Yang Yan, Liu Xing-Ren, Jin Zhao
Department of Respiratory and Critical Care Medicine, Sichuan Academy of Medical Sciences& Sichuan Provincial People's Hospital, Chengdu 610072, China.
Basic Medical College, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2019 Mar;50(2):157-163.
To investigate the effect on β-Catenin pathway by lncRNA urothelial carcinoma associated 1 (UCA1) targeting regulated miR-185-5p in human lung adenocarcinoma A549 cell line.
A549 cell was selected as the study model and were divided into four groups, blank control group, sh-scramble negative control group (sh-scramble), sh- interference group (sh-), miR-185 inhibitor group (miR-185 inhibitor) and sh-+ inhibitor group (sh-+inhibitor). The proliferation-, apoptosis- and autophagy- related protein levels were determined by Western blot. qRT-PCR was employed to detect the mRNA levels of and miR-185-5p. The relationship between lnRNA and miR-185-5p was validated by bioinformatics analysis and luciferase reporter system assays. BrdU staining was used to detect the cell growth, and immunofluorence staining was performed to measure the content of LC3 cells.
sh- significantly decreased expression and increased miR-185-5p expression in A549 cells, and inhibited the cell growth and autophagy, while promoted the cell apoptosis (<0.01). Bioinformatics analysis and luciferase reporter system assays demonstrated that lncRNA and miR-185-5 can combine effectively, indicating that they have a targating relationship. sh- also significantly inhibited the protein levels of β-Catenin/TCF-4, Beclin 1 and LC3 Ⅱ, and decreased the cell growth and autophagy by the miR-185-5p; and down-regulated the LC3 expression (<0.01).
The effect of inhibition for miR-185-5p was decreased by lncRNA inference, and released the β-Catenin/TCF-4, Beclin 1 and LC3 Ⅱ, and further reduced the autophagy and growth in A549 cells.
探讨长链非编码RNA尿路上皮癌相关1（UCA1）靶向调控微小RNA-185-5p（miR-185-5p）对人肺腺癌A549细胞系β-连环蛋白信号通路的影响。
选取A549细胞作为研究模型，分为空白对照组、sh-乱序阴性对照组（sh- scramble）、sh-干扰组（sh- ）、miR-185抑制剂组（miR-185 inhibitor）和sh- +抑制剂组（sh- +inhibitor）。采用蛋白质免疫印迹法检测增殖、凋亡和自噬相关蛋白水平。运用实时荧光定量聚合酶链反应（qRT-PCR）检测lncRNA UCA1和miR-185-5p的信使核糖核酸（mRNA）水平。通过生物信息学分析和荧光素酶报告基因系统实验验证lncRNA UCA1与miR-185-5p之间的关系。采用5-溴脱氧尿嘧啶核苷（BrdU）染色检测细胞生长情况，进行免疫荧光染色检测微管相关蛋白1轻链3（LC3）细胞含量。
sh- 可显著降低A549细胞中lncRNA UCA1表达，增加miR-185-5p表达，抑制细胞生长和自噬，促进细胞凋亡（P<0.01）。生物信息学分析和荧光素酶报告基因系统实验表明lncRNA UCA1与miR-185-5p能够有效结合，提示二者存在靶向关系。sh- 还可显著抑制β-连环蛋白/转录因子4（β-Catenin/TCF-4）、自噬相关蛋白1（Beclin 1）和微管相关蛋白1轻链3Ⅱ型（LC3Ⅱ）的蛋白水平，并通过miR-185-5p降低细胞生长和自噬，下调LC3表达（P<0.01）。
lncRNA UCA1干扰可减弱对miR-185-5p的抑制作用，释放β-Catenin/TCF-4、Beclin 1和LC3Ⅱ，进一步降低A549细胞的自噬和生长。
