miR-214/199a/199a* cluster levels predict poor survival in hepatocellular carcinoma through interference with cell-cycle regulators.

// Peipei Wang 1, 2, * , Song Chen 3, * , He Fang 4, * , Xiaojuan Wu 1 , Dabiao Chen 1 , Liang Peng 1 , Zhiliang Gao 1, 2 , Chan Xie 1, 2 1 Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China 2 Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong Province, China 3 Department of Radiology, Guangzhou Red Cross Hospital/The Fourth Affiliated Hospital of Jinan University Medical College, Guangzhou, Guangdong Province, China 4 Department of Burn Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China * These authors have contributed equally to this work Correspondence to: Chan Xie, e-mail: happyxiechan@hotmail.com Zhiliang Gao, e-mail: gaozl@yahoo.com.cn Keywords: microRNA, human hepatocellular carcinoma, cell cycle Received: July 23, 2015      Accepted: September 30, 2015      Published: October 19, 2015 ABSTRACT Aims: To identify the clinical and functional association of miR-214/199a/199a* cluster in human hepatocellular carcinoma (HCC) and to clarify the mechanism of miR-214. Methods: Kaplan-Meier and Cox proportional regression analyses were used to determine the association of miR-214/199a/199a* cluster levels with the survival of HCC patients. The role of miR-214 in regulating HCC cell proliferation was studied with miR-214 mimics/inhibitor-treated cells. Furthermore, the inhibition effect of miR-214 on E2F2, cyclin-dependent kinase (CDK) 3 and CDK6 expression was assessed in HCC cell lines with miR-214 mimics/inhibitors to increase/decrease miR-214 expression. Direct binding of miR-214 to the 3′-untranslated regions of E2F2, CDK3, and CDK6 was verified by dual-luciferase reporter assay. Results: In analyzing HCC clinical specimens and cell lines, we discovered a uniform decrease in miR-214/199a/199a* expression in comparison with noncancerous tissue or normal liver epithelial cell lines. Higher miR-214 levels were related with improved patient survival. Overexpression of miR-214 in HCC cells inhibited proliferation by inducing G1-S checkpoint arrest. Conversely, RNA interference–mediated silencing of miR-214 promoted cell-cycle progression and accelerated the proliferation of HCC cells. E2F2, CDK3 and CDK6 were each directly targeted for inhibition by miR-214, and restoring their expression reversed miR-214 inhibition of cell-cycle progression. The relationship between expression of miR-214 and its targets was confirmed in HCC tumor xenografts and clinical specimens. Conclusions: Our results demonstrate that miR-214 has tumor-suppressive activity in HCC through inhibition of E2F2, CDK3 and CDK6.