PKCalpha mediated induction of miR-101 in human hepatoma HepG2 cells.
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.
Chiang
Chao-Wei
CW
Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan.
Huang
Yi
Y
Leong
Ka-Wai
KW
Chen
Lih-Chyang
LC
Chen
Hua-Chien
HC
Chen
Shu-Jen
SJ
Chou
Chen-Kung
CK
D020413:3' Untranslated Regions; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008297:Male; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics*; D063151:Polycomb Repressive Complex 2; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics
D020413:3' Untranslated Regions; D017931:DNA Primers; D004268:DNA-Binding Proteins; C491488:EED protein, human; C534072:MIRN101 microRNA, human; C532045:MIRN29a microRNA, human; D035683:MicroRNAs; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D014157:Transcription Factors; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate
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