Lentiviral-mediated miRNA against osteopontin suppresses... : Hepatology (original) (raw)

Hepatobiliary Malignancies

Lentiviral-mediated miRNA against osteopontin suppresses tumor growth and metastasis of human hepatocellular carcinoma

Sun, Bing-Sheng1; Dong, Qiong-Zhu1; Ye, Qing-Hai1*; Sun, Hai-Jing1; Jia, Hu-Liang1; Zhu, Xiao-Qun1; Liu, Dao-Yong1; Chen, Jie1; Xue, Qiong1; Zhou, Hai-Jun1; Ren, Ning1; Qin, Lun-Xiu1*

1_Liver Cancer Institute and Zhongshan Hospital, Institutes of Biomedical Science, Fudan University, Shanghai, China_

* Address reprint requests to: Liver Cancer Institute & Zhongshan Hospital, Fudan University, 180 Feng Lin Road, Shanghai 200032, China

Email:[email protected]

Email:[email protected][email protected]

Received 29 January 2008; Accepted 7 July 2008

Published online in Wiley InterScience (www.interscience.wiley.com).

Supported by China National Natural Science Foundation for Distinguished Young Scholars; Grant Number: 30325041; Grant sponsor: China National “863” Project; Grant Number: 2006AA02Z473; Grant sponsor: Shanghai Science and Technology Developing Program; Grant Number: 03DZ14024; Grant sponsor: Foundation for Outstanding Scholars in New Era of the Ministry of Education of China.

Potential conflict of interest: Nothing to report.

These authors contributed equally to this study.

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Abstract

In our previous study, osteopontin (OPN) was identified as one of the leading genes that promote the metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which OPN promotes metastasis of HCC is not understood. In this study, RNA interference mediated by viral vectors—which could induce a long-lasting down-regulation in gene expression—was applied to analyze the role of OPN in metastasis of HCC. Three lentiviral vectors encoding microRNA against OPN, Lenti.OPNi-1, Lenti.OPNi-2, and Lenti.OPNi-3, were constructed and found to down-regulate the OPN level by 62%, 78%, and 95%, respectively, in HCCLM3 cells which had an overexpression of OPN and a higher metastatic potential. Consequently, both Lenti.OPNi-2 and Lenti.OPNi-3 induced a significant decrease in matrix metalloproteinase (MMP)-2 and urokinase plasminogen activator expression, and led to an obvious inhibition of both in vitro invasion and in vivo lung metastasis of HCCLM3 cells ( P < 0.001). Moreover, Lenti.OPNi-3, rather than Lenti.OPNi-2, could also suppress** **_in vitro_** **proliferation and** **_in vivo_** **tumor growth of HCCLM3. Smaller detectable tumors were found in only 50% of mice after implantation of Lenti.OPNi-3–transfected HCCLM3 cells (341 ± 502.6 mm3 versus >3500 mm3 in controls; P < 0.001). Lenti.OPNi-3, not Lenti.OPNi-2, significantly suppressed the MEK/ERK1/2 pathway in HCCLM3 cells. Recombinant OPN was found to induce translocation of p65 into the nucleus of HCC cells and activation of MMP-2 and MEK/ERK/1/2, which were suppressed by the nuclear factor κB (NF-κB) inhibitor pyrrolidine dithiocarbamate. Conclusion: OPN plays an important role in metastasis as well as tumor growth of HCC, in which different minimum threshold levels of OPN are needed. These effects may occur through activation of the mitogen-activated protein kinase and NF-κB pathways, and MMP-2. OPN could be a hopeful target for the control of HCC. (Hepatology 2008;48:1834-11842.)

Abbreviations: GFP, green fluorescent protein; HCC, hepatocellular carcinoma; IFN, interferon; miRNA, microRNA; MMP, matrix metalloproteinase; MOI, multiplicity of infection; NF-κB, nuclear factor κB; OPN, osteopontin; PDTC, pyrrolidine dithiocarbamate; RNAi, RNA interference; uPA, urokinase plasminogen activator.