下调微小RNA-146b-5p通过增强自噬抑制人甲状腺乳头状癌细胞增殖  

Down - regulation of microRNA - 146b - 5p repress proliferation of thyroid carcinoma cells by en- hancing autophagy activity

作　　者：史阳[1] 卢秀波[2] 刘洋[2] 樊玉霞[2] 袁青领[2] 

机构地区：[1]郑州大学第一临床学院普外科,450052 [2]郑州大学第一附属医院甲状腺外科,450052

出　　处：《中华实验外科杂志》2017年第1期58-60,共3页Chinese Journal of Experimental Surgery

基　　金：河南省医学科技攻关重点计划项目（201402007）

摘　　要：目的探讨通过下调微小RNA（miRNA，miR）-146b-5p促进人甲状腺乳头状癌细胞（TPC-1）自噬功能对其增殖功能的影响。方法脂质体法瞬时转染，实验分为4组：as-miR组（miR-146b-5p inhibitor）、as-NC组、共转染组（as-miR-146b-5p＋siBeclin1）和共转染对照组（as-miR-146b-5p＋siNC）。倒置荧光显微镜和实时定量反转录聚合酶链反应（RT-qPCR）检测转染效率。Western blot和RT-qPCR检测转染后各组TPC-1细胞中Beclin1蛋白、mRNA的表达。透射电镜观察转染后各组TPC-1细胞中自噬小体的变化。细胞计数试剂盒（CCK-8）法和平板克隆形成实验检测转染后各组TPC-1细胞增殖能力变化。结果 与as-NC组比较，转染24 h后as-miR组TPC-1细胞中miR-146b-5p表达下调为0.380±0.021，且Beclin1在蛋白和mRNA水平分别上调为0.612±0.014、0.413±0.005，电镜显示自噬小体明显增多，72 h的吸光度值下降为1.470±0.062（P＝0.007），单个细胞克隆形成率降低为（29.000±3.610）%（P＝0.031），差异均有统计学意义。与共转染对照组比较，转染36 h后共转染组TPC-1细胞中Beclin1在蛋白和mRNA水平分别下调为0.286±0.027、0.265±0.025，电镜显示自噬小体明显减少，72 h的吸光度值上升为2.564±0.050（P＝0.006），单个细胞克隆形成率升高为（49.000±2.821）%（P＝0.027）。结论下调miR-146b-5p通过增强自噬抑制TPC-1细胞的增殖能力。Objective To investigate the influence of autophagy on proliferation of human papillary thyroid carcinoma cells （TPC-1） repressed by down-regulation of microRNA （miRNA, miR）-146b-5p. Methods TPC-1 cells were transient transfected by Lipofectmine 2000, and then cells were devided into four groups： as-miR, as-NC, co-transfection （as-miR-146b-5p＋ siBeclin1） and co-transfection for control （as-miR-146b-5p＋ siNC）. Then the transfection efficiency was determined by invert fluorescence microscope and real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR）. Western blotting and RT-qPCR were used to analyzed the protein and mRNA levels of Beclin1 of the transfected cells, respectively. Transmission electron microscopy was used to evaluated the expression of autophagsome of the transfected cells. And cell counting kit-8 and colony formation assay were used to detected the proliferation of the transfected cells. Results Compared with those treated with as-NC, miR-146b-5p expression was decreased to （0.380±0.021）, and the protein and mRNA levels of Beclin1 were increased to （0.612±0.014）, （0.413±0.005） respectively, and the absorbance at 72 h and amount of colony foci was decreased to （1.470±0.062, P=0.007）, （29.000±3.610）% （P=0.031） respectively, and the autophagsome expression was significantly increased in TPC-1 cells treated with as-miR-146b-5p after 24 h. Compared with those co-transfected with as-miR-146b-5p＋ siNC, the protein and mRNA levels of Beclin1 were decreased to （0.286±0.027）, （0.265±0.025, P=0.006） respectively, and the absorbance at 72 h and amount of colony foci was increased to （2.564±0.050）, （49.000±2.821）% respectively （P=0.027）, and autophagsome expression was significantly decreased in TPC-1 cells co-transfected with as-miR-146b-5p＋ siBeclin1 after 36 h. Conclusion Down-regulation of miR-146b-5p inhibit the viability of TPC-1 cells via strengthening autophagy activity.

关 键 词：微小RNA-146b-5p 甲状腺乳头状癌 自噬 BECLIN1 

分 类 号：R736.1[医药卫生—肿瘤]