵巢癌细胞Skov3增殖与凋亡的机制研究

Effects of miR-147 targeting MDM2 on the proliferation and apoptosis of ovarian cancer cell line Skov3 and its mechanism

目的探讨微小RNA-147(miR-147)靶向调控鼠双微粒体2蛋白(MDM2)的表达及其对卵巢癌细胞株Skov3增殖与凋亡的影响。方法采用Lipo2000脂质体法将miR-147模拟物(mimics)及阴性对照(NC)转染至Skov3细胞并分为miR-147 mimics组和NC组,采用实时荧光定量PCR(QPCR)法检测转染48 h后Skov3细胞的miR-147水平以评价转染效果,分别采用MTT法及流式细胞术检测两组转染后的增殖及细胞凋亡情况,Western blotting检测两组MDM2、p53和caspase-3表达水平,采用双荧光素酶报告实验验证miR-147与靶标MDM2之间的靶向关系及结合位点。结果 QPCR检测显示,miR-147mimics组miR-147的表达水平高于NC组,差异有统计学意义(P<0.05),提示在Skov3细胞中过表达miR-147成功。与NC组相比,miR-147 mimics组的增殖率和MDM2水平均降低,但凋亡率及p53和caspase-3水平均升高,差异有统计学意义(P<0.05);双荧光素酶报告实验检测发现miR-147可显著抑制野生型MDM2-3’端非翻译区(UTR)质粒转染细胞的荧光素酶活性,而对突变型MDM2-3’UTR质粒转染细胞的荧光素酶活性并无影响。结论 miR-147可靶向MDM2的表达,且过表达miR-147表达可抑制Skov3细胞的增殖并诱导凋亡,在卵巢癌防治上有一定参考价值。

Objective To investigate the effect of microRNA-147 （miR-147）on the expression of murine double minute 2 （MDM2） and its effect on the proliferation and apoptosis of ovarian cancer cell line Skov3. Methods The Skov3 cells were transfected with miR-147 mimics（ miR-147 mimics group）or negative control（ NC group）by Lipo2000 liposome method. The real-time quantitative PCR（QPCR） method was used to detect the miR-147 level of Skov3 cells at 48 h post-transfection to evaluate the transfect efficiency. MTY method and flow cytometry were used to detect the proliferation and apoptosis of the two groups after transfection, respectively. The levels of MDM2, p53 and caspase-3 were measured by Western blotting. The dual luciferase reporter assay was employed to verify the relationship between miR-147 and target MDM2 and the binding site. Results QPCR results showed that the level of miR-147 in miR-147 mimics group was significantly higher than that in NC group, and the difference was statistically significant（ P〈0. 05 ）, indica- ting that miR-147 was successfully over-expressed in Skov3 cells. Compared with NC group, the proliferative rate and MDM2 level of miR-147 mimics group were all decreased, but the apoptotic rate and levels of p53 and caspase-3 were all increased（ P〈0. 05）. Dual luciferase reporter assay showed that miR-147 could significantly inhibit the luciferase activity of cells transfected with wild-type MDM2- 3＇ （untranslated region, UTR） plasmid, and had no effect on the luciferase activity of cells transfected with mutant MDM2-3＇ UTR plasmid. Conclusion MiR-147 can target the expression of MDM2, and over-expression of miR-147 can inhibit the proliferation and induce apoptosis of Skov3 cells, and it has a certain reference value in the prevention and treatment of ovarian cancer.