摘要
目的 观察金属硫蛋白1M(MT1M)在三阴性乳腺癌(TNBC)细胞中的表达,探讨其对TNBC细胞迁移、侵袭的影响及机制.方法 利用Ualcan和Kaplan-Meier Plotter在线网站分析MT1M在TNBC和正常乳腺组织中的表达及MT1M表达水平与TNBC患者预后的关系.在TNBC细胞MDA-MB-231、BT-549中转染MT1M过表达质粒为MT1M过表达组,转染空载质粒为对照组.利用蛋白质印迹法(Western blot)验证转染后两组细胞中MT1M蛋白水平;通过划痕实验以及Transwell小室侵袭实验检测两组细胞的迁移和侵袭能力;利用Western blot检测两组细胞中E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B蛋白(Akt)以及磷酸化的蛋白激酶B蛋白(p-Akt)水平,两组间比较采用t检验.结果 网站数据分析显示MT1M mRNA水平在TNBC中异常低表达,其异常低表达的患者预后也较差.MT1M过表达组MDA-MB-231、BT-549 细胞中 MT1M 蛋白表达(11.08±1.90、9.00±0.69)明显高于对照组(1.35±0.52、1.41± 0.43),差异有统计学意义(t=8.569、16.220,P＜0.05).划痕实验结果显示MT1M过表达组MDA-MB-231 细胞划痕愈合率[12 h(25.46±10.31)％,24 h(44.55±23.65)％]均明显低于对照组[12h(33.07±9.28)％,24h(60.68±21.89)％],差异有统计学意义(t=4.426、8.202,P＜0.05);MT1M 过表达组 BT-549 细胞划痕愈合率[24 h(30.40±11.52)％,48 h(64.01±7.28)％]均明显低于对照组[24h(57.21±11.98)％,48h(98.99±0.29)％],差异有统计学意义(t=6.780、8.660,P＜0.05).Transwell小室实验结果显示MT1M过表达组MDA-MB-231、BT-549细胞穿过小室数目[(141.67±1.53)、(173.00±11.27)个]明显低于对照组[(339.67±61.86)、(376.00± 19.00)个],差异有统计学意义(t=5.678、17.390,P＜0.05).MT1M 过表达组 MDA-MB-231、BT-549 细胞中 E-cadherin 蛋白表达(4.80±0.65、4.81±1.20)明显高于对照组(1.29±0.26、1.41± 0.68),差异有统计学意义(t=8.745、4.279,P＜0.05).而 Vimentin、PI3K、Akt 以及 p-Akt 蛋白表达分别为 1.25±0.32、1.11±0.13;1.40±0.52、1.52±0.48;1.05±0.07、1.04±0.05;1.35±0.51、1.06±0.07,明显低于对照组(5.16±1.10、6.35±0.13;4.52±0.50、3.37±0.55;1.90±0.18、1.98±0.19;4.55±0.68、3.10±0.59),差异有统计学意义(t=5.932、48.250;7.525、4.420;7.614、8.466;6.524、5.961,P＜0.05).结论 MT1M在三阴性乳腺癌细胞中呈低表达;MT1M可能通过调控PI3K-Akt/上皮-间充质转化影响三阴性乳腺癌细胞的迁移和侵袭.
Abstract
Objective To observe the expression of metallothionein 1M(MT1M)in triple-negative breast cancer(TNBC)cells,and study the effect and mechanism of MT1M on TNBC cell migration and inva-sion.Methods Ualcan and Kaplan-Meier Plotter online websites were used to analyze the expression of MT1M in TNBC and normal breast tissues and the relationship between MT1M expression level and the prog-nosis of TNBC patients.TNBC cells(MDA-MB-231 and BT-549)were transfected with MT1M overexpression plasmid as MT1M overexpression group,and TNBC cells transfected with empty plasmid as control group.Western blotting was used to verify the MT1M protein level in the transfected cells of the two groups.The mi-gration and invasion ability of cells was detected by scratch test and Transwell chamber test.Western blotting was used to detect the levels of E-cadherin,Vimentin,phosphatidylinositol-3-kinase(PI3K),protein kinase B(Akt)and phosphorylated protein kinase B(p-Akt)in the two groups.The t-test was used for comparison between the two groups.Results Website data analysis showed that MT1M mRNA was abnormally low in TNBC,and patients with abnormally low MT1M expression had poor prognosis.The protein expression of MT1M in MDA-MB-231 and BT-549 cells in MT1M overexpression group(11.08±1.90,9.00±0.69)was significantly higher than that in control group(1.35±0.52,1.41±0.43,t=8.569,16.220,P＜0.05).The wound healing rate of MDA-MB-231 cells in MT1M overexpression group[12 h(25.46±10.31)％;24 h(44.55±23.65)％]was significantly lower than that in control group[12 h(33.07±9.28)％;24 h(60.68±21.89)％,t=4.426,8.202,P＜0.05].The wound healing rate of BT-549 cells in MT1M overex-pression group[24 h(30.40±11.52)％;48 h(64.01±7.28)％]was significantly lower than that in con-trol group[24 h(57.21±11.98)％;48 h(98.99±0.29)％,t=6.780,8.660,P＜0.05].Transwell as-say results showed that the number of MDA-MB-231 and BT-549 cells passing through(141.67±1.53,173.00±11.27)in the MT1M overexpression groups was significantly less than that in the control group(339.67±61.86,376.00±19.00,t=5.678,17.390,P＜0.05).The expression of E-cadherin protein in MDA-MB-231 and BT-549 cells in MT1M overexpression group(4.80±0.65,4.81±1.20)was significantly higher than that in control group(1.29±0.26,1.41±0.68,t=8.745,4.279,P＜0.05).The expression of Vimentin,PI3K,Akt and p-Akt was 1.25±0.32,1.11±0.13;1.40±0.52,1.52±0.48;1.05±0.07,1.04±0.05 and 1.35±0.51,1.06±0.07,significantly lower than the control group(5.16±1.10,6.35± 0.13;4.52±0.50,3.37±0.55;1.90±0.18,1.98±0.19;4.55±0.68,3.10±0.59,t=5.932,48.25;7.525,4.420;7.614,8.466;6.524,5.961,P＜0.05).Conclusion The expression of MT1M was low in TNBC cells.MT1M may affect the migration and invasion of TNBC cells by regulating PI3K-Akt/epithelial mesenchymal transformation.
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