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

Chiang CW, Huang Y, Leong KW, Chen LC, Chen HC, Chen SJ, Chou CK - J. Biomed. Sci. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874775&req=5

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.

Mentions: Previous studies have shown that PKCα may play an important role in TPA-induced growth arrest in HepG2 cells [14]. To re-examine whether the PKCα is required for TPA-mediated cell arrest in HepG2 cells, we knocked down the expression of endogenous PKCα in HepG2 cells using a lentiviral-based PKCα shRNA. We found that TPA induced cell arrest in HepG2 cells was largely abolished in PKCα knockdown HepG2 cells (Fig. 1A). The lentiviral-based PKCα shRNA specifically knocked down only PKCα and has no effect on the expression of two other PKC isoforms, PKCδ and PKCε, in the HepG2 cells (Fig. 1B).


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

Chiang CW, Huang Y, Leong KW, Chen LC, Chen HC, Chen SJ, Chou CK - J. Biomed. Sci. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874775&req=5

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.
Mentions: Previous studies have shown that PKCα may play an important role in TPA-induced growth arrest in HepG2 cells [14]. To re-examine whether the PKCα is required for TPA-mediated cell arrest in HepG2 cells, we knocked down the expression of endogenous PKCα in HepG2 cells using a lentiviral-based PKCα shRNA. We found that TPA induced cell arrest in HepG2 cells was largely abolished in PKCα knockdown HepG2 cells (Fig. 1A). The lentiviral-based PKCα shRNA specifically knocked down only PKCα and has no effect on the expression of two other PKC isoforms, PKCδ and PKCε, in the HepG2 cells (Fig. 1B).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan.

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.

Show MeSH
Related in: MedlinePlus