摘要目的 研究中介体复合物亚基19(midiato complex subunit 19,Med19)基因沉默对人膀胱癌T24细胞株增殖和成瘤作用的影响. 方法 应用shRNA慢病毒载体感染T24人膀胱癌细胞沉默Med19基因,应用实时荧光定量PCR和蛋白质印迹方法检测未感染组、阴性对照感染组以及shMed19感染组细胞的Med19基因表达情况；流式细胞仪分析各组细胞周期的变化；噻唑蓝(MTT)实验、5-溴脱氧尿嘧啶核苷(BrdU)掺入实验、克隆形成实验评估肿瘤细胞增殖和生长能力；将T24细胞接种于裸鼠,进行小鼠成瘤实验,检测肿瘤细胞的致瘤能力. 结果 Med19-shRNA慢病毒感染T24细胞后,感染组Med19 mRNA表达相对含量为0.35 ±0.03,与未感染组0.75±0.07和阴性对照组1.00±0.07比较差异有统计学意义(P＜0.05)；感染组Med19蛋白表达量与未感染组和阴性对照组比较有明显差异；感染组G0/G1期细胞比例为(77.50±0.29)％,与未感染组(69.81±0.81)％和阴性对照组(67.53±0.67)％比较差异有统计学意义(P＜0.05)；MTT和BrdU实验表明感染组细胞存活数量和DNA合成较未感染组和阴性对照组显著降低(P＜0.05)；克隆形成实验中感染组克隆数量为(91.33±6.11)个,较未感染组(179.00±18.36)和阴性对照组(191.00±19.16)显著减少(P＜0.05)；小鼠成瘤实验中,24 d时感染组肿瘤体积为(596.64±485.36)mm3,质量为(0.57±0.44)g,与未感染组(2611.40±396.68) mm3和(1.94±0.60)g,阴性对照组(2463.21±1451.66) mm3和(2.17±1.75)g,组间比较差异有统计学意义(P＜0.05). 结论 Med19基因沉默后人膀胱癌T24细胞的生长、增殖和成瘤能力均受到显著抑制,提示Med19基因在膀胱癌形成过程中具有重要作用.使用shRNA技术抑制Med19基因表达有望成为膀胱癌新的治疗手段之一.

abstractsObjective To study the role of Med19 in bladder cancer by analyzing the effects of lentivirus-mediated suppression of Med19 expression on T24 bladder cancer cells in vitro.Methods The lentivirus vectors containing a small hairpin RNA (shRNA) to target Med19 were constructed.After T24 bladder cancer cells were infected,real-time PCR and Western-blotting were used to study the Med19 expressions in the CON group (non-infected cells),the NC group (Lv-NC-infected cells) and the KD group (Lv-shMed19-infected cells).The influence of Med19 on the proliferation of bladder cancer cells were assessed using MTT,BrdU,colony formation assay and tumorigenicity experiment in mice.Cell cycle was analyzed with flow cytometry assay.Results Med19 relative mRNA level (0.35 ± 0.03) and Med19 protein expressing in the KD group were significantly inhibited (P ＜ 0.05).The KD group displayed an increased proportion of cells (77.50 ± 0.29)％ in the G0/G1 phase compared with the CON group (69.81 ± 0.81)％and NC group (67.53 ± 0.67) ％ (P ＜ 0.05).Compared with the CON group and the NC group,the KD group displayed a significant cell proliferation defect by MTT and BrdU assay and the number of colonies (91.33 ± 6.11) was significant decreased (P ＜ 0.05).On the day 24,the tumor volume (596.64 ± 485.36) mm3 and weight (0.57 ± 0.44) g of the KD group mice were decreased after inoculation into nude mice (P ＜ 0.05).Specific lentivirus-mediated knockdown of Med19 significantly impacted the cell cycle and proliferation of bladder cancer cells.Infected T24 cells nearly lost their tumorigenicity when being inoculated into nude mice.Conclusion Our results provide new evidence of an important role for Med19 in the development of bladder cancer,suggesting that lentiviruses delivering shRNA against Med19 may be a promising tool for bladder cancer therapy.