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

Peipei Wang,Liang Peng,Zhiliang Gao,Chan Xie,Xiaojuan Wu,Song Chen,He Fang,Dabiao Chen

doi:10.18632/oncotarget.6137

Abstract

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. 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. 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. Our results demonstrate that miR-214 has tumor-suppressive activity in HCC through inhibition of E2F2, CDK3 and CDK6.

Highlights

Primary human hepatocellular carcinoma (HCC) remains a major public health threat and the third leading cause of cancer death [1]
Our results demonstrate that miR-214 has tumor-suppressive activity in HCC through inhibition of E2F2, CDK3 and CDK6
Real-time PCR analysis revealed that miR-214/199a/199a* was downregulated in all 14 tested HCC cell lines compared with the immortalized normal liver epithelial cell line THLE3 (Figure 1B) and in 8 HCC samples compared with the matched adjacent nontumor tissues (Figure 1C)

Summary

Introduction

Primary human hepatocellular carcinoma (HCC) remains a major public health threat and the third leading cause of cancer death [1]. It is of great clinical value to investigate the cooperative effect of multiple genes involved in HCC at the same time, and develop effective therapeutic targets for achieving better prognosis of the disease. One of the hallmarks of cancer is uncontrolled cell proliferation, leading to malignant tumor development. The cell cycle is activated by CDK6 early in G1 phase through interactions with cyclins D1 [5], D2 and D3. Distinct cyclins and CDKs play crucial roles in the development and progression of HCC, the effect is often overridden by negative regulators [10,11,12]. Understanding the interactions between multiple cell cycle regulating genes involved in HCC may lead to more effective therapeutic strategies

Methods

Results

Conclusion