[Methylated oligonucleotide inhibiting expression of human MRP2 and reversing multidrug resistance in hepatocellular carcinoma Cell HepG2].
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.
Li
Wen-Huan
WH
Department of Gastroenterology, Shandong Provincial Hospital, Shandong University, Jinan,Shandong, 250021, PR China. wenhuanli@hotmail.com
Cui
Yi
Y
Zhu
Ju-Ren
JR
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology; D016190:Carboplatin / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D015251:Epirubicin / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis* / Q000235:genetics; D009841:Oligonucleotides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D002166:Camptothecin; D016190:Carboplatin; D015251:Epirubicin; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; C000715087:ABCC2 protein, human