Effect Of SiRNA Mediated Down-regulation Of CD147on Human Breast Cancer Cell Line MDA-MB-231

Background and ObjectiveBreast cancer is the most common of women, and the incidence rate is5.01/10000in China. Breast cancer is the mammary epithelial cells in a variety of carcinogenic factor, the occurrence of a genetic mutation, resulting in uncontrolled cell proliferation, even life-threatening. However, early diagnosis and treatment can significantly enhance survival rates. The development of in molecular biology and immunology techniques can move the early diagnosis and treatment forward.CD147is extracellular matrix metalloproteinase inducer (EMMPRIN), existing in body widely. CD147involves different pathophysiological processes, chief among that is participating adhesion between cells and matrix. The aberrant activation of CD147stimulates the production of MMPs, which degrades typelV collagenase and the composition of extracellular matrix (ECM),what results is tumor invasion and metastasis. It seems worthwhile to test whether down-regulation of CD147would suppress the tumor invasion and metastasis. TIMPs are the natural and specific inhibitors of MMPs. The MMPs/TIMPs is balanced dynamically in normal times. In the case of cell transformation of malignancy, MMPs and TIMPs are both over-active, but the elevated level of TIMPs cannot suppress MMPs, what results in disrupting dynamic balance of ECM and promoting invasion and metastasis of tumor.CD147is a hot area of research, but there are not too many reports on the effect of CD147siRNA on human breast cancer cell line MDA-MB-231. In present study, we used RNAi technology to inhibit MDA-MB-231cells’ CD147gene expression, observed the expression changes of MMP-2and TIMP-2genes in mRNA and protein levels, analyzed the possible relationships between them, and discussed the influence of CD147genes silencing on MDA-MB-231cells’ migration and invision ability. Then we established the nude mouse model of breast cancer induce with MDA-MB-231cell line to observe the effects of CD147siRNA on the transplanted carcinoma.Methods1. Detect the protein expressions of CD147, MMP-2, TIMP-2of the MDA-MB-231cells by IHC.2. Determinated the virus titer; construct the lentiviral expression vector of CD147gene and transfected each group MDA-MB-231cells (normal cell group, negative control group and CD147siRNA transfection group). RT-PCR and Western Blot technology were to detect the mRNA and protein level changes of CD147genes after MDA-MB-231cells’ CD147siRNA transfection.3. RT-PCR and Western Blot technology were to detect the mRNA and protein level changes of MMP-2, TIMP-2genes after MDA-MB-231cells’ CD147siRNA transfection.4. CCK-8method and cell scratch test were to detect the proliferation and migration change of MDA-MB-231cells after transfection.5. Established the nude mouse model of breast cancer induce with MDA-MB-231cell line to observe the effects of CD147siRNA on the transplanted carcinoma.6. Statistical analysis:the data was analyzed by SPSS17.0software package, quantitative data was expressed as Mean±SD, the mean difference of groups was compared with single factor analysis of variance (ANOVA), the significant difference level was α=0.05.Results1. The immunocytochemistry experiment results showed that all of CD147, MMP-2, TIMP-2proteins expressed in MDA-MB-231cells and were strong positively expressed.2. Lentiviral vector carrying CD147gene has been successfully constructed. after MDA-MB-231cells’ transfection, the average relative quantity of mRNA and proteins of CD147were decreased obviously in transfection group with time dependence, and highest inhibitory activity at72hours.3.72hours after MDA-MB-231cells’ transfection, the average relative quantity of mRNA and proteins of MMP-2were decreased obviously in transfection group; the average relative quantity of proteins of TIMP-2was decreased obviously in transfection group, but the average relative quantity of TIMP-2mRNA in all experimental groups was not statistically significant (P>0.05).4. The growth inhibition ratio difference of these experimental groups had statistical significance when2d,3d and4d after transfection (P<0.05)5. The migration speed of cells was retarded after MDA-MB-231cells’ CD147siRNA transfection.6. The growth of the transplanted carcinoma was suppressed after siRNA mediated down-regulation of CD147.Conclusions1. CD147may affect MMPs/TIMPs balance in MDA-MB-231cells.2. MDA-MB-231cells’ CD147gene silencing could inhibit the proliferation and migration of MDA-MB-231cells and the growth of the transplanted carcinoma.