miR-138靶向调控SIRT1表达及其对膀胱癌细胞增殖和凋亡的影响

Effects of miR-138 on cell proliferation and apoptosis of bladder cancer via targeting SIRT1

目的探讨微小RNA-138(miRNA-138)在膀胱癌细胞中的表达情况,并研究其对膀胱癌细胞增殖和凋亡的影响及其可能的靶基因。方法采用实时定量PCR(QPCR)法检测膀胱癌细胞T24和正常膀胱上皮细胞SV-HUC-1中miR-138的表达水平。将T24细胞分为3组:未转染组、miR-138对照组(转染阴性对照片段)和miR-138转染组(转染miR-138mimics)。采用MTT法检测细胞的增殖情况,流式细胞术检测细胞的凋亡情况;Western blotting检测细胞中沉默信息调节因子1(SIRT1)蛋白的表达水平;双荧光素酶报告基因实验验证miR-138与SIRT1间的靶向关系。结果膀胱癌T24细胞中miR-138的表达量为0.57±0.19,低于SV-HUC-1细胞的1.00±0.26(P<0.05)。miR-138转染组的miR-138表达量为2.59±0.67,高于未转染组的1.00±0.36和miR-138对照组的1.08±0.49(P<0.05)。miR-138转染组T24细胞的增殖率显著低于未转染组和miR-138对照组(P<0.05)。转染48 h后,miR-138转染组的细胞凋亡率为(29.8±1.9)%,高于未转染组的(5.8±1.2)%和miR-138对照组的(7.7±0.9)%(P<0.05)。miR-138转染组SIRT1的相对表达量为0.59±0.22,低于未转染组的1.00±0.35和miR-138对照组的1.20±0.42(P<0.05)。双荧光素酶报告基因实验证明SIRT1是miR-138的直接作用靶点。结论 miR-138在膀胱癌细胞中低表达,可能通过靶向SIRT1调控膀胱癌细胞的增殖和凋亡。

Objective To investigate the expression of microRNA-138（ miR-138） in bladder cancer T24 cells and to study its effect on proliferation and apoptosis of bladder cancer cells as well as its possible target genes. Methods Real-time quantitative PCR（ QPCR） was used to detect the expression of miR-138 in bladder cancer cell line T24 and normal bladder epithelial cells SV-HUC-1.T24 cells were divided into normal control group,miR-138 negative control group and miR-138 transfection group,which were transfected with none,negative control fragment and miR-138 mimics. MTT assay and flow cytometry were performed to determine the effect of miR-138 on cell proliferation and apoptosis. Western blotting was used to detect silent mating type information regulation 2 homolog1（ SIRT1） protein levels in each group. Dual luciferase reporter assay was used to confirm the target relationship between miR-138 and SIRT1. Results The level of miR-138 in bladder cancer T24 cells was 0. 57 ± 0. 19,lower than 1. 00 ± 0. 26 of normal bladder epithelial cells,and the difference was statistically significant（ P 〈 0. 05）. The miR-138 level of miR-138 transfection group was 2. 59 ±0. 67,higher than 1. 00 ± 0. 36 of normal control group and 1. 08 ± 0. 49 of miR-138 negative control group（ P 〈 0. 05）. The proliferation rate of miR-138 transfection group was lower than that in miR-138 negative control group and normal control group with statistical significance（ P 〈 0. 05）. Forty-eight hours after transfection,the apoptotic rate of miR-138 transfection group was（ 29. 8 ± 1. 9） %,higher than（ 5. 8 ± 1. 2） % of normal control group and（ 7. 7 ± 0. 9） % of miR-138 negative control group（ P 〈 0. 05）. The relative expression of miR-138 in miR-138 transfection group was 0. 59 ± 0. 22,lower than 1. 00 ± 0. 35 of normal control group and 1. 20 ±0. 42 of miR-138 negative control group（ P 〈 0. 05）. The double luciferase reporter assay further confirmed that SIRT1 was a direct target of miR-138. Conclusion MiR-138 is decreased in bladder cancer cells,and the over-expression of miR-138 may inhibit the proliferation and promote apoptosis of bladder cancer cells by targeting SIRT1.