Effect Of RNA Interference-Mediated Silencing Of MTA1 On Expression Of ER_α And Invasion Ability In Breast Cancer Cells

Objective: exploring the roles MTA1 may play in the process of breast cancer progression, invasion and metastasis ability.Lay the academic basis for further implying RNA interference in anti-cancer therapy.Methods:One pair of oligonucleotides targeting MTA1 shRNA was designed and cloned into linearized pGenesil-1 eukaryotic expression vector,the recombinant plasmid was identified by sequencing. Human breast cancer cell lines MDA-MB-231 and MCF-7 were transfected with the recombinant plasmid by Lipofectamine 2000,Fluorescence microscopy was used to observe the effect of RNA interference and evaluate the efficacy of transfection.The transcription of MTA1 gene and MMP-9 gene were determined by reverse transcription-polymerase chain reaction(RT-PCR), the expression of ERαand MMP-9 were determined by immunohistochemical method, the expression of ERαprotein was determined by Western blot,respectively. The invasion and metastasis ability was evaluated in human breast cancer cell lines MDA-MB-231 and MCF-7 by Boyden chamber invasive assay.Results:DNA sequencing showed that the recombinant plasmid pGenesil-1/ MTA1 was successfully constructed.The mean efficacy of transfection was 82.5﹪in MDA-MB-231 cells,and it was 78.2﹪in MCF-7 cells. The transcription of MTA1 gene was down-regulated in the two cancer cells after RNA interference by RT-PCR,the inhibition rates of pGenesil-1/ MTA1 was 80.2﹪,58.7﹪in MDA-MB-231 and MCF-7 cells after transfection and selection three weeks with G418, respectively.The ERαprotein has no expression before transfection and after transfecting pGenesil-1 in MDA-MB-231 cell,but the expression of ERαprotein was positive after selection three weeks with G418 by Western blot and in nucleus of cells by immunohistochemical method. The transcription of MMP-9 gene was down-regulated in MDA-MB-231 cell, but these changes was not come in MCF-7 cells.The invasion-metastasis ability of MDA-MB-231 cells is higher than that of MCF-7 cells in vitro by Boyden chamber invasive assay, its'invasion index were 76.3±2.4﹪, 25.6±1.9﹪, respectively. the difference was distinct(P<0.05). After transfecting pGenesil-1/MTA1, the invasion-metastasis ability of MDA-MB-231 cells was decreased, the invasion index was 27.2±2.1﹪,comparing to before transfecting and transfecting pGenesil-1, the difference was distinct(P<0.05); The invasion index was 23.3±1.6﹪in MCF-7 cells after transfecting pGenesil-1/ MTA1, the difference was no distinct comparing to before transfecting and transfecting pGenesil-1 (P>0.05). Conclusions: 1.The recombinant plasmid pGenesil-1/MTA1 shRNA can effectively suppress the expression of MTA1 in human breast cancer cell lines MDA-MB-231 and MCF-7, the effect of suppression was much effectively in human breast cancer cells that expressed high level MTA1 gene. 2. After interfering MTA1 gene, the expression of ERαprotein was positive in MDA-MB-231 cell, and the expression of MMP-9 mRNA was decreased ,the highly invasion-metastasis ability was also decreased. 3.This results showed RNAi may provide researching on function of MTA1 gene, and innovative approach to cancer therapy.