CAJ | 학술논문

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.
목적 연구중개체복합물아기19(midiato complex subunit 19,Med19)기인침묵대인방광암T24세포주증식화성류작용적영향. 방법 응용shRNA만병독재체감염T24인방광암세포침묵Med19기인,응용실시형광정량PCR화단백질인적방법검측미감염조、음성대조감염조이급shMed19감염조세포적Med19기인표체정황；류식세포의분석각조세포주기적변화；새서람(MTT)실험、5-추탈양뇨밀정핵감(BrdU)참입실험、극륭형성실험평고종류세포증식화생장능력；장T24세포접충우라서,진행소서성류실험,검측종류세포적치류능력. 결과 Med19-shRNA만병독감염T24세포후,감염조Med19 mRNA표체상대함량위0.35 ±0.03,여미감염조0.75±0.07화음성대조조1.00±0.07비교차이유통계학의의(P＜0.05)；감염조Med19단백표체량여미감염조화음성대조조비교유명현차이；감염조G0/G1기세포비례위(77.50±0.29)％,여미감염조(69.81±0.81)％화음성대조조(67.53±0.67)％비교차이유통계학의의(P＜0.05)；MTT화BrdU실험표명감염조세포존활수량화DNA합성교미감염조화음성대조조현저강저(P＜0.05)；극륭형성실험중감염조극륭수량위(91.33±6.11)개,교미감염조(179.00±18.36)화음성대조조(191.00±19.16)현저감소(P＜0.05)；소서성류실험중,24 d시감염조종류체적위(596.64±485.36)mm3,질량위(0.57±0.44)g,여미감염조(2611.40±396.68) mm3화(1.94±0.60)g,음성대조조(2463.21±1451.66) mm3화(2.17±1.75)g,조간비교차이유통계학의의(P＜0.05). 결론 Med19기인침묵후인방광암T24세포적생장、증식화성류능력균수도현저억제,제시Med19기인재방광암형성과정중구유중요작용.사용shRNA기술억제Med19기인표체유망성위방광암신적치료수단지일.
Objective 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.