miR-195启动子甲基化对膀胱癌细胞功能的影响

Effects of miR-195 methylation on the function of bladder cancer cells

目的分析miR-195在膀胱癌组织和细胞系中的表达情况及其对细胞增殖及凋亡的影响,探讨miR-195启动子甲基化与膀胱癌细胞功能的关系。方法实时荧光定量PCR检测miR-195在膀胱癌及癌旁组织、膀胱癌细胞系T24中的表达水平;用寡核苷酸片段NC-mimics和miR-195-mimics转染膀胱癌T24细胞系,噻唑蓝(MTT)法、克隆形成实验、细胞凋亡试验检测转染后对膀胱癌细胞增殖和凋亡的影响;用DAC(甲基化酶抑制剂)处理T24细胞后,实时荧光定量PCR检测miR-195表达水平。结果膀胱癌组织中miR-195的表达水平[-10.35,(-27.58,-4.38)]较癌旁组织[1.01,(1.00,1.02)]明显降低(Z=-7.75,P<0.01),T24细胞中miR-195的表达水平是SV-HUC-1细胞中的[3.81×10^(-5)(3.05×10^(-6),7.63×10^(-6))]倍(Z=-2.09,P<0.01)。与阴性对照组(转染NC-mimics)和空白对照组(只加转染试剂)比较,T24细胞转染miR-195 mimics后第1天(F=15.8,P<0.01)、第2天(F=220.43,P<0.01)和第3天(F=2 779.00,P<0.01)时细胞增殖能力受到明显的抑制,并且细胞出现凋亡。空白对照组、阴性对照组和实验组的细胞克隆形成数分别为(895±5)个、(790±5)个、(390±10)个,差异有统计学意义(F=1 710.27,P<0.01)。与空白对照组比较,1μmol/L DAC处理组及3μmol/L DAC处理组中T24细胞在24 h(F=186.5,P<0.01),48 h(F=120.88,P<0.01),72 h(F=16 275.00,P<0.01)时的miR-195表达水平均明显上调。26例癌组织中miR-195启动子区的甲基化水平较癌旁组织升高(χ2=25.28,P<0.01)。结论膀胱癌组织中miR-195低表达,miR-195可能参与调控食管癌T24细胞的增殖和凋亡,miR-195启动子甲基化与膀胱癌细胞的功能密切相关。

Objective To investigate the expression levels of miR-195 in bladder cancer tissues and T24 cells,and the effects of miR-195 on the proliferation and apoptosis of bladder cancer cells,and elucidate the correlation of miR-195 promoter methylation with the function of bladder cancer cells. Methods The expression levels of miR-195 in bladder cancer and adjacent tissues and T24 cells were detected by real time fluorescence quantitative PCR（ RT-PCR）. The oligonucleotide fragments NC-mimics and miR-195-mimics were transfected into T24 cells,and then the effects of miR-195 on the proliferation and apoptosis of T24 cells were detected by the methyl thiazolyl tetrazolium（ MTT） assay,colony forming test and apoptosis detection kit,respectively. The expression levels of miR-195 in T24 cells treated by methylase inhibitor 5-aza-2＇-deoxycytidine（ DAC） were determined by RT-PCR. Results The expression levels（ median [P25,P75]） of miR-195 in bladder cancer tissues（-10. 35,[-27. 58,-4. 38]） were significantly lower than that in adjacent tissues（ 1. 01,[1. 00,1. 02]; Z =-7. 75,P 〈0. 01）. The expression levels of miR-195 in T24 cells were 3. 81 × 10^-5（ 3. 05 × 10^-6,7. 63 × 10^-6） times of SV-HUC-1 cells（ Z =-2. 09,P〈 0. 01）. Compared with negative control group（ transfected with NC-mimics） and blank group,the proliferation of T24 cells transfected with miR-195 mimics was inhibited obviously at day 1（ F =15. 8,P 〈0. 01）,day 2（ F = 220. 43,P〈 0. 01） and day 3（ F = 2779. 00,P〈 0. 01）,and the apoptosis of T24 cells arose. There was significant difference in the clone formation counts of T24 cells（ F = 1710. 27,P〈 0. 01） among negative control group（ 895 ± 5）,blank group（ 790 ± 5） and treatment group（ 390 ± 10）. Comapred with blank group and treatment group with 1 μmol / L DAC,the expression levels of miR-195 in T24 cells treated with 3 μmol / L DAC increased significantly at 24 h（ F = 186. 5,P 〈0. 01）,48 h（ F =120. 88,P〈 0. 01） and 72 h（ F = 16 275. 00,P 〈0. 01）. The methylation levels of miR-195 promoter in 26 cases of cancer tissues were significantly higher than that in adjacent tissues（ χ2= 25. 28,P〈 0. 01）. Conclusion There is low expression of miR-195 in bladder cancer tissues. miR-195 may participate in the regulation of proliferation and apoptosis of T24 cells and the methylation of miR-195 promoter may be closely related to the function of bladder cancer cells.