Genome-Wide Analysis of miRNA Signature Differentially Expressed in Doxorubicin-Resistant and Parental Human Hepatocellular Carcinoma Cell Lines

Jufeng Zhang,Ying Wang,Wei Zhang,Lin Zhou,Yang Yang,Pingping Zhen,Yanxin Lu,Jun Wan,Chao Zhang,Xia Luo,Karin Dahlman-Wright

doi:10.1371/journal.pone.0054111

Abstract

Chemotherapy regiments have been widely used in the treatment of a variety of human malignancies including hepatocellular carcinoma (HCC). A major cause of failure in chemotherapy is drug resistance of cancer cells. Resistance to doxorubicin (DOX) is a common and representative obstacle to treat cancer effectively. Individual microRNA (miRNA) has been introduced in the evolution of DOX resistance in HCC in recent studies. However, a global and systematic assessment of the miRNA expression profiles contributing to DOX resistance is still lacking. In the present study, we applied high-throughput Illumina sequencing to comprehensively characterize miRNA expression profiles in both human HCC cell line (HepG2) and its DOX-resistant counterpart (HepG2/DOX). A total of 269 known miRNAs were significantly differentially expressed, of which 23 were up-regulated and 246 were down-regulated in HepG2/DOX cells, indicating that part of them might be involved in the development of DOX resistance. In addition, we have identified 9 and 13 novel miRNAs up- and down-expressed significantly in HepG2/DOX cells, respectively. miRNA profiling was then validated by quantitative real-time PCR for selected miRNAs, including 22 known miRNAs and 6 novel miRNAs. Furthermore, we predicted the putative target genes for the deregulated miRNAs in the samples. Function annotation implied that these selected miRNAs affected many target genes mainly involved in MAPK signaling pathway. This study provides us a general description of miRNA expression profiling, which is helpful to find potential miRNAs for adjunct treatment to overcome DOX resistance in future HCC chemotherapy.

Highlights

Hepatocellular carcinoma (HCC) is one of the most common virus-associated cancers resulting in high mortality worldwide [1]
Cell culture The human hepatocarcinoma cell line HepG2 was obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin at 37uC in a humidified atmosphere of 5% CO2
We determined the miRNA expression profiles that were differentially expressed between HepG2/DOX and parental HepG2 cells through deep sequencing, which was followed by validation with qRT-PCR

Summary

Introduction

Hepatocellular carcinoma (HCC) is one of the most common virus-associated cancers resulting in high mortality worldwide [1]. For some patients who are not appropriate for surgical treatments, one has to only rely on chemotherapy. The development of drug resistance towards chemotherapeutic agents often prevents the successful long-term use of chemotherapy for HCC. Whether intrinsic or acquired over time, becomes the main cause of clinical treatment failure. Reversing drug resistance has become an emergent issue in tumor treatment. Drug resistance is a multifactorial phenomenon involving many mechanisms, including gene mutation, DNA methylation, altered metabolism and disposition of drugs, altered quantity or activity of target proteins and so on [2,3,4,5,6]. The key underlying mechanisms of the acquisition of resistance to chemotherapeutic agents still remain largely unexplored [2,7]

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Results

Conclusion