敲减HMGB1表达对TNF-α诱导的乳腺癌细胞侵袭能力的影响

摘要

目的:探讨敲减高迁移率族蛋白B1 (HMGB1) 表达对肿瘤坏死因子α (TNF-α) 诱导的乳腺癌细胞侵袭能力的影响及机制.方法:采用HMGB1小干扰RNA (siRNA) 转染乳腺癌MDA-MB-231细胞, 实时荧光定量PCR和Western blot分别测定细胞中HMGB1的mRNA和蛋白表达情况.用TNF-α处理敲减HMGB1表达的MDA-MB-231细胞, 流式细胞术测定各组细胞凋亡, Transwell小室法测定各组细胞侵袭能力, 细胞划痕实验测定各组细胞的迁移能力, Western blot法测定细胞中上皮钙黏素 (E-cadherin) 、基质金属蛋白酶2 (MMP-2) 、神经钙黏素 (N-cadherin) 、基质金属蛋白酶9 (MMP-9) 和Bax的表达情况.结果:HMGB1 siRNA转染后MDA-MB-231细胞中HMGB1的mRNA和蛋白表达水平明显低于没有转染的细胞 (P <0.05) .与对照组相比, TNF-α处理后的乳腺癌细胞的凋亡水平升高, 细胞侵袭和迁移能力也升高, 细胞中E-cadherin蛋白水平降低, N-cadherin蛋白水平升高, MMP-2、MMP-9和Bax蛋白水平也升高 (P <0.05) .敲减HMGB1表达的MDA-MB-231细胞经TNF-α诱导以后, 细胞凋亡率增加, 侵袭及迁移能力下调, 细胞中E-cadherin蛋白水平升高, N-cadherin蛋白水平下降, MMP-2和MMP-9蛋白水平也下降, Bax蛋白水平升高 (P <0.05) .结论:敲减HMGB1表达可以增强TNF-α诱导的乳腺癌细胞凋亡, 抑制TNF-α诱导的乳腺癌细胞侵袭、迁移和上皮-间充质转化, 其作用机制与MMP-2、MMP-9和Bax蛋白表达有关.%AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α).METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells.The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot.After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test.The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot.RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05).The apoptosis rate in the cells was increased after TNF-αtreatment, and the cell invasion and migration abilities were also increased.The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05).After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-αwithout knockdown of HMGB1 expression (P<0.05).CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α.The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.