MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3 - PubMed (original) (raw)

MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

Guangxian Xu et al. BMC Cancer. 2013.

Abstract

Background: microRNA 21 (miR-21) has been demonstrated to be significantly elevated in many types of cancers, including the hepatocellular carcinoma (HCC). In the present study, we investigated the role of miR-21 in HCC by identifying its novel targets, as well as its underlying molecular mechanism.

Methods: The expression of mitogen-activated protein kinase-kinase 3 (MAP2K3) in human HCC tumor tissues and adjacent non-tumor tissues was determined by immunohistochemistry staining (IHC) analysis. The 3'-untranslated region (3'-UTR) of MAP2K3 combined with miR-21 was experimentally verified by a miRNA luciferase reporter approach. Moreover, the role of miR-21 in regulating HCC cell proliferation was analyzed by an MTT assay infected with miR-21mimics/sponge inhibitor Adenoviral viral vectors.

Results: By immunohistochemistry staining analysis, we found that mitogen-activated protein kinase-kinase 3 (MAP2K3) was strikingly repressed in the human HCC tumor tissues, in comparison with the adjacent non-tumor tissues in clinical settings. More importantly, the repression of MAP2K3 was inversely correlated with the expression of miR-21 in HCC. Further study demonstrated that the MAP2K3 was a novel direct target of miR-21, which was experimentally validated by a miRNA luciferase reporter approach. In HepG2 cells, inhibition of miR-21 expression with an adenoviral miR-21 sponge vector profoundly suppressed cell proliferation by up-regulating MAP2K3 expression at both mRNA and protein levels.

Conclusions: These results provide a clinical evidence that MAP2K3 may be a tumor repressor gene, and it is a direct target of miR-21 in HCC, indicating an underlying mechanism by which miR-21 is able to directly target MAP2K3 and inhibit its expression during the carcinogenesis of HCC, at both transcriptional and post-translational levels. This study also suggests that targeting miR-21-MAP2K3 pathway may be a promising strategy in the prevention and treatment of HCC.

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Figures

Figure 1

Figure 1

Immunohistochemistry (IHC) staining determined MAP2K3 expression in human HCC tumor and matched adjacent tissues. A-D: Representative images of MAP2K3 protein expression determined by IHC staining. A: An image represented a negative (-) expression of MAP2K3 expression; B: An image represented a low level (+) expression of MAP2K3, which showed a weak immunoreactive staining in cytoplasm; C: An image represented a negative (++) expression of MAP2K3 expression; D: An image represented a high level (+++) expression of MAP2K3, which exhibited a strong IHC staining in cytoplasm and perinuclear localization (arrowhead). E: Semi-quantitative analysis of MAP2K3 protein expression using integrated absorbance (IA) in human HCC tissues. Value was expressed as the average values from each individual sample of HCC tumor tissues or its matched adjacent tissue. The total average value of IA in the HCC tumor tissues was significantly greater as compared with the matched adjacent tissues (p<0.05, n = 14). Data was expressed as mean ± SD for 14 sets of samples.

Figure 2

Figure 2

Validation of MAP2K3 mRNA as a target of miR-21. (A): Sequence of potential binding site of miR-21 in the 3’UTR of MAP2K3 mRNA (top panel), mutations were introduced into the binding site for generation of mutated MAP2K3 3’TUR (bottom panel). (B and C): Validation of miR-21 target using MAP2K3 3’UTR luciferase reporter. Cells co-transfected with pMIR-Report/MAP2K3 3’UTR (WT) or pMIR-Report/Mut-MAP2K3 3’UTR (Mut) and pAd/pri-miR-21 (B), pAd/miR-21/inhibitor (C), and pAd/con plasmids showed a decreased luciferase activity in pAd/pri-miR-21 cells (B). Luciferase activity after site directed mutagenesis of the 3’UTR of MAP2K3 mRNA in the miR-21 seed sequence (pMIR-Report/Mut-MAP2K3) was significantly higher with respect to the pMIR-Report/MAP2K3 vector transfected cells (B and C). Results represented the mean ± SD from three independent triplicated experiments (N=9).

Figure 3

Figure 3

miR-21 expression in HepG2 cells. The expression of miR-21 in HepG2 cells infected with Ad/pri-miR-21, Ad/miR-21/inhibitor or Ad/con virus was determined by a qRT-PCR assay. Significantly increased expression of miR-21 was detected in the cells infected with adenovirus Ad/pri-miR-21 (p<0.05); in contrast, a significantly decreased expression of miR-21 was observed in Ad/miR-21/inhibitor infected cells (p<0.05). Compared with pAd/con group, *: p<0.05. Results represented the mean ± SD from three independent triplicated experiments (N=9).

Figure 4

Figure 4

miR-21 targets MAP2K3 mRNA. The HepG2 cells were infected with Ad/pri-miR-21, Ad/miR-21/inhibitor or Ad/con adenoviral vector. The expression of MAP2K3 was detected by immunoblotting analysis against anti-MAP2K3 antibody. Compared with Ad/con group, *: p<0.05. Data in A represented the mean ± SD from three independent triplicated experiments (N=9).

Figure 5

Figure 5

Impact of miR-21 on HepG2 cell proliferation. HCC HepG2 cells were infected with Ad/miR-21/inhibitor (miR-21 sponges) or pAd/con virus at MOI 0f 10, the index of cell proliferation was assessed by an MTT assay from 24 to 72 hours. Compared with Ad/con group, *: p<0.05. Data in A represented the mean ± SD from three independent triplicated experiments (N=9).

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