长链非编码RNA LNC01309对人肝癌细胞增殖和迁移能力的影响及作用机制  

Effect of long non-coding RNA LNC01309 on proliferation and migration abilities of human hepatoma cells and its mechanism of action

作　　者：刘红燕 李婧姣 路泽军 刘咏真 温居一[1,2] LIU Hongyan;LI Jingjiao;LU Zejun;LIU Yongzhen;WEN Juyi(Naval Clinical College of Anhui Medical University,Hefei 230032,China;Department of Oncology,The Sixth Medical Center of PLA General Hospital,Beijing 100048,China)

机构地区：[1]安徽医科大学海军临床学院,合肥230032 [2]解放军总医院第六医学中心肿瘤内科,北京100048

出　　处：《临床肝胆病杂志》2022年第3期563-571,共9页Journal of Clinical Hepatology

基　　金：全军后勤科研重点课题(BHJ14C008)。

摘　　要：目的探讨长链非编码RNA(lncRNA)LNC01309对人肝癌细胞增殖和迁移能力的影响及作用机制。方法收集2018年2月—2019年6月在解放军总医院第六医学中心手术治疗的12例肝细胞癌(以下简称肝癌)患者标本及对应的癌旁组织,采用荧光定量PCR方法检测LNC01309的相对表达量。利用荧光定量PCR法检测LNC01309在人肝癌细胞系(Hep G2、SNU-398和Hep 3B)和人永生化正常肝细胞系(THLE-2)中的表达水平。在肝癌细胞Hep G2中过表达LNC01309,分为质粒对照组(pEXP-control)和过表达组(pEXP-LNC01309)。采用CCK-8检测各组细胞增殖变化,采用划痕愈合试验和Transwell试验检测各组细胞迁移能力。利用RNA免疫共沉淀试验检测LNC01309与RBM38的相互作用(分组为IgG组和RBM38抗体组)。利用放线菌酮追踪分析检测LNC01309对于RBM38蛋白稳定性的影响。在肝癌细胞Hep G2中过表达RBM38进行回复试验,采用CCK-8试验、划痕愈合试验和Transwell试验,分别检测细胞增殖和迁移能力的变化。计量资料两组间比较采用t检验。结果LNC01309在肝癌组织中的平均表达水平高于癌旁组织(4.225±2.285 vs 1.541±0.530,t=3.618,P=0.004)。LNC01309在肝癌细胞(Hep G2、SNU-398和Hep 3B)中的相对表达水平亦均高于正常肝细胞(THLE-2)(t值分别为4.231、6.489、14.480,P值均<0.05)。过表达LNC01309的Hep G2细胞(t=9.172,P<0.001)相对于对照组,细胞的生长速度明显上升(第96小时OD_(450)值:1.885±0.107 vs 2.527±0.234,t=4.330,P=0.012),迁移能力上调(划痕愈合试验:11.65%±2.40%vs 35.66%±4.90%,t=9.837,P<0.001;Transwell试验:100.00%±3.11%vs 161.00%±35.93%,t=4.399,P=0.005);然而过表达LNC01309诱导的肝癌细胞增殖和迁移能力的上调效应,都能够被RBM38部分抑制(第96小时OD_(450)值:2.500±0.227 vs 1.913±0.282,t=2.812,P=0.048;168.00%±9.43%vs 117.20%±18.03%,t=6.622,P<0.001)。相对于IgG对照组,RBM38抗体能够显著富集沉淀LNC01309(t=3.846,P=0.031)。放线菌酮试验结Objective To investigate the effect of long non-coding RNA(lncRNA)LNC01309 on the proliferation and migration abilities of human hepatocellular carcinoma(HCC)cells and its mechanism of action.Methods HCC samples and corresponding adjacent tissue samples were collected from 12 patients with HCC who underwent surgical treatment in The Sixth Medical Center of PLA General Hospital from February 2018 to June 2019,and quantitative real-time PCR was used to measure the relative expression level of LNC01309.Quantitative real-time PCR was also used to measure the expression level of LNC01309 in human hepatoma cell lines(HepG2,SNU-398,and Hep3B)and the human immortalized normal liver cell line THLE-2.After LNC01309 was overexpressed in HepG2 cells,the cells were divided into plasmid control group(pEXP-control)and overexpression group(pEXP-LNC01309).CCK-8 assay was used to observe the change in cell proliferation,and wound healing assay and Transwell assay were used to observe migration ability.RNA co-immunoprecipitation was used to detect the interaction between LNC01309 with RBM38,with cells divided into IgG group and RBM38 antibody group,and cycloheximide chase assay was used to analyze the effect of LNC01309 on the stability of RBM38 protein.RBM38 was overexpressed in HepG2 cells to conduct the recovery experiment,and CCK-8 assay,wound healing assay,and Transwell assay were used to observe the changes in cell proliferation and migration abilities.The t-test was used for comparison of continuous data between two groups.Results The mean expression level of LNC01309 in HCC tissue was significantly higher than that in adjacent tissue(4.225±2.285 vs 1.541±0.530,t=3.618,P=0.004),and the relative expression level of LNC01309 in hepatoma cells(HepG2,SNU-398,and Hep3B)was also significantly higher than that in normal hepatocytes(THLE-2)(t=4.231、6.489、14.480,all P<0.05).Compared with the control group,HepG2 cells with the overexpression of LNC01309 had significant increases in growth rate(OD_(450) value at 96 hours:1.885±0

关 键 词：癌 肝细胞 RNA 长链非编码 RNA结合蛋白质类 

分 类 号：R73[医药卫生—肿瘤]