miR-224 functions as an onco-miRNA in hepatocellular carcinoma cells by activating AKT signaling - PubMed (original) (raw)
miR-224 functions as an onco-miRNA in hepatocellular carcinoma cells by activating AKT signaling
Donglai Ma et al. Oncol Lett. 2012 Sep.
Abstract
microRNAs (miRNAs) are a class of small non-coding RNAs that post-transcriptionally regulate gene expression. Increasing evidence has shown that the deregulation of miRNAs is linked to cancer. The overexpression of miR-224 has been reported in several human cancers. The aim of the present study was to investigate the function of miR-224 in the pathogenetic process of hepatocellular carcinoma (HCC), and the precise mechanism underlying its function. Both gain-of-function and loss-of function assays were conducted through transfection with miR-224 mimics and miR-224 inhibitors in the HepG2 liver carcinoma cell line. The data revealed that miR-224 exerts a significant role in promoting cell proliferation, migration and invasion. Western blot analysis showed that the phosphorylation levels of AKT positively correlated with endogenous levels of miR-224. In addition, results from a dual luciferase reporter assay showed that the expression of the serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A β isoform (PPP2R1B) is inhibited by miR-224; thus, it appears that PPP2R1B is a candidate target of miR-224 in HCC. These data suggest that miR-224 plays a significant role in HCC, possibly through the activation of the AKT signaling pathway by targeting PPP2R1B.
Keywords: AKT signaling; PPP2R1B; hepatocellular carcinoma; miR-224.
Figures
Figure 1
miR-224 regulates HepG2 cell proliferation. Semi-quantitative real-time PCR was performed to measure miR-224. (A) The expression level of miR-224 (normalized to U6 RNA) was significantly decreased by 85% in the miR-224 inhibitor group relative to the scrambled group; (B) miR-224 was notably upregulated by ~14 times in HepG2 cells treated with miR-224 mimics compared to scrambled oligonucleotides. Viable cells were detected by the CCK-8 assay 24, 48 and 72 h after transfection; (C) Cell proliferation was markedly suppressed by downregulation of miR-224 levels compared to the controls; (D) Viable cells in the miR-224 mimic group were significantly higher than in the control groups. Data are expressed as the mean of three separate experiments ± SD. *P<0.05. As-miR-224, antisense miR-224.
Figure 2
miR-224 promotes cell invasion and migration of HepG2 cells. Cell invasion and migration assays were carried out. Forty-eight hours after transfection, miR-224 inhibitors, miR-224 mimics, the two scrambled oligonucleotides and the control HepG2 cells were subjected to the Transwell assay, then incubated for another 36 h (for invasion assay) or 24 h (for migration assay). The number of cells stained with crystal violet was counted under a microscope in five random high-power fields. The average cell number was obtained from three independent experiments. (A) Representative images of the invasive cells fixed and stained on the membrane. (B) Average number of cells that migrated through the matrigel into the lower surface of the membrane in the different experimental groups. (C) Parallel results were obtained in the migration assay. Data are expressed as the mean ± SD. *P<0.05. As-miR-224, antisense miR-224.
Figure 3
miR-224 modulates AKT signaling by targeting PPP2R1B. (A) Schematic representation of the putative binding sites in PPP2R1B 3′UTR for has-miR-224 seeding sequences. (B) Strong inhibition of luciferase activity was detected in HepG2 cells upon transfection of pPPP2R1B 3′UTR-luci-WT compared to cells transfected with either pPPP2R1B 3′UTR-luci-MUT or vector. (Bi) Firefly luciferase activity was standardized to Renilla construct. (Bii) Through downregulation of miR-224 levels with the miR-224 inhibitor, a clear increase in luciferase activity was observed in HepG2 cells upon transfection of pPPP2R1B 3′UTR-luci-WT, while no significant changes were detected in cells transfected with either pPPP2R1B 3′UTR-luci-MUT or vector. Columns, mean of three independent experiments; error bars, standard deviation. *P<0.05. (C) PPP2R1B, total AKT and phosphorylated AKT protein levels were detected by western blot analysis; protein levels were normalized to β-actin. The results suggested that miR-224 reduced the expression of PPP2R1B protein, and restoration of PPP2R1B was observed in miR-224 inhibitor-transfected HepG2 cells, compared with the control group. In addition, a reduction in phosphorylated AKT (but not total AKT) levels was detected in HepG2 cells treated with miR-224 mimics relative to the control group. (D) Schematic description of the possible molecular mechanism of miR-244 in HCC cells. As-miR-224, antisense miR-224.
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