RESUMO
Objective To construct a recombinant adenovirus vector of Hairpin RNA specific for MRP1 gene and study its inhibition of MRP1 gene expression in K562/AS2 cell line resistant to AS_2O_3 (ATO). Methods A MRP1-specific hairpin RNA recombinant adenovirus vector was constructed and used to infected K562/AS2 cells. Expression level of MRP1 mRNA detected by real-time fluorescent quantitative PCR. MRP1 protein detected by flow cytometry. MTT method was used to detected the cytotoxicity of ATO and etoposide. Results MRP1 mRNA and protein expression level in K562/AS2 cells before and after the pAd-MRPl-shRNA adenovirus infection was (34.70±0.28 vs 4.19±0.03, P <0.05) and (26.40±0.16 vs 10.85±0.37, P<0.05), respectively. RR of K562/AS2 to arsenic trioxide and etoposide was (11.4078±0.3183 fold vs 1.6126±0.3015 fold, P<0.05) and (5.9141 ±0.0149 fold vs 1.7664±0.1038 fold, P <0.05), respectively. The reversal fold of ATO and etoposide was (7.2409±1.3668) and (3.3555±0.1886), respectively. Conclusion Successfully constructed pAd-EGFP-U6-shRNA-MRPl adenovirus vector, the vector of infection K562/SA2 cells can inhibit MRP1 gene expression and reverse the resistance of the ATO and etoposide.
RESUMO
Objective To investigate the inhibitional effect of MRPI-shRNA on expression of MRP1gene in K56.2/AS2 cells resistant to arsenic trioxide. Methods Three pieces of MRPI-shRNA were designed,synthesized and transfected into K562/AS2 cells with lipesome. Expression level of MRPI mRNA were determined by real time fluorescent quantitative PCR. MRPI protein expression and intracellular accumulation of DNR were assayed with flow cytometry. Results After treated with MRPI-shRNA, the expression level of MRPI mRNA and MRPI protein in K562/AS2 cells decreased significantly(79.1±0.07) % and (62.48±0.86) %,respectively (P <0.05). The intracellular accumulation of DNR increased significantly(P < 0.05). Conclusion MRPI-shRNA can down-regulate the expression of MRPI gene in K562/AS2 cell line.