敲减HMGB1表达对TNF-α诱导的乳腺癌细胞侵袭能力的影响

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

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-α treatment,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-α without 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.