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  • 李莎,朱怡卿,石荟,戈霞晖,许靖,商艳,王芳,白冲.长链非编码RNA-H19特异性吸附微RNA-760调控nanog基因表达促进非小细胞肺癌细胞增殖和迁移[J].第二军医大学学报,2019,40(8):854-859    [点击复制]
  • LI Sha,ZHU Yi-qing,SHI Hui,GE Xia-hui,XU Jing,SHANG Yan,WANG Fang,BAI Chong.Long non-coding RNA-H19 promotes proliferation and migration of non-small cell lung cancer cells through sponging microRNA-760 to regulate nanog gene expression[J].Acad J Sec Mil Med Univ,2019,40(8):854-859   [点击复制]
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长链非编码RNA-H19特异性吸附微RNA-760调控nanog基因表达促进非小细胞肺癌细胞增殖和迁移
李莎1△,朱怡卿2△,石荟1,戈霞晖3,许靖3,商艳1,3*,王芳2,白冲1
0
(1. 海军军医大学(第二军医大学)长海医院呼吸与危重症医学科, 上海 200433;
2. 海军军医大学(第二军医大学)基础医学院医学遗传学教研室, 上海 200433;
3. 上海中医药大学附属第七人民医院呼吸科, 上海 200137
共同第一作者
*通信作者)
摘要:
目的 探讨长链非编码RNA(lncRNA)-H19对非小细胞肺癌(NSCLC)细胞增殖、迁移的影响及分子机制。方法 收集2015年10月至2016年5月海军军医大学(第二军医大学)长海医院确诊的20例NSCLC患者手术切除的NSCLC组织及相应的癌旁正常组织。通过实时荧光定量PCR(qPCR)检测NSCLC组织及癌旁正常组织,人NSCLC细胞系A549、NCI-H1299和人正常肺上皮细胞系BEAS-2B中lncRNA-H19的表达。在A549细胞中过表达lncRNA-H19后,采用CCK-8法和Transwell实验分别检测细胞的增殖和迁移能力。利用生物信息学分析预测lncRNA-H19与微RNA(miRNA)-760的结合位点,通过双荧光素酶报告基因实验分析lncRNA-H19对miRNA-760的吸附作用,采用qPCR和蛋白质印迹法分别检测lncRNA-H19过表达对miRNA-760及下游靶基因nanog表达的影响。通过对A549细胞转染miRNA-760模拟剂过表达miRNA-760后,检测过表达miRNA-760对lncRNA-H19促进NSCLC细胞增殖和迁移作用的影响。结果 LncRNA-H19在NSCLC组织和A549、NCI-H1299细胞中均呈高表达,分别与其在癌旁正常组织和BEAS-2B细胞中的表达水平相比差异均有统计学意义(P均<0.01)。与对照组相比,过表达lncRNA-H19后A549细胞的增殖能力增强(P<0.05)、迁移能力提高(P<0.01)。在线数据库starBase v3.0预测分析结果显示lncRNA-H19能特异性吸附miRNA-760。双荧光素酶报告基因检测结果显示lncRNA-H19与miRNA-760之间存在特异性结合。与对照组相比,过表达lncRNA-H19可抑制A549细胞中miRNA-760的表达(P均<0.05),并促进其下游基因nanog在mRNA和蛋白水平的表达(P均<0.01)。过表达miRNA-760可抑制lncRNA-H19对A549细胞增殖和迁移的促进作用,与lncRNA-H19过表达组相比差异均有统计学意义(P均<0.05)。结论 LncRNA-H19可通过特异性吸附miRNA-760调控nanog基因表达,从而促进NSCLC细胞增殖和迁移。
关键词:  非小细胞肺癌  长链非编码RNA-H19  微RNA-760  nanog基因
DOI:10.16781/j.0258-879x.2019.08.0854
投稿时间:2019-03-17修订日期:2019-07-17
基金项目:国家自然科学基金(81570020,81300018),浙江省公益技术应用研究计划项目(2016C33216),浦东新区科技发展基金(PKJ2016-Y49).
Long non-coding RNA-H19 promotes proliferation and migration of non-small cell lung cancer cells through sponging microRNA-760 to regulate nanog gene expression
LI Sha1△,ZHU Yi-qing2△,SHI Hui1,GE Xia-hui3,XU Jing3,SHANG Yan1,3*,WANG Fang2,BAI Chong1
(1. Department of Respiratory and Critical Care Medicine, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Medical Genetics, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
3. Department of Respiratory Medicine, Seventh People's Hospital of Shanghai, Shanghai University of Traditional Chinese Medicine, Shanghai 200137, China
Co-first authors.
* Corresponding author)
Abstract:
Objective To explore the role of long non-coding RNA (lncRNA)-H19 in the proliferation and migration of non-small cell lung cancer (NSCLC) cells and the molecular mechanisms. Methods The expressions of lncRNA-H19 in 20 NSCLC tissues and paired non-tumor tissues, which were collected from Changhai Hospital of Naval Medical University (Second Military Medical University) from Oct. 2015 to May 2016, were detected by real-time quantitative PCR (qPCR). We also examined lncRNA-H19 expressions in NSCLC cell lines A549 and NCI-H1299 and normal lung epithelial cell line BEAS-2B by qPCR. The proliferation and migration of A549 cells overexpressing lncRNA-H19 were detected by CCK-8 assay and Transwell assay, respectively. Bioinformatics analysis and duel-luciferase reporter assay were conducted to predict and confirm the interaction between microRNA (miRNA)-760 and lncRNA-H19. Western blotting analysis and RT-qPCR were performed to observe the influence of lncRNA-H19 overexpression on the expression of miRNA-760 and target gene nanog. MiRNA-760 was overexpressed in A549 cells, and its role in lncRNA-H19 promoting proliferation and migration of NSCLC cells was observed. Results The expressions of lncRNA-H19 in NSCLC tissues and A549 and NCI-H1299 cells were significantly upregulated compared with those in normal tissues and BEAS-2B cells (all P<0.01). Overexpression of lncRNA-H19 significantly improved the proliferation ability (P<0.05) and migration ability (P<0.01) of A549 cells compared with the negative control group. The results of starBase v3.0 showed that lncRNA-H19 could specifically adsorb miRNA-760, and duel-luciferase reporter assay showed that lncRNA-H19 directly bound to miRNA-760. Compared with the negative control group, overexpression of lncRNA-H19 significantly inhibited miRNA-760 expression in A549 cells (P<0.05) and promoted the expression of the downstream gene nanog at mRNA and protein levels (all P<0.01). Overexpression of miRNA-760 significantly inhibited lncRNA-H19-induced proliferation and migration of A549 cells (all P<0.05). Conclusion LncRNA-H19 can promote the proliferation and migration of NSCLC cells through sponging miRNA-760 to regulate nanog gene expression.
Key words:  non-small cell lung carcinoma  long non-coding RNA-H19  microRNA-760  nanog gene