高表达ITGB1促进人乳腺上皮MCF10A细胞EMT及迁移
|
摘要
目的探讨ITGB1基因过表达对人乳腺上皮MCF10A细胞迁移和侵袭的影响。方法构建重组质粒p BABEpuro-myc-ITGB1,在人胚肾上皮细胞系293T细胞中包装反转录病毒,转染人乳腺上皮MCF10A细胞系,构建ITGB1基因过表达的稳定细胞系;实验设空白对照组(MCF10A)、阴性对照组(MCF10A-vector)和ITGB1基因过表达组(MCF10A-ITGB1);经嘌呤霉素筛选后,实时荧光定量PCR和蛋白质印迹法检测ITGB1 mRNA及蛋白的表达;Transwell迁移和侵袭实验分别检测细胞株迁移和侵袭能力;蛋白质印迹法进一步检测上皮间质转换(EMT)相关蛋白E-cadherin、vimentin、fibronectin、N-cadherin以及ITGB1下游ILK、p-FAK和FAK蛋白的表达。结果成功构建ITGB1基因稳定过表达人乳腺上皮MCF10A细胞系;ITGB1基因过表达组(MCF10A-ITGB1)与空白对照组(MCF10A)和阴性对照组(MCF10A-vector)细胞相比,迁移能力(P<0.05)和侵袭能力(P<0.01)显著增强;EMT相关蛋白E-cadherin显著下调(P<0.05),vimentin、fibronectin、N-cadherin以及ITGB1下游ILK,p-FAK蛋白显著上调(P<0.05)。结论在人乳腺上皮MCF10A细胞中,过表达ITGB1基因诱导细胞EMT并促进其迁移和侵袭。
Objective To investigate the effect of overexpressed-integrin beta 1( ITGB1) gene on epithelial-mesenchymal transition( EMT) and cell migration in breast epithelial cell line MCF10 A. Methods Recombinant plasmid p BABE-puro-myc-ITGB1 was constructed and retrovirus was packaged in HEK293 T cells. MCF10 A cells were transfected with the retrovirus carrying ITGB1 gene or the controls. Blank group( MCF10A),negative control group( MCF10A-vector),ITGB1 overexpression group( MCF10A-ITGB1) were set up. After treatment with puromycin for 3 days,the expression of ITGB1 in MCF10 A cells was determined by quantitative real-time PCR and Western blot assays. The ability of migration and invasion were analyzed by Transwell assays. The expression of EMT marker proteins( E-cadherin,vimentin,fibronectin and N-cadherin) and ITGB1 downstream proteins( ILK,p-FAK and FAK) in MCF10 A cells were detected by Western blot. Results ITGB1 gene was successfully deliveredinto MCF10 A cells,resulting in a stable expression of ITGB1 mRNA and protein. Compared with MCF10 A and MCF10A-vector cells,the ability of migration and invasion were notably enhanced in MCF10A-ITGB1 cells( P <0. 05). The expression of the epithelial marker E-cadherin protein was downregulated( P < 0. 01),the mesenchymal marker proteins( vimentin,fibronectin and N-cadherin) and ITGB1 downstream proteins( ILK and p-FAK)were upregulated in MCF10A-ITGB1 cells( P < 0. 01). Conclusions ITGB1 induces EMT and promotes cell migration and invasion in breast epithelial cell line MCF10 A.
Objective To investigate the effect of overexpressed-integrin beta 1( ITGB1) gene on epithelial-mesenchymal transition( EMT) and cell migration in breast epithelial cell line MCF10 A. Methods Recombinant plasmid p BABE-puro-myc-ITGB1 was constructed and retrovirus was packaged in HEK293 T cells. MCF10 A cells were transfected with the retrovirus carrying ITGB1 gene or the controls. Blank group( MCF10A),negative control group( MCF10A-vector),ITGB1 overexpression group( MCF10A-ITGB1) were set up. After treatment with puromycin for 3 days,the expression of ITGB1 in MCF10 A cells was determined by quantitative real-time PCR and Western blot assays. The ability of migration and invasion were analyzed by Transwell assays. The expression of EMT marker proteins( E-cadherin,vimentin,fibronectin and N-cadherin) and ITGB1 downstream proteins( ILK,p-FAK and FAK) in MCF10 A cells were detected by Western blot. Results ITGB1 gene was successfully deliveredinto MCF10 A cells,resulting in a stable expression of ITGB1 mRNA and protein. Compared with MCF10 A and MCF10A-vector cells,the ability of migration and invasion were notably enhanced in MCF10A-ITGB1 cells( P <0. 05). The expression of the epithelial marker E-cadherin protein was downregulated( P < 0. 01),the mesenchymal marker proteins( vimentin,fibronectin and N-cadherin) and ITGB1 downstream proteins( ILK and p-FAK)were upregulated in MCF10A-ITGB1 cells( P < 0. 01). Conclusions ITGB1 induces EMT and promotes cell migration and invasion in breast epithelial cell line MCF10 A.
引文
[1]Bill R,Christofori G.The relevance of EMT in breast cancer metastasis:Correlation or causality?[J].FEBS Lett,2015,589:1577-1587.
[2]庞云,程勇.结直肠癌合并糖尿病患者组织中EMT相关蛋白的表达[J].基础医学与临床,2016,36:799-804.
[3]Yang J,Hou Y,Zhou M,et al.Twist induces epithelialmesenchymal transition and cell motility in breast cancer via ITGB1-FAK/ILK signaling axis and its associated downstream network[J].Int J Biochem Cell Biol,2016,71:62-71.
[4]Liu M,Casimiro MC,Wang C,et al.p21CIP1 attenuates Ras-and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo[J].Proc Natl Acad Sci U S A,2009,106:19035-19039.
[5]周明莉,杨佳佳,唐石伏,等.高表达Twist基因促进人乳腺癌细胞MCF-7干细胞样细胞富集和迁移[J].第三军医大学学报,2014,36:2083-2086.
[6]Pegoraro S,Ros G,Piazza S,et al.HMGA1 promotes metastatic processes in basal-like breast cancer regulating EMT and stemness[J].Oncotarget,2013,4:1293-1308.
[7]Mock K,Preca BT,Brummer T,et al.The EMT-activator ZEB1 induces bone metastasis associated genes including BMP-inhibitors[J].Oncotarget,2015,6:14399-14412.
[8]李洪利,尹崇高.PTTG1通过上皮-间质转化促进非小细胞肺癌的迁移和侵袭[J].基础医学与临床,2014,34:1376-1380.
[9]Wang L,Zhang Y,Lv W,et al.Long non-coding RNA Linc-ITGB1 knockdown inhibits cell migration and invasion in GBC-SD/M and GBC-SD gallbladder cancer cell lines[J].Chem Biol Drug Des,2015,86:1064-1071.
[10]Hunt S,Jones AV,Hinsley EE,et al.MicroRNA-124suppresses oral squamous cell carcinoma motility by targeting ITGB1[J].FEBS Lett,2011,585:187-192.
[11]Li XL,Hara T,Choi Y,et al.A p21-ZEB1 complex inhibits epithelial-mesenchymal transition through the microRNA 183-96-182 cluster[J].Mol Cell Biol,2014,34:533-550.
[2]庞云,程勇.结直肠癌合并糖尿病患者组织中EMT相关蛋白的表达[J].基础医学与临床,2016,36:799-804.
[3]Yang J,Hou Y,Zhou M,et al.Twist induces epithelialmesenchymal transition and cell motility in breast cancer via ITGB1-FAK/ILK signaling axis and its associated downstream network[J].Int J Biochem Cell Biol,2016,71:62-71.
[4]Liu M,Casimiro MC,Wang C,et al.p21CIP1 attenuates Ras-and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo[J].Proc Natl Acad Sci U S A,2009,106:19035-19039.
[5]周明莉,杨佳佳,唐石伏,等.高表达Twist基因促进人乳腺癌细胞MCF-7干细胞样细胞富集和迁移[J].第三军医大学学报,2014,36:2083-2086.
[6]Pegoraro S,Ros G,Piazza S,et al.HMGA1 promotes metastatic processes in basal-like breast cancer regulating EMT and stemness[J].Oncotarget,2013,4:1293-1308.
[7]Mock K,Preca BT,Brummer T,et al.The EMT-activator ZEB1 induces bone metastasis associated genes including BMP-inhibitors[J].Oncotarget,2015,6:14399-14412.
[8]李洪利,尹崇高.PTTG1通过上皮-间质转化促进非小细胞肺癌的迁移和侵袭[J].基础医学与临床,2014,34:1376-1380.
[9]Wang L,Zhang Y,Lv W,et al.Long non-coding RNA Linc-ITGB1 knockdown inhibits cell migration and invasion in GBC-SD/M and GBC-SD gallbladder cancer cell lines[J].Chem Biol Drug Des,2015,86:1064-1071.
[10]Hunt S,Jones AV,Hinsley EE,et al.MicroRNA-124suppresses oral squamous cell carcinoma motility by targeting ITGB1[J].FEBS Lett,2011,585:187-192.
[11]Li XL,Hara T,Choi Y,et al.A p21-ZEB1 complex inhibits epithelial-mesenchymal transition through the microRNA 183-96-182 cluster[J].Mol Cell Biol,2014,34:533-550.