[Methylated oligonucleotide inhibiting expression of human MRP2 and reversing multidrug resistance in hepatocellular carcinoma Cell HepG2]

Abstract

Background & objective: Accumulating evidences showed that the overexpression of multidrug resistance-associated protein 2 (MRP2) was the main cause of multidrug resistance in hepatocellular carcinoma (HCC); and the methylated cytosine at cytosine-guanine (CpG) dinucleotides might silence the gene expression. This study was to evaluate the inhibiting effect of methylated oligonucleotide (MON) on MRP2 gene transcription.

Methods: MON complementary to human MRP2 promoter region was designed. The non- methylated oligonucleotide (NON) carried same nucleotide acid sequence with MON, but the cytosines were not methylated. Cells from human HCC cell line HepG2 were divided into 3 groups: control group, NON group, and MON group. HepG2 cells were transfected with liposome-encapsulated oligonucleotide, cytotoxicity was determined by MTT assay. Reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry (FCM) were used to analyze the expression of MRP2.

Results: MON specifically inhibited MRP2 gene transcription and down-regulated the expression of MRP2 in a concentration- dependent manner in vitro, whereas NON had no effect. The positive rates of MRP2 protein were 18.0%, 9.15%, 5.73%, 3.73%, and 2.56% respectively after transfected with 1, 2, 4, 8, and 16 micromol/L MON. It was 24.5% in NON group. The 50% inhibitory concentration (IC50) of epirubicin, hydroxyl- camptothecin, and carboplatin were reduced in MON group.

Conclusions: MON can inhibit MRP2 gene transcription, enhance chemosensitivity, and reverse multidrug resistance of HCC cells,it may become a new gene therapeutic agent for HCC.