MiR-520b targeted MEKK2 and cyclin D1 contribute to hepatoma cell growth <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The expression levels of MEKK2 and cyclin D1 were examined by western blot analyses in HepG2 and H7402 cells treated with two different siRNAs targeting MEKK2 (termed siMEKK2-1 and siMEKK2-2) or two different siRNAs targeting cyclin D1 (termed sicyclinD1-1 and sicyclinD1-2). (B) The effect of transient transfection of siRNAs targeting MEKK2 (siMEKK2-1 or siMEKK2-2) or cyclin D1 (sicyclin D1-1 or sicyclin D1-2) on the growth of HepG2 and H7402 cells was examined by colony formation assays. (C) The effect of transient transfection of sicyclin D1-1 or siMEKK2-1 on the growth of HepG2 and H7402 cells was examined by EdU incorporation assays. (D) HepG2 and H7402 cells were transfected with miR-NC, miR-520b, NC, sicyclin D1-1 and/or siMEKK2-1, respectively. The effects of miRNAs or siRNA targeting cyclin D1 or MEKK2 on hepatoma cell proliferation were determined by MTT assays at 24 h, 48 h and 72 h after transfection. *<i>P</i><0.05, **<i>P</i><0.01, Student's <i>t</i> test. (E) Tumor growth measured after subcutaneous injection of HepG2 cells transient transfected with NC, siMEKK2-1 or sicyclinD1-1. The tumor volume was calculated every 3 days. Points, mean (n = 6); bars, SD.</p

Cyclin D1 overexpression rescues miR-520b depressed growth of hepatoma cells.

<p>(A) The effect of miR-520b or sicyclinD1-1 on phosphorylation levels Rb (p-Rb) was examined in HepG2 and H7402 cells by western blot analyses. (B) Western blot analyses showed the expression levels of cyclin D1 and p-Rb in HepG2 and H7402 cells treated by miR-520b or both pcDNA3-cyclin D1 and miR-520b. (C) The effect of cyclin D1 overexpression on the miR-520b-inhibited proliferation of HepG2 cells was examined by flow cytometry analyses. (D) The effect of cyclin D1 overexpression on the miR-520b-inhibited proliferation of HepG2 cells was examined by MTT assays at 24 h, 48 h and 72 h after transfection. *<i>P</i><0.05, ** <i>P</i><0.01, Student's <i>t</i> test.</p

MiR-520b directly inhibits expression of MEKK2 and cyclin D1 <i>via</i> their 3′UTR.

<p>(A) Sequence alignment between miR-520b and the 3′UTR of human MEKK2 and cyclin D1 mRNA. Solid line, seed match region; dashed line, seed-mutated region. (B) The effect of miR-520b on the activity of firefly luciferase reporter containing either wild type (WT) or mutant type (Mut) 3′UTR was tested by luciferase reporter gene assays. (C) The effect of miR-520b or Inh-520b on the endogenous expression levels of MEKK2 and cyclin D1 was examined in HepG2 and H7402 cells by western blot analyses. β-actin was used as an internal control. The intensity for each band was quantified densitometrically.</p

MiR-520b inhibits growth of hepatoma cells <i>in vivo</i>.

<p>(A) The curve of tumor growth is shown. (B) Photograph of excised tumors 4 weeks after implantation. (C) qRT-PCR analysis of miR-520b expression in excised tumors was performed and normalized against an endogenous control (U6 RNA).</p

MiR-520b is downregulated in HCC tissues and hepatoma cell lines.

<p>(A) The relative expression of miR-520b in clinical HCC patients was examined by qRT-PCR. (B) The relative expression of miR-520b in hepatoma cells and normal liver cells was examined by qRT-PCR. *<i>P</i><0.05, **<i>P</i><0.01, Student's <i>t</i> test.</p