摘要
目的探讨长链非编码RNA(lncRNA)人去泛素化酶含有卵巢肿瘤结构域的6B反义RNA 1(OTUD6B-AS1)通过微小RNA(microRNA,miR)-122-5p对肝癌细胞增殖、侵袭和迁移的影响。方法反转录-聚合酶链反应(RT-PCR)检测2010年1月至2013年1月河南科技大学第一附属医院手术切除的98例肝癌组织标本及其配对的癌旁组织、肝癌细胞系(Hhu7、Hep3B、HCCLM3、MHCC97H)和人正常肝细胞(LO2)中OTUD6B-AS1的相对表达量。用脂质体2000(Lipofectamine^TM2000)分别将sh-NC(sh-NC组)和sh-OTUD6B-AS1(sh-OTUD6B-AS1组)转染入Hep3B细胞。克隆形成实验和细胞计数试剂盒(CCK-8)法检测细胞增殖;Transwell检测细胞侵袭;细胞划痕实验检测细胞迁移。用TargetScan预测OTUD6B-AS1与miR-122-5p的互补结合位点,随后用双荧光素酶报告基因实验确定两者的靶向关系。为了进一步验证两者调控关系,将Hep3B细胞分别分为sh-NC+miR-NC组、sh-OTUD6B-AS1+miR-NC组、sh-OTUD6B-AS1+anti-miR-122-5p组,比较3组细胞增殖、侵袭和迁移。两两比较采用t检验;多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验;生存分析采用Log-rank检验。结果肝癌组织中OTUD6B-AS1相对表达量为2.42±0.96,明显高于癌旁组织(0.92±0.39,t=14.331,P<0.05),差异有统计学意义。肝癌细胞系Hhu7(2.84±0.23)、Hep3B(3.80±0.68)、HCCLM3(1.89±0.25)和MHCC97H(2.01±0.71)的OTUD6B-AS1相对表达量明显高于正常肝细胞LO2(1.04±0.23,F=51.827,P<0.05),差异有统计学意义。sh-OTUD6B-AS1组细胞增殖、侵袭和迁移能力均低于sh-NC组(t=5.556、2.454,P值均<0.05),差异均有统计学意义。双荧光素酶报告基因实验结果证实OTUD6B-AS1可以靶向调控miR-122-5p。sh-NC+miR-NC组和sh-OTUD6B-AS1+anti-miR-122-5p组增殖、侵袭和迁移能力明显高于sh-OTUD6B-AS1+miR-NC组(F=111.908、0.301,P值均<0.05),差异均有统计学意义。结论OTUD6B-AS1可能通过负调控miR-122-5p促进肝癌细胞的增殖、侵袭和迁移。
Objective To explore the effect of long non-coding RNA(lncRNA)OTUD6B-AS1 on the proliferation,invasion and migration of hepatocellular carcinoma(HCC)cells through microRNA(miRNA,miR)-122-5p.Methods Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to detect the relative expression of OTUD6B-AS1 in 98 cases of HCC tissue specimens and their paired adjacent tissues,liver cancer cell lines(Hhu7,Hep3B,HCCLM3,MHCC97H)and human normal liver cells(LO2).Lipidosome-2000 was used to transfect sh-NC(sh-NC group)and sh-OTUD6B-AS1(sh-OTUD6B-AS1 group)into Hep3B cells.Clone formation test and cell counting kit-8(CCK-8)method were used to examine the cell proliferation;Transwell test cell invasion;cell scratch test cell migration.TargetScan was used to predict the complementary binding site of OTUD6B-AS1 and miR-122-5p,and then the dual luciferase reporter gene experiment was used to determine the target relationship between the two.In order to further verify the regulatory relationship between the two,Hep3B cells were divided into sh-NC+miR-NC group,sh-OTUD6B-AS1+miR-NC group and sh-OTUD6B-AS1+anti-miR-122-5p group,and cell proliferation,invasion and migration of three groups were compared.Results The relative expression of OTUD6B-AS1 in HCC tissues was(2.42±0.96),which was significantly higher than that in adjacent tissues(0.92±0.39,t=14.331,P<0.05).The relative expression of OTUD6B-AS1 in Hhu7(2.84±0.23),Hep3B(3.80±0.68),HCCLM3(1.89±0.25)and MHCC97H(2.01±0.71)was significantly higher than in LO2(1.04±0.23,F=51.827,P<0.05).The cell proliferation,invasion and migration abilities in sh-OTUD6B-AS1 group were significantly reduced as compared with those in sh-NC group(t=5.556,2.454,all P<0.05).The results of dual luciferase reporter gene experiments confirmed that OTUD6B-AS1 could target miR-122-5p.Compared with the sh-OTUD6B-AS1+miR-NC group,the sh-NC+miR-NC group and the sh-OTUD6B-AS1+anti-miR-122-5p group had significantly increased proliferation,invasion and migration abilities(F=111.908,0.301,all P<0.05).Conclusion OTUD6B-AS1 may negatively regulate miR-122-5p to promote the proliferation,invasion and migration of HCC cells.
作者
张小博
李艳会
马鹏飞
郑幼伟
Zhang Xiaobo;Li Yanhui;Ma Pengfei;Zheng Youwei(Department of Hepatobiliary Surgery,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang 471003,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第1期156-159,共4页
Chinese Journal of Experimental Surgery
基金
河南省科技厅重点攻关项目(172102310589)
河南省医学科技攻关计划项目(2018020284)。
关键词
长链非编码RNA
癌
肝细胞
微小RNA
增殖
侵袭
迁移
Long non-coding RNA
Carcinoma,hepatocellular
MicroRNA
Proliferation
Invasion
Migration