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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 |
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(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) |
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| 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）. |
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| 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 |
