Expetiment Study On Adenovirus Vector Mediated Hairpin RNA Inhibition Of MRP1 Gene Expression In K562/AS2 Cell Line Resisrant To AS 2 O 3

[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 As2O3 (ATO).[Methods]A MRP1–specific Hairpin RNA recombinant adenovirus vector was constructed and used to infected K562/AS2 cells. Expression level of MRP1 mRNA was detected by real-time fluorescent quantitative PCR. MRP1 protein was detected by flow cytometry. MTT method was used to detected the cytotoxicity of ATO and etoposide.[Result]Part I: The recombinant plasmid pGenesi l.1-MRP1-shRNA for PCR was amplified, and inserted into the entry vector pDONR, and then the homologous recombination was carried out. The positive clones were selected and forward and reverse sequencing was performed. Positive pDONR-MRP1-shRNA shuttle plasmid with correct sequence and adenovirus backbone plasmid pAd /BLOCK–iTTM–DEST were homologously recombined and screened.. The selected strains were amplified by culture, the plasmid was purified by agarose gel electrophoresis purification using DNA extraction kit. and recombinant adenovirus plasmid pAd-shRNA-MRP1 expressing shRNA-MRP1 was obtained. The final identification and determination of the titer was carried out. 4-7 days after infection of 293A cells green fluorescent protein expression can be observed in the culture bottle, which indicated the infective recombinant adenovirus packaging was successful.Part II: K562/AS2 cells were infected by recombinant adenovirus vector, pAd-MRP1-shRNA. MRP1 mRNA and protein expression level in K562/AS2 cells before and after the pAd-MRP1-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),respetively. 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]pAd-EGFP-U6-shRNA-MRP1 recombinant adenovirus vector is successfully constructed. The expression of MRP1 gene in K562/AS2 cell infected by the vector can be down-regulated and the resistance of the the K562/AS2 cell to either ATO or etoposide can be reversed..