Effect Of Silencing ATX By RNA Interference On Biologicactivity Of Human Breast Cancer Cell Lines

Objective: Study the effects of ATX gene expression by small interfering RNA in human breast cancer cells on autotaxin,to explore the application of autotaxin in treatment of breast cancer.Methods: Experiment was divided into three groups: negative control group, blank plasmid group,interference fragment transfection group。Reference ENPP2 human melanoma gene sequence,design and synthesis ATX-siRNA sequence(Justice chain:5'-GGU CCU CCU ACA GUG CUA UTT-3' ,Antisense strand:5'-AUA GCA CUG UAG GAG GAC CTT3')and random negative control sequence(Justice chain :5'-GGU CUC CCA AAC GGU CUA UTT-3' ,Antisense strand:5'-AUG AGC CGU GAU GAG GAC CTT-3')。In cationic liposomes-mediated transfection of human breast cancer cells MCF-7, transfection efficiency was observed after 48h , collecte the cells, each group extracte RNA and protein respectively, (1) Semi-quantitative RT-PCR detecte the expression of ATX mRNA after transfection , (2) western blot detecte expression of transfected ATX protein, (3) interfere sequence was transfected to human breast cancer cells MCF-7 by cationic liposome,after 48h, observe of transfection efficiency and collecte the cells,to determinate cells the ability of tumor to penetrate matrigel, invasiveness of tumor cells changes was detected; Compare the differences between sets of data through variance analysis and T test.Results: ATX-siRNA which was synthesised in vitro was transfected to breast cancer cells MCF-7, was observed by fluorescence microscopy after 48h, transfection efficiency was up to 60%.(1) ATX-mRNA content was measured by semi-quantitative RT-PCR in three groups of cells respectively: negative control group(0.965±0.076), blank plasmid group(1.097±0.184),interference fragment transfection group(0.546±0.721), P<0.05; (2) ATX protein expression of three groups of cells wasdetected by Western blot respectively: negative control group(0.713±0.027), blank plasmid group(0.680±0.052),interference fragment transfection group(0.501±0.023), P<0.05; (3)Tanswell small room was used to detecte invasiveness changes of three groups cells: negative control group(141.2±6.693), blank plasmid group(130.0±4.690),interference fragment transfection group(26.0±1.532), P<0.05. Through the T test and analysis of variance of data in each group, through the dtas of interference fragment transfection group which were compared to datas of blank plasmid group and negative control group, P<0.05, there was a significant difference. Conclusion:ATX-siRNA inhibits the expression of ATX-mRNA and protein, degrades invasion of human breast cancer cell.