title: [Role of inhibition of nuclear factor-kappa B gene transcription by specific miRNA in reversing multi-drug resistance of liver cancer].
abstract: Objective:To investigate the reversal effect of inhibition of nuclear factor-kappa B (NF-κB) gene transcription by specific miRNA on multi-drug resistance (MDR)of liver cancer.Methods:The expression of P-glycoprotein (P-gp) and NF-κB in hepatoma cells, drug-resistant HepG2/ADM cells, and liver cells (LO2 cells) was analyzed. Specific NF-κB miRNA plasmids were constructed, screened, and transfected into HepG2 or HepG2/ADM cells. Western blot was used to measure the concentrations of P-gp and NF-κB, and FQ-PCR was used to measure gene expression; Cell Counting Kit-8 assay was used to measure cell proliferation and the influence of drugs on cell proliferation; flow cytometry and Annexin-V-PE/7-ADD double staining were used to observe cell cycle and apoptosis. The t-test was used to compare means between groups, and a one-way analysis of variance was used to compare means between multiple groups.Results:After being treated by adriamycin, hepatoma cells showed increased expression of P-gp and an increased level of NF-κB phosphorylation. At 24, 48, and 72 hours, the resistance index of the HepG2/ADM cells (IC50= 4.166, 1.522, and 1.380 μmol/L) was 8.519, 6.874, and 6.166 times that of the HepG2 cells (IC50= 0.489, 0.221, and 0.224 μmol/L). The HepG2/ADM cells showed significantly higher relative mRNA expression (∆ct value) of mdr1 and NF-κB than the HepG2 cells (3.310±0.154/2.580±0.040 vs 0.084±0.038/0.6067±0.032, bothP< 0.01). After being transfected with miRNA1, the HepG2/ADM cells showed significantly lower mRNA expression of mdr1 than the cells in the miRNA-negative group (2-∆∆ct= 0.326±0.011 vs 0.804±0.057,t= 14.262,P< 0.01), as well as significant reductions in the expression of intracellular t-p65, nuclear p-p65, and P-gp compared with the cells in the miRNA-negative group (P< 0.01), with inhibited cell proliferation, G1 phase arrest, and increased apoptosis.Conclusion:Abnormal expression of MDR1/P-gp is closely associated with MDR, and inhibition of NF-κB activation by specific miRNA can significantly inhibit MDR1/P-gp gene transcription and reverse MDR of liver cancer.
authors: Medical School of Nantong University, Nantong 226001, China.
authors: L
authors: L
authors: Wang
authors: Affiliated Hospital of Nantong University, Nantong 226001, China.
authors: Y
authors: Y
authors: Cai
authors: Affiliated Hospital of Nantong University, Nantong 226001, China.
authors: Y
authors: Y
authors: Shi
authors: Medical School of Nantong University, Nantong 226001, China.
authors: M
authors: M
authors: Yao
authors: Affiliated Hospital of Nantong University, Nantong 226001, China.
authors: L H
authors: LH
authors: Pan
authors: Affiliated Hospital of Nantong University, Nantong 226001, China.
authors: J J
authors: JJ
authors: Gu
authors: Affiliated Hospital of Nantong University, Nantong 226001, China.
authors: X L
authors: XL
authors: Yang
authors: Affiliated Hospital of Nantong University, Nantong 226001, China.
authors: W
authors: W
authors: Wu
authors: Affiliated Hospital of Nantong University, Nantong 226001, China.
authors: D F
authors: DF
authors: Yao
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D049109:Cell Proliferation; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D016328:NF-kappa B / Q000235:genetics*; D010766:Phosphorylation; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D035683:MicroRNAs; D016328:NF-kappa B; D004317:Doxorubicin