microRNA-34a靶向抑制Notch-1基因对膀胱癌J82细胞增殖和迁移的影响

microRNA-34a inhibits cellular proliferation and migration in bladder cancer cell line J82 by targeting notchl

目的探讨microRNA-34a（miR-34a）通过靶向抑制Notch-1基因表达对人膀胱癌J82细胞增殖和迁移的影响。方法查询基因组数据库，应用基因预测软件进行生物信息学分析，预测miR-34a可能靶向调控Notchl基因；实时定量逆转录聚合酶链反应和蛋白印迹法分别检测16例浸润性膀胱癌组织及质粒转染的膀胱癌J82细胞的miR-34a和Notchl表达情况；萤光素酶实验检测miR-34a与Notchl基因的结合位点；将miR-34a表达质粒转染J82细胞，新型四唑氮盐化合物比色法和迁移实验评估miR-34a对细胞增殖和迁移的影响。结果浸润性膀胱癌组织miR-34a的表达中位数为0．016，四分位数间距0．018；Notchl基因表达中位数为2．765，四分位数间距2．156；癌旁组织miR．34a表达中位数为0．042，四分位数间距0．059；Notchl基因表达中位数为2．312，四分位数间距1．365，组间差异均有统计学意义（P〈0．01）；浸润性膀胱癌组织的Notchl蛋白表达量为0．857±0．197，癌旁组织为0．648±0．171，组间差异有统计学意义（P〈0．01）。质粒转染J82细胞，miR-34a表达质粒组miR-34a的表达量为（2．408±0．789）×10-4，对照质粒组为（0．153±0．029）×10-4（P=0．0026）；miR-34a表达质粒组Notchl基因表达量为3．001±0．106，对照组为4．998±1．053（P=0．0308）；miR-34a表达质粒组Notchl蛋白表达量为0．747±0．050，对照组为0．988±0．102（P=0．0215）；miR-34a表达质粒组细胞萤光素酶活性为0．422±0．028，对照组为2．392±0．148（P〈0．0001），组间差异均有统计学意义；miR-34a表达质粒组细胞增殖受到抑制，细胞迁移数量为179．3±21．02，对照组为269．7±23．71，组间差异有统计学意义（P=0．0078）。结论miR-34a可与Notchl基因的3’非翻译区（3’UTR）结合靶向抑制Notchl基因表达，进而抑制了膀胱癌J82细胞的增殖和迁移。

assessed by transwell migration assay. Results The expression level of miR-34 in invasive TCCB tissues was lower than in adjacent bladder tissues （0.016（0.018） vs 0. 042 （0. 059）, N = 16; P =0.0006）. On the contrary, the average levels of Notchl mRNA and protein were higher in tumors than in adjacent bladder tissues （2.765（2.156） vs 2.312（1.365）, N =16; P=0.0025 and 0.857 ±0.197 vs 0.648 ±0.171; P 〈 0. 0001 ）. After the transfection of miR-34a, the expressive level of miR-34a in J82 was highly induced （（2.408±0.789） ×10-4 vs（0.153 -＋0.029） ×10-4; p=0.0026）. However, the expressive levels of Notchl mRNA and protein were obviously decreased （3. 001± 0. 106 vs 4. 998 ~ 1. 053 ; P = 0. 0308 and 0. 747± 0. 050 vs 0. 988± 0. 102; P = 0. 0215 ）. The results of luciferase assay showed that firefly activity was highly dimished （0. 422 ± 0. 028 vs 2. 392±0. 148 ; P 〈 0. 0001 ）. Cellular proliferation was inhibited after the transfection of miR-34a in J82 （P 〈 0. 0001 ）. Moreover, number of migration cells of J82 was significantly reduced after the ectopic expression of miR-34a （ 179.3 ± 21.02 vs 269.7 ± 23.71 ; P = 0. 0078 ）. Conclusions miR-34a inhibits the cellular proliferation and migration of bladder cancer cell line J82 via binding to the 3＇TR of Notchl mRNA.