MicroRNAs (miRNAs) and methionine adenosyltransferase 1A (MAT1A) are dysregulated in hepatocellular carcinoma (HCC), and reduced MAT1A expression correlates with worse HCC prognosis. Expression of miR-664, miR-485-3p, and miR-495, potential regulatory miRNAs of MAT1A, is increased in HCC. Knockdown of these miRNAs individually in Hep3B and HepG2 cells induced MAT1A expression, reduced growth, and increased apoptosis, while combined knockdown exerted additional effects on all parameters. Subcutaneous and intraparenchymal injection of Hep3B cells stably overexpressing each of this trio of miRNAs promoted tumorigenesis and metastasis in mice. Treatment with miRNA-664 (miR-664), miR-485-3p, and miR-495 siRNAs reduced tumor growth, invasion, and metastasis in an orthotopic liver cancer model. Blocking MAT1A induction significantly reduced the antitumorigenic effect of miR-495 siRNA, whereas maintaining MAT1A expression prevented miRNA-mediated enhancement of growth and metastasis. Knockdown of these miRNAs increased total and nuclear level of MAT1A protein, global CpG methylation, lin-28 homolog B (Caenorhabditis elegans) (LIN28B) promoter methylation, and reduced LIN28B expression. The opposite occurred with forced expression of these miRNAs. In conclusion, upregulation of miR-664, miR-485-3p, and miR-495 contributes to lower MAT1A expression in HCC, and enhanced tumorigenesis may provide potential targets for HCC therapy.
Authors
Heping Yang, Michele E. Cho, Tony W.H. Li, Hui Peng, Kwang Suk Ko, Jose M. Mato, Shelly C. Lu
(A) 5-mC levels in tumors derived from Hep3B cells stably expressing lower (with siRNA or si) or higher levels of miRNAs. *P < 0.05; **P < 0.01; ***P < 0.0001 vs. SC; †P < 0.05 vs. EV. n = 8 per condition. (B) Effect of varying miRNA expression on nuclear H3K27me3 levels in tumors (Western blots) and SAMe levels. Results are mean ± SEM from 8 per condition. *P < 0.05; **P < 0.01 vs. respective controls. (C) Diagram shows HpaII and MspI sites between PvuII and NdeI in human LIN28B promoter. Black squares, CCGG sites; TSS, transcriptional start site. Numbers are relative to TSS. Southern blot analysis of LIN28B promoter region between –1576 and +2432 (right). DNA samples from liver tumor derived from stably transfected Hep3B containing miR-664, miR-485, and miR-495 and their siRNAs were digested as indicated. MspI digestion results in a band size of 1359 bp as control for HpaII digestion. (D) Effect of overexpressing miR-664, miR-485, and miR-495 and their siRNAs on MAT1A and LIN28B protein expression (top) and let-7a mRNA expression (bottom). Numbers below the blots are densitometric values expressed as percentage of respective controls. *P < 0.01 vs. EV; †P < 0.05 vs. miR-495; ‡P < 0.01 vs. SC; §P < 0.05 vs. miR-495si. (E) Increased nuclear localization of MAT1A protein is seen after knockdown of miR-664, miR-485-3p, and miR-495. Original magnification, ×630 (oil immersion).