微小RNA-143和微小RNA-29a对膀胱癌细胞的影响及其与Ras相关结构域家族1A基因甲基化的关系

Cell proliferation and invasion in microRNA-143 and microRNA-29a transfected bladder cancer cells and its relationship of ras association domain family 1A methylation

目的 观察微小RNA（miR）-143和miR-29a对T24膀胱癌细胞株生长、侵袭能力的影响,并探讨其与Ras相关结构域家族1A（RASSF1A）基因甲基化的关系.方法 化学合成miR-143和miR-29a的模拟物（mimics）,并转染T24膀胱癌细胞株.通过逆转录-聚合酶链反应（RT-PCR）验证miRNA mimics的转染效果.根据转染mimics的不同,分为WT组（不作任何处理的T24细胞）、NC组（转入不含miRNA的mimics的阴性对照）、M1组（转入含miR-143的mimics）、M2组（转入含miR-29a的mimics）.利用流式细胞仪检测各组细胞凋亡率和细胞周期改变,Transwell法检测细胞侵袭能力,RT-PCR和Western blot检测RASSF1A基因转录水平和蛋白质表达,甲基化特异性PCR（MSP）检测RASSF1A基因甲基化状态.结果 RT-PCR证实两组mimics转染成功.WT、NC、M1和M2组的细胞凋亡率分别为2.5％、3.4％、12.4％和13.3％.M1和M2组S期细胞有所增加.NC、M1和M2组细胞侵袭抑制率分别为2.0％、16.5％和13.2％,M1和M2转染组高于NC组,差异有统计学意义（P＜0.05）.RT-PCR和Western blot检测显示,转染miR-29a的M2组RASSF1A基因转录水平和蛋白质含量较NC组增加,但与M1组比较差异无统计学意义（P＞0.05）.MSP检测结果显示,WT、NC和M1组RASSF1A基因均呈完全甲基化,M2组呈部分甲基化状态.结论 转染miR-143和miR-29a可使T24膀胱癌细胞株细胞凋亡增加、侵袭能力减弱.转染miR-29a可以部分抑制RASSF1A基因甲基化,从而使该基因转录水平和蛋白质表达增加.

Objective To evaluate the relationship among bladder cancer cell proliferation and invasion,microRNA-143 and 29a,and ras association domain family 1A （RASSF1A） methylation.Methods MiR-143 and miR-29a mimics were transfected to T24 cell lines.Transfection efficiency was confirmed by reverse transcription polymerase chain reaction （RT-PCR）.All cell lines were divided into four groups：WT group （wild T24 cell lines without any treatment）,NC group （transfected by negative control mimics）,M1 group （transfected by miR-143 mimics） and M2 group （transfected by miR-29a mimics）.Apoptosis rates,cell cycle,cell invasive inhibitor rates,RASSF1 A gene expression and methylation were detected.Results Transfection was successful confirmed by RT-PCR.Apoptosis rates of WT,NC,M1 and M2 group were 2.5%,3.4%,12.4% and 13.3% respectively.S-phase cells in M1 and M2 group increased significantly.Cell invasive inhibitor rates of NC,M1 and M2 group were 2.0%,16.5% and 13.2% respectively which showed the rates in M1 and M2 group were higher than NC group （P 〈0.05）.RASSF1A gene expression was upgraded in M2 group and no changes were found in M1 group.What＇s more,MSP results showed that RASSF1 A gene was partial methylated in M2 group while it was completely methylated in other groups.Conclusion T24 bladder cancer cell proliferation and invasion decreased after miR-143 and miR-29a transfected.miR-29a transfection may inhibit RASSF1 A gene methylation and lead to gene expression uploaded.