Construction Of Eukaryotic Expression Vector Of ShRNA Specific For HOXA1O Gene And Its Effects On K562Cells

Objective To obtain effective siRNA fragement of HOXA10gene and vertify its function, then shRNA plasmid pGPHI/GFP/Neo-HOXA10targeting HOXA10was constructed and sequenced from this siRNA.To investigate the effect of plasmid mediated RNAi targeting HOXA10on the proliferation,apoptosis and drug resisetance of human Chronic Myeloid Leukemia K562Cell line.Methods1.Three pairs of small interfering RNA targeting to the different sites of HOXA10were designed and introduced into A549.The mRNA expression of HOXA10was detected by semi-quantiatative RT-PCR,the cell proliferation was assayed by MTT and the apoptosis was measured by flowcytometry.The most effective siRNA assay was screened and was tested the relationship between it and cell proliferation and apoptosis.2. ShRNA oligo was designed and compounded according to the effective and specific siRNA strand.Then plasmid pGPHI/GFP/Neo-HOXA10targeting HOXA10was constructed.3. The plasmid was transfected into K562cells by positive ion liposome, and K562cells was effected by RNAi and chemotherapeutic.The mRNA expression of HOXA10of K562was detected by RT-PCR,the cell proliferation and drug sensitivity were assayed by MTT,the apoptosis was measured by flowcytometry and Hoechst33258.Results1. The mRNA of HOXA10was inhibited by three siRNAs in A549cells, among which siRNA1gave the srongest inhibiting of HOXA10by ODR=(20.190±1.698)%.The inhibitory rate of cell proliferation is(69.793±2.092)%and the apoptosis rate was (29.593±2.670)%. 2. The plasmid pGPHI/GFP/Neo-HOXA10was successfully constructed by restriction enzyme identification and sequencing.3. The mRNA of HOXA10was inhibited by this plasmid in K562cells by ODR=(38.86±4.49)%.The inhibitory rate of cell proliferation of experimental group was(66.30±1.26)%(P<0.05) and the apoptosis rate was (22.29±1.67)%(P<0.05),which was enhanced combining low dose Ara-C.The IC50of stable transfection cell to VCR and VP16was significantly reduced as compared with that of the control(P <0.05),the apoptosis morphological changes and rates were different from the control groups obviously(P<0.05).Conclusions1. We successfully validated siRNAl,which can specifically degrade HOXA10mRNA and inhibit the proliferation of A549cell and promote its apoptosis.2. pGPHI/GFP/Neo-HOXA10that tartet HOXA10was successfully constructed.3. pGPHI/GFP/Neo-HOXA10can inhibit the proliferation of K562cell and promote its apoptosis,can increase the sensitivity of K562to VCR and VP16.This vector can reverse the multidrug resisetance of leukemia cells in some degree.