Study On RNA Interference Reversing The Multidrug Resistance Of Leukemia Cell

[Background and Objective]Multidrug resistance (MDR) is one of the major causes for chemotherapeutic treatment failure in leukemia patients. The mdr1 gene product, P-glycoprotein (P-gp), is an energy dependent drug efflux pump, which play an important role in the mechanism of MDR, and makes the leukemia refractory and relapsed. Although early studies have shown that P-gp antagonists such as verapamil, cyclosporin A, antibiotics, suppressor of enzyme, could reverse MDR at the protein level, unfortunately, clinical studys have been impeded by their significant toxicity. New methods to treat MDR is desired in urgent.RNA interference is a conserved nature biological response to double-stranded RNA, which can catalyze and cleavage target mRNA, thus results in sequence-specific post-transcriptional gene silencing (PTGS). RNAi become a reseach hot in gene therapy of tumor and disease caused by viruses. To downregulate the expression of mRNA in cytoplasm, RNAi is the best of all antisense technologies. It can silence gene for a long time by shRNA vector, and cause specific silencing in different tissues by different promoters.To start the mdrl reversing research, we must get a proper research modle. Nowerdays, the most common modles are drug-resistance cell strains induced by anti-cancer drugs, which cannot work accurate in feedback us thefunctions of reversing factors.So, we cloned four small hairpin RNA (shRNA) plasmids, transfected them into K562/A02 to reverse MDR. And then we constructed a cell modle K562/mdil by transfecting whole cDNA of mdrl to K562. [Method]We cloned four shRNA eukaryotic expression vectors aimed at three mdrlmRNA target sequences, then, transfected them into drug resistance cell line K562/A02 by liposome-induced gene transfection, 48 hours later, we harvested cells to detect.The leukemia sensitive cell line K562 was transfected with mdrl cDNA by liposome-induced gene transfection and selected by G418.The mdrl phenotype was identified by RT-PCR and Q-PCR, P-gp expression was detected by flow cytometry, the function of P-gp was measured by Rhl23 or daunonibicin efflux experiment and the sensitivity of cell lines to the drug was detected by MTT test. [Results]l.We designed and cloned three shRNA plasmids targeted mdrl: Pmdrlshl, Pmdrlsh2, Pmdrlsh3. Owing to a mistake, we got an anti-mdrl shRNA plasmid Pmdrlsh2W, which lose a base in the sense strand of transcription.2.? All mdrl-targeted shRNA could downregulate mdrl mRNA markedly. mdrlmRNA were quantified by Q-PCR,the results read Pmdrlshl(0.08 ± 0.02), Pmdrlsh2(0.07 ± 0.01), Pmdrlsh2W(0.07 ± 0.03), Pmdrlsh3(0.10±0.01), compared with each other P>0.05: Phk(0.57±0.12), Blank(0.78 ± 0.21), compared with each other P>0.05, compared withmdrl-targeted group P<0.01. Pmdrlshl, Pmdrlsn2, Pmdrlsh2W, Pmdrlsh3 in K562/A02 cell reduced mdrlmRNA by 86%, 88%, 88%, 82% respectively(compared with Phk). ?Daunorubicin efQux experiment showed that the daunorubicin efflux ratio at 60min were Pmdrlshl(13%), Pmdrlsh2(6%), Pmdrlsh2W(10%), Pmdrlsh3(22%), Phk(45%)? Blank(40%); at 90min, we found under fluorescence microscope no daunorubicin fluorescence in Phk and Blank cells, but dominant fluorescence in Pmdrlshl, Pmdrlsh2, Pmdrlsh2W, Pmdrlsh3 cells. Inhibition of P-gp expression by shRNA enhanced the intracellular accumulation of drug. (3)MTT test demonstrated the RI of Pmdrlshl, Pmdrlsh2, Pmdrlsh2W, Pmdrlsh3, Phk, Blank cells were 2.39, 1.91, 2.19, 3.07, 8.92, 9.90.?A mismatch in sense strand of transcription like Pmdrlsh2W cannot affect the function of shRNA.3. (DRT-PCR demonstrated the cell modle K562/mdrl constructed by gene transfection can transcript mdrl; Q-PCR verified the mdrlmRNA quantity were K562/mdrl (0.09 ± 0.01), K562/neo(0.00046 ± 0.000006), K562/A02(0.21±0.02), compared with each other P<0.01. ?P-gp espression detected by FCM resulted in K562/mdrl(FI 4.41), K562/neo(FI 0.32), K562/A02(FI 6.18), compared with each other P<0.01. ?Rhl23 efflux experiment showed that the Rhl23 efflux ratio at 90min were K562/mdrl(60%), K562/neo(6%), K562/A02(57%).@MTT test demonstrated the RI of K562/mdrl, K562/neo, K562/A02 were 6.5,1,10.1. [Conclusion]l.Four shRNA targeted three different targets can all knock down the expression of mdrl.2.A mismatch in the sense strand of transcription will not affect thefunction of shRN A.3.Drug resistance cell line established by gene transfection with mdrl cDNAcan provide a better cell modle for mdrl reversing research.4 jndrl play an important role in drug resistance.