[Regulatory effect of microRNA-141 on synthesis of high mobility group protein B1 in mouse carcinoma cell line hepal-6]

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2016 Jul;41(7):661-7. doi: 10.11817/j.issn.1672-7347.2016.07.001.
[Article in Chinese]

Abstract

Objective: To explore the regulatory effect of microRNA-141 (miR-141) on expression of high mobility group protein B1 (HMGB1) in mouse hepatoma carcinoma cell line (hepal-6 cells).

Methods: The hepal-6 cells were divided into 4 groups: a miR-141 mimic group, a miR-141 mimic control group, a miR-141 inhibitor group and a miR-141 inhibitor control group. The miR-141 was transfected into hepal-6 cells with lipofectamine 2000. The levels of miR-141 and HMGB1 mRNA in the hepal-6 cells were detected by quantitative real-time PCR (qRT-PCR), and then HMGB1 protein was examined by Western blot. The regulatory effect of miR-141 on 3'-UTR of candidate target gene (HMGB1) was determined by dual-luciferase reporter assay.

Results: Compared with the control group, the level of miR-141 was up-regulated in the miR-141 mimic group, while down-regulated in the miR-141 inhibitor group. Moreover, the levels of HMGB1 mRNA and protein decreased in the miR-141 mimic group, while increased in the miR-141 inhibitor group. The dual-luciferase reporter assay showed that miR-141 could target the 3'-UTR of HMGB1 gene.

Conclusion: MiR-141 can up-regulate the expression of HMGB1 protein at the post-transcriptional level by targeting to the specific sequence of 3'-UTR of HMGB1 gene, which suggests that HMGB1 gene may be a target gene of miR-141.

目的:探讨微小RNA-141(miR-141)对小鼠肝癌细胞株hepal-6高迁移率族蛋白B1(high mobility group protein B1,HMGB1)表达的调控作用。方法:将小鼠肝癌细胞株hepal-6分为miR-141拟似物转染组、miR-141拟似物转染对照组、miR-141抑制剂转染组、miR-141抑制剂转染对照组。分别用脂质体lipofectamine 2000将miR-141转染hepal-6细胞。用实时定量PCR (quantitative real-time PCR,qRT-PCR)检测miR-141和HMGB1 mRNA表达,然后用Western印迹检测HMGB1蛋白表达。采用双荧光素酶报告基因检测miR-141对靶基因HMGB1的调控作用。结果:与相应对照组比较,miR-141拟似物转染组中miR-141表达水平上调,而miR-141抑制剂转染组中miR-141表达水平下调。同时,与相应对照组比较,miR-141拟似物转染组中HMGB1 mRNA和蛋白表达明显下调,而miR-141抑制剂转染组中HMGB1 mRNA和蛋白表达均明显上调。双荧光素酶报告基因分析表明miR-141能够作用于HMGB1基因的3'-UTR。结论:miR-141可以作用于HMGB1基因的3'-UTR,在转录后水平可上调HMGB1蛋白的表达,HMGB1可能是miR-141直接调控的靶基因。.

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Carcinoma, Hepatocellular*
  • Cell Line, Tumor
  • Down-Regulation
  • Gene Expression Regulation, Neoplastic
  • HMGB1 Protein
  • Liver Neoplasms*
  • Luciferases
  • Mice
  • MicroRNAs
  • RNA, Messenger
  • Real-Time Polymerase Chain Reaction
  • Transfection
  • Up-Regulation

Substances

  • 3' Untranslated Regions
  • HMGB1 Protein
  • HMGB1 protein, mouse
  • MicroRNAs
  • RNA, Messenger
  • Luciferases