相关成纤维细胞自噬调控对乳腺癌MCF-7细胞上皮间质转化影响

目的 研究报道,乳腺癌相关成纤维细胞(cancer associated fibroblasts,CAFs)在乳腺癌的侵袭、转移中起到促进作用,而机制未明.本研究探讨乳腺癌CAFs自噬调控对MCF-7细胞EMT进程的影响.方法 采用胶原酶消化法对CAFs及配对人乳腺癌正常成纤维细胞(NBFs)进行原代培养,采用免疫荧光和蛋白质印迹法进行鉴定;建立CAFs和NBFs与MCF-7条件培养基(CM)共培养及Transwell小室共培养模型,共分为3组进行实验,空白组即MCF-7单独培养组(CON),对照组即正常乳腺癌成纤维细胞与MCF-7共培组(NBFs),实验组即乳腺癌相关成纤维细胞与MCF-7共培养组(CAFs).Transwell侵袭及迁移实验检测各组MCF-7细胞侵袭和迁移能力.蛋白印迹法检测各组MCF-7细胞EMT上皮标志分子E-cadherin蛋白和间质标志分子N-cadherin和Vimentin蛋白表达情况;蛋白质印迹法检测CAFs与NBFs自噬相关蛋白LC3B、Beclin1和p62表达差异;自噬抑制剂3MA处理CAFs后蛋白质印迹法检测CAFs上述自噬相关蛋白表达差异;予以自噬抑制剂3MA预处理CAFs后,收集CM与MCF-7共培养,分3组进行实验,空白组即MCF-7单独培养组(CON);对照组即MCF-7与CAFs-CM共培养组(CAFs-CM);实验组即MCF-7与3MA-CAFs-CM共培养组(3MA-CAFs-CM).蛋白质印迹法检测各组MCF-7细胞EMT上皮标志分子E-cadherin蛋白和间质标志分子N-cadherin和Vimentin蛋白表达情况.结果 通过酶消化法原代培养可获取α-SMA(+)、Vimentin(+)E-cadherin(-)具有CAFs典型特征的肌成纤维细胞;Transwell侵袭、迁移实验证实CAFs可促进MCF-7细胞侵袭、迁移;蛋白质印迹法检测显示,CAFs组MCF-7 E-cadherin蛋白表达量为0.432±0.012,比NBFs组的0.585±0.02及空白组的0.659±0.016表达水平低,F=120.098,P<0.05.而CAFs组间质蛋白标志物Vimentin蛋白表达量为0.309±0.065,比NBFs组的0.103±0.011及空白组的0.062±0.009表达低,F=42.395,P均<0.05.CAFs组N-cadherin蛋白表达量为1.439±0.07比BNFs组的1.3347±0.028及空白组的1.176±0.021表达低,F=22.578,P<0.05,提示CAFs促进MCF-7发生EMT;CAFs细胞的Beclin1蛋白表达量为2.950±1.261,高于NBFs的0.862±0.727,t=-3.21,P<0.05;CAFs细胞的LC3BⅡ/LC3BⅠ灰度值比值为2.937±0.194,高于NBFs的2.314±0.224,t=-3.638,P<0.05;而CAFs p62蛋白表达量为0.342±0.093,低于NBFs的0.750±0.021,t=7.432,P<0.05,提示CAFs自噬水平高于NBFs.予以自噬抑制剂3MA处理CAFs后,处理组CAFs细胞的Beclin1蛋白表达量为0.211±0.008,低于未处理组的0.266±0.009,t=8.094,P<0.05;处理组CAFs细胞的LC3BⅡ/LC3BⅠ灰度值比值为0.496±0.004,低于未处理组的0.618±0.022,t=9.312,P<0.05;而处理组的p62蛋白表达量为0.307±0.043,高于未处理组的0.143±0.008,t=-6.471,P<0.05,说明3MA可以抑制CAFs自噬水平.自噬调控后共培养,各组E-cadherin蛋白表达有统计学意义,F=44.157,P<0.05;与对照组MCF-7细胞E-cadherin蛋白表达量1.209±0.010相比,CAFs-CM组的1.062±0.027表达下调,而3MA-CAFs-CM组MCF-7细胞E-cadherin蛋白表达量为1.104±0.019,较CAFs-CM组表达上调,均P<0.05;3组MCF-7细胞Vimentin和N-cadherin表达差异有统计学意义,P<0.05.结论 CAFs能够促进MCF-7发生EMT并增强其侵袭转移能力,而抑制CAFs自噬后,其诱导的MCF-7发生EMT进程受到抑制.

Influences of autophagic regulations of carcinoma associated fibroblasts on the EMT process of breast cancer MCF-7 cell line

OBJECTIVE To investigate the influences of autophagic regulations of CAFs (carcinoma associated fibroblasts) on the EMT process of breast cancer MCF-7 cell line.METHODS Breast cancer tissue and para-carcinoma tissue were isolated from 3 breast cancer patients to obtain CAFs and NBFs (normal breast fibroblasts)by primary culture cell in digestion methods and identified by immunofluorescence assay and western blot.The fibroblasts and cell lines MCF-7 co-culture model were established by conditioned medium co-culture and transwell co-culture.CAFs co-culture with MCF-7 as experimental group (CAFs),NBFs co-culture with MCF-7 as control group(NBFs) and MCF-7 cultured in DMEM was blank group (CON).Transwell matrigel invasion assay and transwell migration assay were used to test the ability of MCF-7 cells to invade and migrate.Expressions of EMT-related markers(such as E-cadherin,N-cadherin,Vimentin) in MCF-7 cell were detected via western-blot.The differences of autophagy-related protein (LC3B,beclin1,P62)expression were tested between CAFs and NBFs via western-blot.Treated CAFs with 3MA,then the protein expression change of autophagy-related markers was tested via western-blot.Conditioned medium of cultured CAFs(CAF-CM) or CAFs pre-treated with 3MA(3MA-CAFs-CM) was collected then co-cultureed with MCF-7.The protein expression differences of EMT-relatedmarkers (E-cadherin,N-cadherin,Vimentin) in CAF-CM treated MCF-7 cells,3MA-CAFs-CM treated MCF-7 and MCF-7 cultured in DMEM were detected via western-blot.RESULTS CAFs and NBFs can be obtained by primary culture from breast cancer tissue using enzyme digestion method.The harvested cells were identified as CAFs with a typical SMA(+)vimentin(+) Ecadherin(-) phenotype.The transwell matrigel invasion assay and migration assay showed that CAFs could promote the invasion and migration ability of MCF-7 cells.Western blot was used to test the expression of EMT related protein,compared to the NBFs group(0.585±0.02) and Control(0.659±0.016),the level of epithelial markers such as E-cadherin(0.432±0.012) of MCF-7 cells was downregulated in CAFs group(F=120.098,P<0.05).But the levels of mesenchymal markers such as N-cadherin(0.309±0.065) in CAFs group were upregulated than in NBFs group(0.103±0.011) and Control(0.062±0.009)(F=42.359,P<0.05).The level of Vimentin in CAFs group(1.439±0.07) was also upregulated than in NBFs group(1.3347±0.028) and Control(1.176±0.021)(F=22.578,P<0.05).The change of EMT related protein indicated CAFs promote the EMT process of MCF-7 cells.Western blot showed the level of autophagy-related protein beclin1 in CAFs was 2.950±1.261 that is higher than 0.862±0.727 in NBFs(t=-3.21,P<0.05),and the level of LC3BⅡ/LC3BⅠ in CAFs was 2.937±0.194 that was also higher than 2.314±0.224 in NBFs (t=-3.666,P<0.05),On the contrary,the level of p62 in CAFs was 0.342±0.093 and was lower than 0.750±0.021 in NBFs(t=7.432,P<0.05)which indicated that the autophagy level of CAFs was higher than that of NBFs.After treated with 3MA,Western blot showed the level of autophagy-related protein beclin1 was 0.211±0.008 in treated group which was lower than 0.266±0.009 in untreated CAFs(t=8.094,P<0.05).And the level of LC3B Ⅱ/LC3B Ⅰ in treated group was 0.496±0.004 which was lower than 0.618±0.022 in CAFs group((t=9.312,P<0.05)).The level of p62 in treated group was 0.307±0.043 which was higher than 0.143±0.008 in CAFs group((t=-6.471,P<0.05).The change of autophagy-related protein between treated and untreated CAFs showed autophage inhibited in CAFs.Western blot showed the levels of epithelial markers E-cadherin were different in Control,CAFs-CM and 3MA-CAFs-CM(F=44.157,P<0.05).The expression of E-cadherin in CAFs-CM group was 1.062±0.027,which was lower than that in control group,1.209±0.010 (P<0.05),but the expression of E-cadherin in 3MA-CAFs-CM was 1.104±0.019 which was higher than that of CAFs-CM group(P<0.05).The expression of Vimentin,N-cadherin in Control,CAFs-CM and 3MA-CAFs-CM group were different,P<0.05.CONCLUSIONS CAFs induces EMT process of MCF-7 and enhances the migration and invansion ability of MCF-7 cell.Autopahagic inhibition of CAFs suppressed MCF-7 EMT process.