lncRNA SNHG1结合miR-145促进胃癌细胞增殖的机制研究

doi:10.3969/j.issn.1673-8640.2020.10.023

摘要/Abstract

摘要：

目的探讨小核仁RNA宿主基因1（SNHG1）作为竞争性内源RNA（ceRNA）竞争性结合微小RNA-145（miR-145）,从而促进胃癌细胞生长的机制。方法利用Kaplan-Meier Plotter网站分析SNHG1与胃癌生存期的相关性;生物信息分析和实时荧光定量聚合酶链反应（qRT-PCR）检测SNHG1在胃癌及癌旁组织中的表达及SNHG1与miR-145表达的关系;CCK8实验检测过表达及敲低SNHG1对胃癌细胞生长情况的影响;荧光素酶报告基因实验检测SNHG1与miR-145的结合情况;分别及同时上调SNHG1、miR-145后采用免疫印迹法检测β-连环蛋白（CTNNB1）、骨髓瘤病毒原癌基因（MYC）蛋白表达的变化。结果在胃癌患者中,SNHG1表达水平低者较高者总生存期延长（P=0.015）。胃癌组织SNHG1表达明显升高（P<0.05）。过表达SNHG1可以促进胃癌细胞增殖,敲低SNHG1可抑制细胞增殖;荧光素酶报告基因实验结果显示,SNHG1和miR-145能有效结合;体外实验表明,miR-145下调CTNNB1、MYC蛋白表达,SNHG1促进CTNNB1、MYC蛋白表达,且SNHG1能抑制miR-145下调CTNNB1和MYC蛋白。结论 SNHG1在胃癌组织中高表达,且通过结合miR-145上调CTNNB1、MYC蛋白表达,促进胃癌细胞生长,是参与胃癌生物学行为的机制之一。

关键词: 小核仁RNA宿主基因1, 微小RNA-145, 细胞生长, 胃癌

Abstract:

Objective To investigate small nucleolar RNA host gene 1（SNHG1） promoting the growth of gastric cancer through binding to microRNA-145（miR-145）. Methods Kaplan-Meier Plotter database analyzed the correlation of SNHG1 with gastric cancer overall survival. Bioinformatics analysis and real-time fluorescence quantitation polymerase chain reaction（qRT-PCR） were used to investigate the expression level and the expression relationship of SNHG1 and miR-145 in gastric cancer. The influence of overexpression and knock-down of SNHG1 on the growth of gastric cancer cell was determined by CCK8 assay. Luciferase reporter gene assay studied the interaction between SNHG1 and miR-145. Western blot was used to determined the change of CTNNB1 and MYC protein with the transfection of SNHG1,miR-145 and SNHG1 with miR-145. Results The overall survival of patients with low SNHG1 expression was longer than that of patients with high SNHG1 expression（P=0.015）. Compared to adjacent tissues,the expression level of SNHG1 in gastric cancer tissues was increased（P<0.05）. The overexpression of SNHG1 promoted the growth of gastric cancer cells,while knock-down SNHG1 inhibited cell growth. Luciferase reporter assay showed that SNHG1 could bind to miR-145 effectively. The miR-145 down-regulated the expressions of CTNNB1 and MYC proteins,SNHG1 promoted the expressions of CTNNB1 and MYC proteins,and SNHG1 inhibited the down-regulation of CTNNB1 and MYC proteins by miR-145. Conclusions The over-expressed SNHG1 in gastric cancer binding to miR-145 enhances the expression of CTNNB1 and MYC proteins that could promote gastric cancer growth.

Key words: Small nucleolar RNA host gene 1, MicroRNA-145, Cell growth, Gastric cancer

中图分类号:

R446.7

周锋利, 褚以忞, 李吉, 杨大明, 彭海霞, 徐莹. lncRNA SNHG1结合miR-145促进胃癌细胞增殖的机制研究[J]. 检验医学, 2020, 35(10): 1062-1069.

ZHOU Fengli, CHU Yimin, LI Ji, YANG Daming, PENG Haixia, XU Ying. LncRNA SNHG1 promoting the growth of gastric cancer through binding to miR-145[J]. Laboratory Medicine, 2020, 35(10): 1062-1069.

图/表 5

图1 SNHG1对胃癌OS期的影响及在胃癌及癌旁组织中的表达情况注:（a）Kaplan-Meier Plotter数据库中SNHG1高表达组和低表达组中位OS, SNHG1低表达, SNHG1高表达;（b）starBase v3.0数据库中375例胃癌组织与32例正常胃组织中SNHG水平比较;（c）17例胃癌患者的胃癌及癌旁组织SNHG1水平比较

图2 SNHG1对胃癌细胞生长的影响注:（a）胃癌细胞株SGC7901中过表达SNHG1;（b）CCK8实验显示SGC7901中SNHG1过表达能促进细胞生长;NC; sh-SNHG1;与NC组比较,*P<0.05;（c）在胃癌细胞株BGC823中过表达SNHG1;（d）CCK8实验显示BGC823中过表达SNHG1能促进细胞生长; pcDNA3.0; SNHG1;与pcDNA3.0组比较,*P<0.05;（e）在胃癌细胞株SGC7901中敲低SNHG1的表达;（f）CCK8实验发现SGC7901中敲低SNHG1,细胞株的生长受到抑制; NC; siSNHG1-1; siSNHG1-2;与NC组比较,*P<0.05

表1 TANRIC数据库显示在胃癌中与SNHG1表达水平呈负相关的microRNA

肿瘤	位置/注释	基因	r值	P 值
TCGA-STAD	ENSG00000255717.2	hsa-mir-103-1	0.564	9.01×10^-8
TCGA-STAD	ENSG00000255717.2	hsa-mir-106b	0.477	1.01×10^-5
TCGA-STAD	ENSG00000255717.2	hsa-mir-107	0.579	3.51×10^-8
TCGA-STAD	ENSG00000255717.2	hsa-mir-128-1	0.486	6.47×10^-6
TCGA-STAD	ENSG00000255717.2	hsa-mir-128-2	0.474	1.14×10^-5
TCGA-STAD	ENSG00000255717.2	hsa-mir-139	–0.427	9.07×10^-5
TCGA-STAD	ENSG00000255717.2	hsa-mir-145	–0.421	1.17×10^-4
TCGA-STAD	ENSG00000255717.2	hsa-mir-151	0.539	3.89×10^-7
TCGA-STAD	ENSG00000255717.2	hsa-mir-15b	0.435	6.42×10^-5

图3 SNHG1与miR-145在胃癌中表达的相关性及SNHG1与miR-145结合情况注:（a）miR-145与SNHG1的相关性（r=-0.37,P=3.81×10-13）;（b）在GES-1细胞中过表达SNHG1; pcDNA3.0; SNHG1;（c）在SGC7901细胞中过表达SNHG1; pcDNA3.0; SNHG1;（d）通过RNA22 v2 microRNA target detection网站预测到SNHG1与miR-145存在的结合位点;（e）荧光素酶报告基因实验结果

图4 SNHG1与miR-145结合对CTNNB1、MYC蛋白表达的影响注:（a）SNHG1 mRNA表达量与CTNNB1 mRNA表达量的相关性（r=0.12、P=0.025）;（b）SNHG1表达与MYC mRNA表达量的相关性（r=0.28,P=2.16×10-8）;（c）在GES-1细胞中过表达miR-145;（d）在GES-1细胞中过表达SNHG1;（e）在GES-1细胞中同时过表达miR-145和SNHG1; pcDNA3.0+NC; SNHG1+miR-145;（f）免疫印迹法检测CTNNB1和MYC蛋白的表达

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