ILP-2-ECM1(P85)促进乳腺癌细胞的生长

凋亡抑制蛋白-2（inhibitor of apoptosis protein-like protein-2, ILP-2）是新发现的凋亡抑制蛋白质，其抑制肿瘤细胞凋亡促进其生长的分子机制有待阐明，而细胞外基质蛋白1(extracellular matrix protein 1, ECM1)所介导的信号通路与肿瘤细胞的生长密切相关。本研究通过免疫共沉淀法，检测到乳腺癌MCF-7细胞中ILP-2与ECM1（P85）存在相互作用。分别用化学合成的ILP-2-siRNA及ECM1-siRNA干扰处理MCF-7细胞。以未转染的MCF-7细胞和转染阴性对照siRNA的细胞分别作为空白和阴性对照，利用蛋白质印迹法，检测ILP-2-siRNA干扰后ECM1、FAK、Akt蛋白的表达，以及ECM1-siRNA干扰后ILP-2蛋白的表达。其结果显示，与空白对照组相比，ILP-2-siRNA-5 (0.32 ± 0.095)及ECM1-siRNA-1 (0.42 ± 0.024)干扰效率较高(均P<0.001)；ILP-2-siRNA-5组待测蛋白质的相对表达量均显著下调 (ECM1, 0.19 ± 0.013, P<0.001), FAK (0.64 ± 0.069, P<0.01), Akt (0.35 ± 0.120, P<0.01))，ECM1-siRNA-1组ILP-2 (0.48 ± 0.060) 蛋白表达也显著下调，表明ILP-2与ECM1-mTOR信号通路联系密切。分别在ILP-2-siRNA和ECM1-siRNA转染24、48和72 h时，使用CCK-8法检测乳腺癌细胞的增殖，并用TUNEL标记荧光法和吖啶橙/溴化乙啶双荧光染色法(AO/EB)检测其凋亡。结果显示，与空白对照组相比，ILP-2-siRNA-5组和ECM1-siRNA-1组的存活率均显著下降（P<0.001），凋亡率均明显升高（P<0.001）。利用共转染技术同时敲低ILP-2和ECM1表达,检测细胞的凋亡情况。结果显示，在干扰处理后24 h（0.55±0.122），48 h（0.80 ± 0.107）和72h（0.73 ± 0.091）的凋亡率显著均高于阴性对照组（P＜0.05）。但与只敲低ILP-2或ECM1相比，无显著性差异（P＞0.05）。表明ILP-2可能是通过与ECM1作用激活FAK-mTOR信号通路，影响MCF-7细胞的增殖和凋亡，对乳腺癌细胞MCF-7的生长发挥了积极的作用。

ILP-2-ECM1 (P85)Promotes the Proliferation of Breast Cancer Cells

Inhibitor of apoptosis protein-like protein-2 (ILP-2) is a newly discovered protein that inhibits cell apoptosis, and its molecular mechanism of inhibiting apoptosis and promoting growth of tumor cells remains to be elucidated. The signaling pathway mediated by extracellular matrix protein 1 (ECM1) is related to the growth of tumor cells closely. In this study, the interaction of endogenous ILP-2 with ECM1 was found in the MCF-7 cell line by co-immunoprecipitation. Respectively, two groups of siRNA targeting ILP-2 and ECM1 were transfected into MCF-7 cells with the non-transfected cells and cells transfected with the negative siRNAs as controls. Western blotting was used to detect the expression of ECM1, FAK and Akt proteins in cells treated with ILP-2-siRNA interference as well as the expression of ILP-2 protein in cells treated with ECM1-siRNA interference. The results showed that the ILP-2-siRNA-5 (0.32 ± 0.095) and ECM1-siRNA-1(0.42 ± 0.024) have a higher interference efficiency (P<0.001). The relative expression levels of the tested proteins in the ILP-2-siRNA-5 group were significantly down-regulated, ECM1 (0.19 ± 0.013, P<0.001), FAK (0.64 ± 0.069, P<0.01), Akt (0.35 ± 0.120, P<0.01). The expression of ILP-2 (0.48 ± 0.060) protein in ECM1-siRNA-1 group was also significantly down-regulated, which indicates that ILP-2 is closely relevant to the ECM1-mTOR signaling pathway. The two groups of MCF-7 cells transfected respectively by interference aimed at ILP-2 and ECM1 were processed for 24, 48 and 72 h. The proliferation of breast cancer cells was detected by CCK-8 assay, and cell apoptosis rates were analyzed by TUNEL method and AO/EB staining. It was showed that compared with the control group, the survival rates of ILP-2-siRNA-5 group and ECM1-siRNA-1 group were apparently decreased while the cell apoptosis rates of which were significantly increased. The apoptosis of cells was detected after knockdown of ILP 2 and ECM1 expression by co-transfection, the results showed that the apoptotic rate at 24 h (0.55±0.122), 48 h (0.80±0.107), and 72 h (0.73±0.091) after interference treatment was significantly higher than that of the negative control group (P<0.05), but the difference was not significant when compared with that of ILP-2 or ECM1 knock down only (P>0.05). It is indicated that ILP-2 may activate FAK-mTOR signaling pathway by interacting with ECM1, affecting the proliferation and apoptosis of breast cancer MCF-7 cell line and playing a positive role in the growth of MCF-7.