微小RNA-96对宫颈癌细胞HeLa增殖凋亡及Spindlin1表达的影响

Effect of microRNA-96 on proliferation,apoptosis and expression of spindlin1 in cervical cancer cell line HeLa

目的探讨微小RNA-96(miR-96)对宫颈癌细胞HeLa增殖凋亡及Spindlin1(SPIN1)表达的影响。方法采用脂质体法将miR-96模拟物(mimics)及阴性对照分别转染至宫颈癌HeLa细胞(miR-96转染组和miR-96对照组),以未行转染的HeLa细胞为未转染组,实时定量PCR(QPCR)检测各组转染48 h后细胞中miR-96的表达情况,MTT及流式细胞仪分别检测各组转染48 h后的增殖及凋亡情况,分别采用QPCR和Western blotting检测转染48 h后各组细胞的SPIN1 mRNA和蛋白水平,通过双荧光素酶靶标实验验证miR-96与SPIN1之间的关系。结果转染48 h后未转染组、miR-96对照组和miR-96转染组的miR-96水平依次为1.068±0.053、1.175±0.084和2.434±0.088,miR-96转染组的miR-96水平高于其余两组,差异有统计学意义(P<0.05);未转染组、miR-96对照组和miR-96转染组的增殖率依次为(99.633±5.059)%、(97.727±3.079)%和(62.023±5.425)%,凋亡率依次为(7.515±0.924)%、(8.123±1.247)%和(26.845±4.126)%,与其余两组相比,miR-96转染组的增殖率降低而凋亡率升高,差异有统计学意义(P<0.05);未转染组、miR-96对照组和miR-96转染组的SPIN1 mRNA水平依次为0.965±0.046、0.917±0.044和0.549±0.039,SPIN1蛋白水平依次为0.667±0.042、0.715±0.045和0.384±0.038,miR-96转染组的SPIN1 mRNA和蛋白水平均低于其余两组,差异有统计学意义(P<0.05)。miR-96抑制含有野生型SPIN1-3’UTR质粒细胞的荧光素酶活性,却对含有突变型质粒细胞的荧光素酶活性无影响。结论过表达miR-96可抑制宫颈癌细胞的增殖凋亡并降低SPIN1表达水平,miR-96对SPIN1有靶向调控作用,可作为治疗宫颈癌的有效靶点。

Objective To investigate the effect of microRNA-96 （miR-96） on proliferation, apoptosis and expression of spin- dlinl （SPIN1） in cervical cancer cell line HeLa. Methods The miR-96 analog（ mimics ） and negative control were transfected into cervical cancer HeLa cells （miR-96 transfection group and miR-96 control group） by liposome, and the HeLa cells without transfection were chosen as non-transfection group. Real time quantitative PCR （QPCR） was used to detect the expression of miR-96 in each group at 48 h after transfection. MTY and flow cytometry were used to detect the proliferative and apoptotic rates at 48 h after transfection in each group. The mRNA and protein levels of SPIN1 in each group were detected by QPCR and Western blotting at 48 h after transfec- tion, respectively. The relationship between miR-96 and SPIN1 was verified by double luciferase target test. Results At 48 h after transfection, the miR-96 levels were 1. 068±0. 053, 1. 175±0. 084 and 2. 434±0. 088 in non-transfection group, miR-96 control group and miR-96 transfection group. The miR-96 level in miR-96 transfection group was higher than those of other two groups （P〈0. 05 ）. The proliferative rates were （99. 633 ±5. 059）%, （97. 727± 3. 079）% and （62. 023±5.425）%, and the apoptotic rates were （7.515± 0. 924）%, （8. 123±1. 247）% and （26. 845±4. 126）% in non-transfection group, miR-96 control group and miR-96 transfection group, respectively. Compared with other two groups, the proliferative rate of miR-96 transfection group decreased and the apoptotic rate increased （P〈0. 05）. The mRNA levels of SPIN1 were 0. 965±0. 046, 0. 917±0. 044 and 0. 549±0. 039, and the protein levels of SPIN1 were 0. 667±0. 042, 0. 715±0. 045 and 0. 384±0. 038 in non-transfeetion group, miR-96 control group and miR-96 transfectiongroup, respectively. The mRNA and protein levels of SPIN1 in miR-96 transfection group were lower than those of other two groups, and the difference was statistically significant （P〈0. 05）. MiR-96 inhibited luciferase activity of cells with wild-type SPIN1-3＇ UTR plasmid, but had no effect on luciferase activity in cells with mutant plasmid. Conclusion Overexpression of miR-96 can inhibit the proliferation and apoptosis of cervical cancer ceils and reduce the expression level of SPIN1. MiR-96 can target SPIN1 effectively and can be used as an effective target for the treatment of cervical cancer.