miR-101诱导上皮性卵巢癌细胞SKOV3凋亡及其与细胞内Ca^(2+)水平关系的研究

Study on miR-101 induced apoptosis and its relationship to intracellular Ca^(2+) level in epithelial ovarian cancer SKOV3 cells

目的:研究microRNA-101(miR-101)对上皮性卵巢癌细胞SKOV3诱导凋亡的作用,并探讨凋亡发生与细胞内Ca^(2+)浓度的关系。方法:采用阳离子脂质体为载体将miR-101 mimics转染SKOV3细胞,随机分为空白对照组、miR-101 NC组和miR-101转染组;实时定量RT-PCR法检测细胞中miR-101表达量;流式细胞术检测细胞凋亡及细胞内Ca^(2+)浓度;Western blot法检测凋亡相关蛋白caspase-3表达。结果:实时定量RT-PCR检测结果显示,miR-101转染组的miR-101表达(5.302±0.069)较空白对照组(1.038±0.036)和miR-101 NC组(1.126±0.062)分别上调了5.10倍和4.70倍(F=5326.276,P=0.000)。流式细胞术检测结果显示,miR-101转染组的细胞凋亡率、细胞内Ca^(2+)浓度分别为(66.36±13.54)%和(228.94±19.51)nmol/L,较空白对照组[(40.60±5.97)%,(62.91±7.73)nmol/L]和miR-101 NC组[(41.54±3.89)%,(59.09±10.66)nmol/L]明显增加,差异有统计学意义(P=0.019);Western blot结果显示,miR-101转染组的细胞凋亡相关蛋白caspase-3表达(1.325±0.052)较空白对照组(0.817±0.073)和miR-101 NC组(0.820±0.062)增加,差异有统计学意义(P=0.000)。结论:miR-101过表达具有诱导上皮性卵巢癌细胞SKOV3凋亡的作用,其诱导凋亡与细胞内Ca^(2+)浓度增加有关。

Objective： To study the effect of microRNA-101（ miR-101） on the apoptosis of epithelial ovarian cancer cell line SKOV3 and to investigate the relationship between the apoptosis and the intracellular Ca2＋concentration. Methods： miR-101 mimics was transfected into SKOV3 cells by using cationic liposomes as a carrierular,and was randomly divided into the control group,the miR-101 negative control group（ miR-101 NC group） and the miR-101 group. The expression level of miR-101 in the three groups was measured by RT-PCR after transfection. The effects of miR-101 on SKOV3 cells apoptosis and on the concentration of intracellular Ca2＋were evaluated by flow cytometry. Also,the expression of caspase-3 protein was analyzed by Western blot. Results： RT-PCR results revealed that the expression of miR-101 in miR-101 group（ 5. 302 ±0. 069） was increased by 5. 10 fold and 4. 70 fold respectively compared with the control group（ 1. 038 ±0. 036） and the miR-101 NC group（ 1. 126 ±0. 062）,with statistical correlations of F = 5326. 276 and P = 0. 000. The flow cytometry suggested that the percentage of apoptosis cells and the concentration of intracellular calcium was increased significantly in the miR-101 group[（ 66. 36 ±13. 54） %,（ 228. 94 ±19. 51） nmol/L]compared with the control group[（ 40. 60 ±5. 97） %,（ 62. 91 ±7. 73） nmol/L]and the miR-101 NC group[（ 41. 54±3. 89） %,（ 59. 09±10. 66） nmol/L]（ P = 0. 019）. In addition,Western blot analysis suggested that caspase-3 expression in the miR-101 group（ 1. 325 ± 0. 052） was up-regulated compared with the control group（ 0. 817 ±0. 073） and the miR-101 NC group（ 0. 820 ±0. 062）（ P =0. 000）. Conclusions： Overexpression of miR-101 could induce apoptosis of epithelial ovarian cancer SKOV3 cells,and the induction of apoptpsis may be related to the increase of intracellular Ca2＋level.