干扰UCA1及抑制miR-185-5p对非小细胞肺癌β-Catenin通路的活化、自噬和存活影响

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英文篇名：Effects of Interference with UCA1 and Inhibition of miR-185-5p on Activation, Autophagy and Survival of β-Catenin Pathway in Non-small Cell Lung Cancer 作者：杨雁 ; 刘行仁 ; 金钊 英文作者：YANG Yan;LIU Xing-ren;JIN Zhao;Department of Respiratory and Critical Care Medicine,Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital;Basic Medical College,Chengdu University of Traditional Chinese Medicine; 关键词：长链非编码RNA ; UCA1 ; miR-185-5p ; A549肺癌细胞 ; β-Catenin 英文关键词：LncRNAUCA1;;miR-185-5p;;A549cells;;β-Catenin 中文刊名：HXYK 英文刊名：Journal of Sichuan University(Medical Science Edition) 机构：四川省医学科学院·四川省人民医院呼吸与危重症医学科;成都中医药大学基础医学院; 出版日期：2019-03-15 出版单位：四川大学学报(医学版) 年：2019 期：v.50 基金：四川省干部保健科研项目(No.川干研2017-223)资助 语种：中文; 页：HXYK201902003 页数：7 CN：02 ISSN：51-1644/R 分类号：18-24

摘要

目的探究在非小细胞肺癌中,长链非编码RNA(long non-coding RNA,lncRNA)尿路上皮癌抗原1(urothelial carcinoma associated 1,UCA1)敲降miR-185-5p对β-Catenin通路的影响。方法选取非小细胞肺癌A549细胞为研究材料,设置A549空白对照组、sh-scramble阴性对照组、sh-UCA1干扰组、miR-185 inhibitor组和sh-UCA1+inhibitor组。Western blot验证体系中增殖、凋亡和自噬相关分子表达;qRT-PCR鉴定体系中lncRNA UCA1和miR-185-5p的表达;生物信息学软件和荧光素酶实验检测lncRNA UCA1和miR-185-5p之间结合性。BrdU染色鉴定细胞增殖,免疫荧光染色检测细胞中LC3~+细胞含量。结果在A549细胞中,lncRNA UCA1干扰后,UCA1表达量显著下降并促进miR-185-5p表达,有效抑制肺癌细胞增殖和自噬,促进凋亡发生(P<0.01);经生物信息学和双荧光素酶报告系统验证lncRNA UCA1和miR-185-5p有较强结合性;并进一步验证lncRNA UCA1可以有效抑制β-Catenin/TCF-4及Beclin 1和LC3Ⅱ表达,降低miR-185-5p对肺癌细胞增殖和自噬效果,减少细胞内LC3含量(P<0.01)。结论 lncRNA UCA1干扰后,可以有效降低UCA1对miR-185-5p的抑制效果,进而解除β-Catenin/TCF-4、Beclin 1和LC3Ⅱ受到的抑制作用,从而减少肺癌细胞自噬发生和减缓增殖。
        Objective To investigate the effect on β-Catenin pathway by lncRNA urothelial carcinoma associated 1(UCA1) targeting regulated miR-185-5 p in human lung adenocarcinoma A549 cell line. Methods 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-UCA1 interference group(sh-UCA1), miR-185 inhibitor group(miR-185 inhibitor) and sh-UCA1+ inhibitor group(sh-UCA1+inhibitor). The proliferation-, apoptosis-and autophagy-related protein levels were determined by Western blot. qRT-PCR was employed to detect the mRNA levels of UCA1 and miR-185-5 p. The relationship between lnRNA UCA1 and miR-185-5 p 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. Results sh-UCA1 significantly decreased UCA1 expression and increased miR-185-5 p expression in A549 cells, and inhibited the cell growth and autophagy, while promoted the cell apoptosis(P<0.01). Bioinformatics analysis and luciferase reporter system assays demonstrated that lncRNA UCA1 and miR-185-5 can combine effectively, indicating that they have a targating relationship. sh-UCA1 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-185-5 p; and down-regulated the LC3 expression(P<0.01). Conclusion The effect of UCA1 inhibition for miR-185-5 p was decreased by lncRNA UCA1 inference, and released the β-Catenin/TCF-4, Beclin 1 and LC3 Ⅱ, and further reduced the autophagy and growth in A549 cells.

引文

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