微小RNA-137调控Notch1基因介导自噬在肝癌细胞增殖和迁移中的作用  

Role of miR-137 in Notchl mediated autophagy in proliferation and migration of hepatocellular carcinoma cells

作　　者：宋虎[1] 张建军[2] 李世朋[1] 王振[1] 杜晨阳[1] 郑虹[2] 沈中阳[2] 

机构地区：[1]天津医科大学一中心临床学院,300192 [2]天津市第一中心医院东方器官移植中心

出　　处：《中华肝胆外科杂志》2018年第1期43-48,共6页Chinese Journal of Hepatobiliary Surgery

基　　金：国家高技术研究发展计划（863）（2012AA021001）;卫生公益性行业科研专项（201302009）;天津市器官移植临床医学研究中（15ZXLCSY00070）

摘　　要：目的探讨微小RNA-137（miR-137）调控Notchl基因并介导自噬对肝癌细胞增殖和迁移的影响。方法体外培养人SMMC7721肝癌细胞系，用miR-137模拟物、miR-137抑制物以及Notchl基因的干扰RNA（siRNA）转染细胞，分为正常对照组（NC组）、miR-137模拟物组、miR-137抑制物组、NotchlsiRNA组。采用实时定量-PCR（RT—PCR）检测SMMC7721细胞转染后miR-137与NotchlmRNA的表达。细胞迁移实验（Transwell）分析miR-137及Notchl基因对SMMC7721细胞迁移侵袭的影响。细胞免疫组化观察SMMC7721细胞B-链蛋白（13-catenin）和波形蛋白（vimentin）的表达情况，自噬双标腺病毒检测SMMC7721细胞自噬体个数的变化。采用蛋白印迹法（Westernblot）检测Notchl基因、上皮型钙黏蛋白（E-Cadherin）、神经型钙黏蛋白（N—Cadherin）、vimentin、选择性自噬接头蛋白（P62）及微管相关蛋白1轻链3（LC3）的表达。结果实时荧光定量PCR（RT—PCR）结果显示miR-137抑制物组Notchl基因的相对表达含量（5．71±0．45）明显高于miR-137模拟物组（0．21±0．06），差异有统计学意义（P〈0．05）。Transwell实验显示miR-137模拟物组（66．00±4．55）和NotchlsiRNA组（88．00±6．78）肝癌细胞侵袭转移的个数较miR-137抑制物组（515．00±35．12）明显降低（P〈0．05）。细胞组化检测显示miR-137模拟物组及NotchlsiRNA组B—catenin表达明显增加且vimentin表达明显下降（P〈0．05）。自噬双标腺病毒检测结果显示miR-137模拟物组自噬体数量（5．50±3．70）明显低于miR-137抑制物组（32．75±4．11），差异有统计学意义（P〈0．05）。Westernblot检测显示，与miR-137抑制物组、NC组比较，miR-137模拟物组Notchl基因、N—Cadherin、vimentin和LC3表达水平明显降低，E—Cadherin、P62表达水平明显增高。NotchlsiRNA组Notchl基因、N-Cadherin、LC3表达水平明显低于NC组，E—Cadherin、P62表达水平明显高于NC�Objective To explore the role of miR-137 in the proliferation and migration of hepato- cellular carcinoma （HCC） cells by regulating Notch1 and mediating autophagy. Methods The human SMMC7721 hepatoma cell line was transfected with miR-137 mimics, miR-137 inhibitor and Notch1 interfe- ring RNA （siRNA） , and divided into normal control group （NC group） , miR-137 mimics group, miR-137 inhibitor group, Notch1 siRNA group. The expression levels of miR-137 and Notch1 mRNA after the trans- fection were detected by RT-PCR in SMMC7721 cells. Transwell experiments were performed to analyze the effect of miR-137 and Notchl on the migration and invasion of SMMC7721 cells. The expression levels of 13- catenin and vimcntin in SMMC7721 cells were detected by immunohistochemistry. The number of autophago- somes was detected by double labeled adenovirus. Western blot was utilized to detect the expression of Notchl, E-Cadherin, N-Cadherin, vimentin, P62, and LC3. Results The results of RT-PCR showed that the relative expression level of Notch1 in miR-137 inhibitor group （ 5.71 ± 0.45 ） was significantly higher than that in miR-137 mimics group （0.21 ± 0.06） with statistical significance （P 〈 0.05 ）. The Transwell experiments showed that there were fewer invasive metastatic hepatoma cells in miR-137 mimics group （66. 00 ± 4. 55） and Notchl siRNA group （88. 00 ± 6. 78） than that in the miR-137 inhibitor group （515.00 ± 35.12） （P 〈 0.05 ）. The expression levels of 15-catenin in miR-137 mimics group and Notch l siRNA group were significantly increased and the expression level of vimentin was decreased （ P 〈 0.05 ）. The results of autophagy double labeled adenovirus test showed that the number of autophagosomes in miR- 137 mimics group （5.50±3.70） was significantly fewer than that in miR-137 inhibitor group （32. 75 ± 4. 11 ）, and the difference was statistically significant （P 〈 0.05 ）. The expression levels of Notchl, N-cadherin, vimentin, and LC3 protein in miR-13

关 键 词：肝细胞癌 微小RNA-137 Notchl基因 侵袭和转移 自噬 

分 类 号：R735.7[医药卫生—肿瘤]