209 MICRORNA-519D IS UP-REGULATED IN HEPATOCELLULAR CARCINOMA AND SENSITIZES HCC CELLS TO DOXORUBICIN TREATMENT BY TARGETING CDKN1A/P21

Abstract

Background: MicroRNAs (miRNAs) are aberrantly expressed in cancers and evidences support their potential use in cancer treatment. MiR-519d belongs to the Chromosome 19 MiRNA Cluster (C19MC), which was recognized as overexpressed in placenta and breast cancer. This study aims to assay miR-519d expression pattern in Hepatocellular Carcinoma (HCC), to identify miR-519d targets and to characterize its role in hepatocarcinogenesis and response to anti-cancer treatments. Methods: Real-Time RT-PCR analysis of miR-519d was performed in 56 HCCs and cirrhosis, in normal livers and HCC cells. Transfection experiments with miR-519d or anti-miR-519d were performed in HCC cells and modulation of CDKN1A/p21 expression was evaluated by western blot (WB) and RT-PCR. Luciferase assay was performed to evaluate the interaction between miR-519d and 3’UTR region of CDKN1A/p21. Cell cycle modulation was analysed by FACS in miR-519d overexpressing and CDKN1A/p21 silenced cells. Apoptotic markers were evaluated by WB and FACS analysis in miR-519d and CDKN1A/p21 siRNA transfected cells following Doxorubicin treatment. Results: MiR-519d is overexpressed in 51.7% of HCCs. Upon miR-519d enforced expression, a decrease of CDKN1A/p21 was observed in HepG2 cells. Conversely, miR-519d silencing determined an increase of CDKN1A/p21 in SNU475 cells. Co-transfection of CDKN1A/p21-3’UTR reporter vector with miR-519d determined a decrease of the luciferase activity in HepG2 cells, proving a direct interaction between miR-519d and CDKN1A/p21 3’UTR. Cell cycle analysis showed a decrease of G1 phase in both miR-519d overexpressing and CDKN1A/p21 silenced HepG2 cells. Conversely, a cell cycle arrest in G1 phase was observed following miR-519d silencing in SNU475 cells. Doxorubicin treatment determined an increased apoptotic cell death, assessed by FACS-Annexin V and WB, following miR-519d overexpression and CDKN1A/p21 inhibition in HepG2 cells. Conclusions: Here we report the overexpression of miR-519d in HCC and identify CDKN1A/p21 among its targets. We provide evidences that miR-519d exerts an oncogenic activity by promoting proliferation of HCC cells through CDKN1A/p21 inhibition. We demonstrated that miR-519d overexpression is associated with a stronger response to Doxorubicin, suggesting miR-519d as a promising molecular marker for the optimization of HCC treatment.