siRNA-mediated Stably Knockdown of MRJ Up-regulates Expression of MMP-9 and Leads to Cytoskeleton Rearrangement in Breast Cancer

Abstract: MRJ knockdown stable cell line of breast cancer MDA-MB-231 cells were constructed and screened with plasmid of MRJ siRNA. The MRJ knockdown stable cell line was used to investigate the effects of MRJ on cytoskeleton rearrangement and the expression of matrix metalloproteinase 9 (MMP-9). We transfected the MDA-MB-231 cells with the plasmid of MRJ siRNA (pRNAT-U6.1/neo-MRJsi) and used 600 μg/mL G418 to screen the stable knockdown cell line. The selected cell line was then subjected to Real-time PCR and Western blot assay to quantitate the mRNA level and protein level. Then munnofluorence was used to investigate the distribution of microtubes and microfilaments using anti-α-tublin antibody and phalloidin dyes， respectively.Moreover， the enzyme activity of MMP-9 was measured using gelatin zymography and its mRNA level was detected using quantitative Real-time PCR. The results showed that we successfully established MDA-MB-231/MRJsi stable cell line that knockdowned MRJ expression and named it MDA-MB-231/MRJsi cell line. The gene expression of MRJ in MDA-MB-231/MRJsi cells reduced 50% compared to the wild type MDA-MB-231 cells (P<0.05) and the protein level reduced 70%. Moreover， the cytoskeleton proteins in MDA-MB-231/MRJsi cells were redistributed when compared to wild type MDA-MB-231 cells. Furthermore， the enzyme activity of MMP-9 was enhanced after MRJ suppression. In addition， the mRNA level of MMP-9 was increased significantly (P<0.01) which was consistant with its enzyme activity. In conclusion， the establishment of stable MDA-MB-231/MRJsi cell line is a fundamental cell model for further research of MRJ function in oncogenesis and development of breast cancer.

CSTR: 32200.14.cjcb.2013.09.0012