Original Article
Copyright ©2014 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. May 14, 2014; 20(18): 5493-5504
Published online May 14, 2014. doi: 10.3748/wjg.v20.i18.5493
Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism
Zhong-Hui Cui, Shi-Qiang Shen, Zu-Bing Chen, Chao Hu
Zhong-Hui Cui, Shi-Qiang Shen, Zu-Bing Chen, Chao Hu, Department of General Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
Author contributions: Cui ZH performed the majority of experiments and contributed to this work; Chen ZB designed the study; Hu C contributed analytic tools; Cui ZH analyzed data and wrote the paper; and Shen SQ were involved in editing the manuscript.
Correspondence to: Shi-Qiang Shen, MD, Department of General Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China. swsw2212@sina.com
Telephone: +86-27-88041911 Fax: +86-27-88041911
Received: October 28, 2013
Revised: January 3, 2014
Accepted: February 26, 2014
Published online: May 14, 2014
Processing time: 197 Days and 15.9 Hours
Abstract

AIM: To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma (HCC) tumor endothelial cells (TECs).

METHODS: Flow cytometry was used to identify HCCTECs and analyze their purity. Differentially expressed miRNAs in HCC TECs as compared to normal hepatic sinusoidal endothelial cells (HSECs) were examined using the HmiOA v4 Human miRNA OneArray® microarray. miR-204-3p showed the most significant decrease in expression and was further studied. Over-expression of miR-204-3p was achieved using lentiviral transduction into TECs of HCC. The biological changes in HCC TECs before and after transduction were detected using MTT and apoptosis assays. The association between miR-204-3p and fibronectin 1 (FN1) was determined using the dual luciferase activity assay. Changes in FN1 protein expression before and after transduction were detected using Western blot analysis.

RESULTS: Microarray results showed that compared to normal HSECs, 15 miRNAs were differentially expressed in HCC TECs, including 6 miRNAs with increased expression and 9 miRNAs with decreased expression. Among them, miR-204-3p showed the most significant decrease in expression (log2 = -1.233477, P = 0.000307). Over-expression of miR-204-3p in HCC TECs via lentiviral transduction significantly inhibited the proliferation of HCC TECs and promoted apoptosis. Results from the dual luciferase activity experiment showed that the luciferase intensity in the wild type FN1 group was significantly inhibited (P < 0.05), while that in the mutant FN1 group was not obviously affected. This observation indicated that FN1 was one of the potential targets of miR-204-3p. After over-expression of miR-204-3p in HCC TECs, Western blot analysis showed that the expression of FN1 protein was significantly inhibited.

CONCLUSION: MiR-204-3p acts on its potential target gene, FN1, and inhibits its expression, thus blocking the adhesion function of FN1 in promoting the growth of TECs.

Keywords: Tumor vascular endothelial cells of hepatocellular carcinoma; Hepatic sinusoidal endothelial cells; MiRNA microarray; Mir-204-3p; Fibronectin 1

Core tip: This study first employed a microarray to detect differentially expressed miRNAs in hepatocellular carcinoma (HCC) tumor endothelial cells (TECs), as compared to hepatic sinusoidal endothelial cells with the goal of identifying specific miRNAs that play important roles in the angiogenesis of HCC. Our study proved that fibronectin 1 (FN1) is a potential target gene of miR-204-3p, suggesting that FN1 regulates the growth of HCC TECs via the miR-204-3p/FN1 signaling pathway. The underlying mechanism was also investigated to provide new targets and a theoretical basis for the anti-angiogenic gene therapy of HCC.