MicroRNA-224 down-regulates Glycine N-methyltransferase gene expression in Hepatocellular Carcinoma

Jung-Hsien Hung,Yen-Fu Chen,Chung-Hsien Li,Anya Maan-Yuh Lin,Chih-Hung Chou,Hsien-Da Huang,Chia-Hung Yen,Yu-Chang Tyan,Marcelo Chen,Kuo-Jui Lee,Hsin-Yun Cheng,Ching-Hua Yeh,Pin-Cheng Huang,Ting-Fen Tsai,Ann-Ping Tsou,Cheng-Chieh Fang,Yi-Ming Arthur Chen

doi:10.1038/s41598-018-30682-5

Abstract

Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.

Highlights

Hepatocellular carcinoma (HCC) was the fifth most frequently diagnosed cancer and the second most frequent cause of cancer death in men worldwide in 20121
Identification of miR-224 as a putative target of GNMT. We used both miRWalk[25] and miRTar[26] software to identify potential 112 microRNAs targeting at the coding sequences (CDS) and 3′untranslated region (3′UTR) of human GNMT mRNA (Supplementary Table S1), while up-regulated 67 miRNAs in HCC were found through literature review[17,27,28]
We identified miR-224 as the key regulator of gene expression of GNMT, mapped its seeding site and further created a GNMT cDNA mutant which can resist to the miR-224 regulations

Summary

Introduction

Hepatocellular carcinoma (HCC) was the fifth most frequently diagnosed cancer and the second most frequent cause of cancer death in men worldwide in 20121. We identified a novel PTEN inhibitor-PREX2 as another GNMT-interacting protein We showed that such interaction enhanced degradation of PREX2 through an E3 ligase HectH9-mediated proteasomal ubiquitination pathway[11]. These studies implicate that GNMT is actively involved in maintaining normal liver function and further support the role of GNMT as a tumor suppressor gene in HCC. MiR-224 as an onco-miR plays a role in HCC development and progression based on the fact that it binds to 3′UTR regions of multiple genes that are involved in apoptosis, cell proliferation, migration, invasion and autophagy[18,19,20,21,22,23,24]. We hypothesized that GNMT is regulated by miR-224 in tumorigenesis of HCC

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