| ObjectiveTo construct and identify the siRNA eukaryotic expression vector targeting gene CXC chemokine receptor-4 and study its role in invasion and metastasis ability of bread cancer cells.And study the effects of the RNA secondary structure on the RNA interference efficacy and to optimize the siRNAs selection and design.Method1 Two siRNAs were designed and synthesized according to the coding the cDNA sequence of CXCR4 gene and cloned into eukaryotic expression plasmid pGE-1-U6/kna,digested plasmids with restriction enzyme and sequenced the plasmids to confirm the presence of the siRNA insert.2 There were 4 groups in our study,Normal control group,CXCR4-siRNA1 experimental group,CXCR4-siRNA2 experimental group,CXCR4-siRNA1+2 experimental group.The constructed CXCR4-siRNA expression vectors were transfected into MDA-MB-231 cells,which could highly erpress CXCR4,by liposome. Then drawing growth curve in vitro of cell viability of MDA-MB-231 cells before and after transfected with recombinant plasmids,a statistical comparison was done to determine whether the impact of the transfected cell activity and tumorigenicity. Western blotting were used to evaluate the suppression of CXCR4 expression in different groups.The invasion and migration of MDA-MB-231 cells were evaluated by cell invasion assay in vitro,siRNA that interfere efficiency is the strongest was chosen.In order to find the correlation between the siRNA efficacy and the local structure of its target sequence,the secondary structure of mRNA were predicted professional software.Results1 The recombinant plasmids identified by the enzyme cutting method and the sequence analysis completely coincided with the designs.It show that the expression vectors named CXCR4-siRNA1 and CXCR4-siRNA1 were constructed successfully.2 The cell growth curves were delineated to analyze the cells proliferating dynamic values,the cell growth curves erery groups cells before and after transfection were basically consistent(P＞0.05),and we could come to the conclusion that the activity and tumorigenicity of MDA-MB-231 cells had not been changed by transfection. According to the results of Normal control group,CXCR4-siRNA1 experimental group,CXCR4-siRNA2 experimental group and CXCR4-siRNA1+2 experimental group.CXCR4/β- actin values were 0.4532±0.0012,0.2103±0.0030,0.3922±0.0021 and 0.1089±0.0011.CXCR4-siRNA expression vectors could obviously inhibit the expression of CXCR4 in prostate cancer cells.In terms of Normal control group,CXCR4-siRNA1 experimental group,CXCR4-siRNA2 experimental group and CXCR4-siRNA1+2 experimental group,through the basal plasma membrane of cells were(48.3±1.7)/mm~2,(18.7±2.4)/mm~2,(24.5±1.9)/mm~2 and(9.1±3.6) /mm~2.Knock-down efficiency was recorded in all of the 3 experiment groups. CXCR4-siRNA1 presented higher silencing efficiency than CXCR4-siRNA 2,and the combination of CXCR4-siRNA 1 and CXCR4-siRNA 2(CXCR4-siRNA 1+2) achieved was more efficient in reduction of CXCR4 expression than CXCR4-siRNA 1 or CXCR4-siRNA 2 only.The secondary structure of the mRNA analysis shows that target of siRNA1 located in the secondary structure of CXCR4 mRNA annular region and siRNA2 located stems region.ConclusionThe shRNA eukaryotic expression vectors against CXCR4 mRNA have been successfully constructed,and they effectively reduce the expression of CXCR4 in MDA-MB-231 cells and decrease the exoteric invasive ability of breast cancer effectually.The siRNA targeting none-secondary structure region presented higher silencing efficiency than those targeting secondary structure region.The same target gene for different targets at the same time interfere with the effectiveness of better than a single target.Recognizing that the secondary structure of mRNA closely relate t o the RNA interference efficiency,which should be considered in the processing of the target sequence selection.A better understanding of breast cancer metastases molecular mechanisms and the mechanism of RNA interference,is expected to metastasis of breast cancer gene therapy has opened new ways. |
