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, both P < 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.