PKCalpha mediated induction of miR-101 in human hepatoma HepG2 cells

Abstract

Background: Protein Kinase C (PKC) is a serine/threonine kinase that involved in controlling of many cellular processes such as cell proliferation and differentiation. We have observed previously that TPA (12-O-tetradecanoylphorbol 13-acetate) induces cell cycle arrest in G0/G1 phase in human hepatoma HepG2 cells. However, is there any miRNA involved in PKCalpha mediated cell growth arrest is still unknown.

Methods: We first surveyed 270 miRNA expression profiles in 20 pairs of human hepatoma tissues. We identified 11 up-regulated and 23 down-regulated miRNAs (FDR < = 0.01; fold-change > = 2) in human hepatoma tissue after Student's T-test and Mann-Whitney rank test. We then examined miRNAs expression profile in TPA treated HepG2 cells. Two miRNAs, miR-101, and miR-29c, were shown to be significantly down regulated in human hepatoma tissues and induced over 4-fold in HepG2 cells under TPA treatment.

Results: In this study, we examined TPA regulated miRNA expression profile in human hepatoma HepG2 cells. We identified two miRNAs, 101 and 29c, were induced by TPA and down regulated in human hepatoma tissues suggest that they might play as tumor suppressor gene and in tumor formation of HCC. Since induction kinetics of miR-101 by TPA was much faster than miR-29c suggests that the induction of miR-101 may be the primary response of TPA treatment. We then further investigated how miR-101 was regulated by TPA. MiR-101 targets two subunits of PRC2 complex, enhancer of zeste homolog 2 (EZH2) and EED, and was shown to play as a tumor suppressor gene in human prostate, breast and liver cancers. The target sequence of miR-101 located in the 3' UTR of both EZH2 and EED's mRNA was identified by bioinformatic analysis and was validated by reporter luciferase activity assay. Then we showed that TPA not only up regulated miR-101 expression, but also reduced protein level of EZH2, EED and H3K27me3 in HepG2 cells. Using lenti-virus-mediated shRNA to knockdown endogenous PKCalpha expression, we observed that TPA induced growth arrest, elevation of miR-101 and reduction of EZH2, EED and H3K27me3 proteins were all PKCalpha dependent. Specific inhibitor of ERK completely blocked TPA induced miR-101 expression.

Conclusions: Therefore, this is the first time to show that PKCalpha and ERK pathway play important role to activate miR-101 expression, reduce PRC2 complex and H3K27me3 level. This epigenetic regulatory pathway may represent a novel mechanism of carcinogenesis and deserve further investigation.

Figure 1

TPA-induced growth arrest in HepG2 is PKCα-dependent. (A) 1 × 10⁵ of parental HepG2 and PKCα knockdown HepG2 cells were seeded in 24 well plates. After culturing in serum-free medium for 24 hrs, cells were treated with TPA 100 nM in serum-free medium for indicated time before MTT analysis. The cell proliferation rate were analyzed using Day6 as 100%. Results shown are averages of three independent experiments performed in triplicates. (B) The expression profile of different PKC isoforms in parental HepG2 and HepG2 PKCα knockdown stable lines were examined by Western blot analysis.

Figure 2

Identification of key regulatory miRNA in TPA induced growth arrest in HepG2 cells. (A) Expression levels of 270 miRNAs in 20 pairs of human HCC tissues. The labeled miRNAs were inversely modulated in TPA-treated HepG2 cells and dotted lines indicate the 2-fold change threshold. Expression levels of miRNA were presented as 39-Ct. (B) Expression levels of miR-29c and miR-101 in 20 pair of human HCC tissues and their adjacent normal tissues. Expression levels of miRNA were presented as 39-Ct. p-values were calculated using T-test. (C) Time-dependent changes in miR-101, miR-29c and miR-122 expression levels. HepG2 cells were treated with 100 nM TPA for indicated time periods and the total RNAs were collected for stem-loop RT-qPCR. Expression levels of miR-101, miR-29c and miR-122 were normalized to miR-16 and expressed as fold-change using time 0 as baseline.

Figure 3

TPA-induced miR-101 and its downstream effects are all PKCα-dependent in HepG2 cells. (A) HepG2 and HepG2 PKCα knockdown cells were treated with 100 nM TPA for 48 hours. Expression levels of miR-101 was normalized to miR-16 and expressed as fold change using DMSO-treated sample as baseline. (B) Alignment of miR-101 sequence and the predicted miR-101 target sites in the 3'UTR of EZH2 and EED. (C) HEK293 and HepG2 cells were co-transfected either pMIR-REPORT constructs of EED-3'UTR(+1~+ 211) and EZH2-3'UTR(+1~+263) 10 ng with 4 μg of miR-LacZ, miR-101-1, and miR-101-2 for 48 hours. Forty-eight hours after transfection, cells were harvest for luciferase activity analysis. Results shown are averages of three independent experiments performed in triplicate. (D) HepG2 and HepG2 PKCα knockdown cells were treated with 100 nM TPA for 48 hours. Cell lysates were collected for immunoblotting assay using the PKCα, EZH2, SUZ12, EED, p21, and H3K27me3 antibodies.

Figure 4

TPA-induced miR-101 in HepG2 is mediated by ERK signaling pathway. (A) HepG2 cells were cultured serum-free medium for 24 hrs, pre-treatment of specific MAPK signaling inhibitors U0126 10 μM for 0.5 h, and treated with TPA 100 nM for 8 hrs. Total RNAs were isolated from indicated samples with standard procedures. The expression level of miR-101 was normalized to miR-16 and expressed as fold change using control sample as baseline. Results shown are averages of three independent experiments performed in triplicates. (B) Protein levels of p-ERK, ERK, p21 and β-actin after TPA and ERK inhibitor treatment.