miR-133a-3p通过抑制氧化还原因子1抑制肝癌细胞恶性生物学行为  

miR-133a-3p inhibits malignant biological behavior of hepatoma cells by inhibiting redox factor 1

作　　者：李天雄 孙志鹏 尹刚 闫巍 阿民布和 张能维 LI Tian-Xiong;SUN Zhi-Peng;YIN Gang;YAN Wei;A Min-Buhe;ZHANG Neng-Wei(Surgery of Gastrointestinal Hepatobiliary Tumors,Beijing Century Temple Hospital Affiliated to Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属北京世纪坛医院胃肠肝胆肿瘤外科,北京100038

出　　处：《中国免疫学杂志》2020年第10期1217-1223,共7页Chinese Journal of Immunology

基　　金：中国铁路总公司科技研究开发计划课题基金(J2017Z609)资助。

摘　　要：目的:通过多种实验途径探讨miR-133a-3p通过调节氧化还原因子1(Ref-1)对肝癌细胞恶性生物学行为的影响。方法:收集2017年2月~2018年12月来本院就诊的肝癌手术患者的癌组织和癌旁相邻正常组织标本,用qRT-PCR检测miR-133a-3p在癌组织和癌旁相邻正常组织中以及在肝癌细胞系HepG2、Huh-7、SK-Hep1、SMMC-7721和肝正常细胞Hep-3B中的表达;构建miR-133a-3p-mimics(miR-mimics)组、miR-NC转染质粒组和正常对照组(Control),qRT-PCR检测转染前后miR-133a-3p在HepG2中的表达;CCK-8和克隆形成实验检测HepG2细胞转染前后增殖能力变化;细胞划痕和Transwell实验分别检测转染前后HepG2细胞的迁移和侵袭能力变化;RNA转录组测序检测HepG2细胞转染前后转录组RNA表达差异并作通路富集分析;qRT-PCR检测筛选RNA-seq中RNA表达差异较大的基因;qRT-PCR和Western blot检测Ref-1在HepG2细胞转染前后中的表达。免疫组化和qRT-PCR分别检测Ref-1在肝癌组织和肝癌细胞系中表达;最后HepG2细胞同时转染了miR-mimics和Ref-1 miR-inhibitors,进行联合干预,随后CCK-8、细胞划痕和Transwell分别检测了联合干预后细胞增殖、迁移和侵袭能力变化。结果:miR-133a-3p在肝癌患者癌组织中的表达显著低于癌旁组织,同时其在肝癌细胞HepG2中的表达最低;qRT-PCR结果表明miR-mimics可有效促进miR-133a-3p在HepG2中的表达;CCK-8和克隆形成实验结果显示miR-133a-3p过表达后可以显著抑制HepG2细胞的增殖、迁移和侵袭等恶性生物学行为;RNA-seq结果显示HepG2转染前后Ref-1的表达差异最大;qRT-PCR和Western blot结果显示HepG2转染成功后,Ref-1表达随miR-133a-3p的过表达而降低。免疫组化和qRT-PCR结果显示Ref-1在肝癌组织和肝癌细胞系中的表达均显著高于阴性对照组;联合干预后HepG2细胞的增殖、迁移和侵袭等恶性生物学抑制作用更加强烈。结论:miR-133a-3p可能通过抑制Ref-1表达从而抑制肝癌细胞的增Objective:Effects of miR-133 a-3 p on the malignant biological behavior of hepatocellular carcinoma cells by regulating redox factor 1(Ref-1) were investigated by various experimental approaches.Methods: Collection of cancer tissues and adjacent normal tissue specimens of patients with liver cancer surgery who visited our hospital from February 2017 to December 2018.Expression of miRNA-133 a-3 p in cancer tissues and adjacent normal tissues,as well as in hepatocellular carcinoma cell lines HepG2,Huh-7,SK-Hep1,SMMC-7721 and Hep-3 B were detected by qRT-PCR.mi-133 a-3 p-mimics(miRNA-mimics),miRNA-NC transfection plasmids and control group(Control)were constructed.Expression of miRNA-133 a-3 p in HepG2 was detected by qRT-PCR before and after transfection.CCK-8 and clonogenic assay were used to detect the proliferation of HepG2 cells before and after transfection,cell scratch and Transwell assay were used to detect the migration and invasion of HepG2 cells before and after transfection.RNA transcriptome sequencing was used to detect the difference of RNA expression in HepG2 cells before and after transfection and to analyze the pathway enrichment.qRT-PCR detection of genes with large differences in RNA expression in RNA-seq.Detection of Ref-1 expression in HepG2 cells before and after transfection by qRT-PCR and Western blot.Expression of Ref-1 in hepatocellular carcinoma tissues and hepatocellular carcinoma cell lines were detected by immunohistochemistry and qRT-PCR.Finally,HepG2 cells were simultaneously transfected with miR-mimics and Ref-1 miR-inhibitors for combined intervention.CCK-8,cell scratches and Trans-well were used to detect changes in cell proliferation,migration and invasion after combined intervention.Results: Expression of miR-133 a-3 p in hepatocellular carcinoma tissues was significantly lower than that in adjacent tissues,and the expression of miR-133 a-3 p in hepatocellular carcinoma cells HepG2 was the lowest.Results of qRT-PCR showed that miRNA-mimics could effectively promote the expression

关 键 词：miR-133a-3p REF-1 HEPG2 增殖 迁移 侵袭 

分 类 号：R73[医药卫生—肿瘤]