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

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.

MiR‐151a is involved in the pathogenesis of atopic dermatitis by regulating interleukin‐12 receptor β2

“…RNA samples was then examined for sample quality and subjected to deep sequencing. Small RNA library construction and sequencing were conducted as previously described …”

“…For quantitative real‐time PCR (qRT‐PCR) experiments, 500 μL of plasma was thawed on ice and used for RNA isolation with TRIZOL (Invitrogen) in accordance with the manufacture's protocol. cDNAs were prepared by Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) . For RNA extraction of blood cells, PBMCs were separated from 500 μL peripheral blood by Ficoll‐Hypaque density gradient centrifugation, of which the RNA was isolated with TRIZOL (Invitrogen) in accordance with the manufacture's protocol.…”

Mechanisms of doxorubicin resistance in hepatocellular carcinoma

“…Although non-coding RNAs have diverse functions, much recent focus has been on miRNAs, particularly examining their ability to modulate known mechanisms of resistance through suppression of mRNA translation. By comparing miRNA expression patterns in doxorubicin-sensitive and doxorubicin-resistant tumors a number of resistance-associated miRNA changes have been observed [104,105]. Some of these have been shown to cause resistance while others have a plausible mechanism for inducing resistance that has not yet been proven.…”

MiRNA expression profile of ionizing radiation‐induced liver injury in mouse using deep sequencing

“…The clean reads were then blasted against GeneBank and Rfam database for classification and annotation of non‐coding RNAs other than miRNAs (Figure ). Both of the total rRNA proportion in the two libraries were less than 40%, which showed that the animal samples were of high quality (Zhang et al, ). In terms of reads number, rRNA was the most common one, followed in descending order by snoRNA, tRNA, and snRNA.…”