雌激素受体α基因沉默介导转化生长因子-β通路对乳腺癌细胞活性的影响

Effect of estrogen receptor alpha gene silencing on the activity of breast cancer cells through transforming growth factor-βsignaling pathway

目的探讨雌激素受体α(ESR1)基因沉默介导转化生长因子-β(TGF-β)信号通路对乳腺癌细胞活性的影响及其作用机制。方法选取南华大学附属第一医院58例乳腺癌患者的癌组织及癌旁组织标本。免疫组织化学法检测组织中ESR1蛋白表达阳性率差异。细胞分为5组,Blank、NC、HA-ESR1、ESR1-siRNA、SRI-011381 hydrochloride+ESR1-siRNA组。实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)法分别检测各组ESR1、TGF-β1、TGF-βRⅠ、TGF-βRⅡ、E-cadherin、N-cadherin和Vimentin的mRNA和蛋白表达水平。噻唑蓝(MTT)法检测各组细胞增殖活性,多组间比较采用单因素方差分析。结果癌组织中ESR1阳性表达率显著高于癌旁组织,差异有统计学意义[癌组织:阳性表达40例,阳性率68.97%(40/58)比25.86%(15/58),χ^(2)=7.877,P<0.05]。ESR1-siRNA组ESR1(mRNA:0.98±0.01比0.54±0.02,t=34.08;蛋白:0.82±0.04比0.36±0.04,t=14.08)及TGF-β1、TGF-βRⅠ和TGF-βRⅡ的mRNA和蛋白表达量均明显低于Blank组和NC组(mRNA:TGF-β11.00±0.04比0.54±0.06,t=11.05;TGF-βRⅠ1.00±0.04比0.63±0.03,t=12.82;TGF-βRⅡ1.00±0.05比0.44±0.04,t=15.15;蛋白:TGF-β11.53±0.07比0.50±0.06,t=19.35;TGF-βRⅠ1.03±0.08比0.42±0.04,t=11.81;TGF-βRⅡ0.84±0.09比0.37±0.03,t=8.58,P<0.05),N-cadherin和Vimentin mRNA和蛋白表达量明显低于Blank组和NC组,而E-cadherin表达明显高于Blank组和NC组(mRNA:E-cadherin 1.00±0.03比0.55±0.04,t=15.59;N-cadherin 1.00±0.03比0.67±0.04,t=11.43;Vimentin 1.00±0.02比1.83±0.11,t=12.86;蛋白:E-cadherin 1.33±0.09比0.78±0.05,t=9.25;N-cadherin 2.11±0.12比1.36±0.08,t=9.01;Vimentin 0.56±0.05比1.05±0.07,t=9.87,P<0.05),细胞增殖活性低于Blank组和NC组(24 h:0.56±0.05比0.35±0.03,t=6.24;48 h:0.88±0.08比0.48±0.04,t=7.75;72 h:1.09±0.10比0.74±0.07,t=4.97,P<0.05),差异均有统计学意义;而HA-ESR1组的各指标变化趋势逆转;SRI-011381 hydrochloride+ESR1-siRNA组仅ESR1表达低于ESR1-siRNA组(mRNA:0.54±0.02比0.97±0.02,t=26.33;蛋白:0.36±0.04比0.80±0.04,t=13.47,P<0.05),差异有统计学意义。结论沉默ESR1基因可能抑制TGF-β信号通路的激活,进而抑制乳腺癌上皮-间充质转化及肿瘤细胞活性。

Objective To investigate the effect of estrogen receptor alpha(ESR1)gene silencing on the activity of breast cancer cells and its mechanism by mediating transforming growth factor-β(TGF-β)signaling pathway.Methods By collecting 58 breast cancer tissues and adjacent tissues from the department of breast and thyroid surgery of our hospital,the positive rate of ESR1 protein expression was detected by immunohistochemistry.Cells were divided into 5 groups:Blank group,NC group,HA-ESR1 group,ESR1-siRNA group,SRI-011381 hydrochloride+ESR1-siRNA group.The expression levels of ESR1,TGF-β1,TGF-βRⅠ,TGF-βRⅡ,E-cadherin,N-cadherin and Vimentin were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the cell proliferation activity in each group.SPSS 22.0 software was used for statistical analysis.Results The positive expression rate of ESR1 in cancer tissues was significantly higher than that in paracancerous tissues,with statistical difference(Cancer tissue:positive expression in 40 cases,positive rate of 68.97%(40/58);Paracancerous tissue:positive expression in 15 cases,positive rate of 25.86%(15/58),χ^(2)=7.877,P<0.05).Compared with Blank group and NC group,the expression of ESR1(mRNA:0.98±0.01 vs.0.54±0.02,t=34.08;protein:0.82±0.04 vs.0.36±0.04,t=14.08),TGF-β1,TGF-βRⅠand TGF-βRⅡ(mRNA:TGF-β11.00±0.04 vs.0.54±0.06,t=11.05;TGF-βRⅠ1.00±0.04 vs.0.63±0.03,t=12.82;TGF-βRⅡ1.00±0.05 vs.0.44±0.04,t=15.15;protein:TGF-β11.53±0.07 vs.0.50±0.06,t=19.35;TGF-βRⅠ1.03±0.08 vs.0.42±0.04,t=11.81;TGF-βRⅡ0.84±0.09 vs.0.37±0.03,t=8.58)in ESR1-siRNA group was significantly decreased,N-cadherin and Vimentin were significantly decreased,E-cadherin expression was significantly increased(mRNA:E-cadherin 1.00±0.03 vs.0.55±0.04,t=15.59;N-cadherin 1.00±0.03 vs.0.67±0.04,t=11.43;Vimentin 1.00±0.02 vs.1.83±0.11,t=12.86;protein:E-cadherin 1.33±0.09 vs.0.78±0.05,t=9.25;N-cadherin 2.11±0.12 vs.1.36±0.08,t=9.01;Vimentin 0.56±0.05 vs.1.05±0.07,t=9.87),accompanied by decreased cell proliferation(24 h:0.56±0.05 vs.0.35±0.03,t=6.24;48 h:0.88±0.08 vs.0.48±0.04,t=7.75;72 h:1.09±0.10 vs.0.74±0.07,t=4.97),with statistical differences(all P<0.05);However,the change trend of each index in HA-ESR1 group was contrary to that in ESR1-siRNA group.There existed statistically significant differences in the expressions of ESR1 in SRI-011381 hydrochloride+ESR1-siRNA group than those in ESR1-siRNA group(mRNA:0.54±0.02 vs.0.97±0.02,t=26.33;protein:0.36±0.04 vs.0.80±0.04,t=13.47),with statistical differences(both P<0.05).Conclusion Silencing ESR1 gene may inhibit the activation of TGF-βsignaling pathway,thereby inhibiting epithelial-mesenchymal transformation and tumor cell activity in breast cancer.