MiR-222 overexpression promotes proliferation of human hepatocellular carcinoma HepG2 cells by downregulating p27 - PubMed (original) (raw)

. 2014 Apr 15;7(4):893-902.

eCollection 2014.

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MiR-222 overexpression promotes proliferation of human hepatocellular carcinoma HepG2 cells by downregulating p27

Yue-Feng Yang et al. Int J Clin Exp Med. 2014.

Abstract

Objective: Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related death worldwide. Increasing evidence suggests that microRNAs, a novel class of non-coding RNAs that function as endogenous suppressors of gene expression, are deregulated in HCC. Although microRNA-222 (miR-222) overexpression has been described in HCC, the role of miR-222 and its target genes in the proliferation of hepatocellular carcinoma cells remain poorly elucidated.

Methods: HepG2 cells were transfected with miR-222 mimic, inhibitor or their negative controls. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and EdU incorporation assay. Flow cytometry was performed to assess the effects of miR-222 on HepG2 cell cycle progression. MiR-222 and putative targets genes (p27 and p57) expression levels were determined using qRT-PCR and/or Western blot.

Results: MiR-222 overexpression induced an enhancement of HepG2 cell proliferation in vitro, paralleling with an altered cell cycle progression via increased cell population in S phase. P27 expression, other than p57, was negatively regulated by miR-222 overexpression at post-transcriptional level in HepG2 cells. Transfection of either small interfering RNA (siRNA) for p27 or miR-222 mimic increased HepG2 cell proliferation rate, whereas co-transfection of p27 siRNA and miR-222 mimic did not further enhance HepG2 cell proliferation in comparison with the cells transfected with p27 siRNA or miR-222 mimic alone, validating that p27 is a target gene of miR-222 during HepG2 cell proliferation.

Conclusion: This study suggests that miR-222 overexpression promotes HepG2 cell proliferation by downregulating p27.

Keywords: Hepatocellular carcinoma; MicroRNA-222; P27; proliferation.

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Figures

Figure 1

Figure 1

MiR-222 overexpression promotes HepG2 cell proliferation. A: The expression level of miR-222 was significantly increased by miR-222 mimic, while decreased by miR-222 inhibitor in HepG2 cells (n = 4). B: MiR-222 mimic increased HepG2 cell proliferation, while miR-222 inhibitor decreased their proliferation (n = 6). **P < 0.01.

Figure 2

Figure 2

Quantitative analysis of EdU-positive cells confirms that miR-222 overexpression promotes HepG2 cell proliferation. A: MiR-222 overexpressing cells were 39% EdU-positive compared with 26% in negative control cells (n = 4). B: MiR-222 inhibiting cells were 16% EdU-positive compared with 27% in negative control cells (n = 4). **P < 0.01.

Figure 3

Figure 3

MiR-222 regulates HepG2 cell cycle progression. A: MiR-222 mimic increased the percentage of HepG2 cells in S phase and decreased that in G2 phase (n = 3). B: MiR-222 inhibitor decreased the percentage of HepG2 cells in S phase and increased that in G2 phase (n = 3). **P < 0.01.

Figure 4

Figure 4

P27 is a target gene of miR-222 in HepG2 cells. A: MiR-222 mimic or inhibitor did not regulate p27 or p57 expression at mRNA level (n = 4). B: P27 protein expression, other than p57, was negatively regulated by miR-222 in HepG2 cells (n = 4). C: Transfection of p27 siRNA led to efficient knockdown of p27 in HepG2 cells (n = 4). D: The cells transfected with either p27 siRNA or miR-222 mimic had higher proliferation rate than negative control cells, while co-transfection of p27 siRNA and miR-222 mimic did not further enhance cell proliferation compared with the cells transfected with p27 siRNA or miR-222 mimic alone (n = 6). **P < 0.01.

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