三阴性乳腺癌细胞脂肪非典型钙黏蛋白4表达下调的生物学作用及机制

Biological actions of down-regulation of FAT atypical cadherin 4 in triple negative breast cancer cells and the mechanism

背景与目的三阴性乳腺癌(TNBC)容易发生侵袭转移,恶性程度极高。笔者团队前期研究发现脂肪非典型钙黏蛋白4(FAT4)在TNBC组织中呈低表达且与预后相关,进而本研究进一步探讨FAT4对TNBC细胞生物学行为的影响,及其相关机制。方法用qRT-PCR及Western blot检测正常人乳腺上皮细胞系(MCF-10A)及不同TNBC细胞系(BT-549、MDA-MB-231、MDA-MB-468、MDA-MB-436)中FAT4的表达水平。选择其中合适的TNBC细胞系,分别转染FAT4-shRNA(FAT4敲低组)、阴性对照序列(阴性对照组),以未处理的TNBC细胞为空白对照组,分别采用CCK-8实验、流式细胞术、Transwell小室实验检测细胞增殖能力、凋亡率、侵袭与转移能力的变化,并用Western blot检细胞中Hippo信号通路下游靶点YAP及上皮间质转化(EMT)相关蛋白的变化。结果与正常人乳腺上皮细胞比较,各TNBC细胞系中FAT4的mRNA与蛋白表达量均不同程度的降低(均P<0.05)。功能实验选用BT-549细胞和MDA-MB-436细胞。无论在BT-549细胞或MDA-MB-436细胞中,与空白对照组比较,FAT4敲低组在转染后24、48、72 h的增殖能力均明显升高、凋亡率明显降低、侵袭与转移能力明显增强(均P<0.05);YAP蛋白表达量无明显变化(P>0.05),但磷酸化YAP(p-YAP)表达量明显降低,上皮标志物E-cadherin水平明显降低,而间质标志物N-cadherin水平明显升高(均P<0.05);阴性对照组与空白对照组间以上指标差异均无统计学意义(均P>0.05)。结论FAT4表达水平在TNBC细胞中普遍降低,FAT4表达的降低能增强TNBC细胞增殖和侵袭迁移能力、减少TNBC细胞凋亡,其机制可能与FAT4降低后Hippo信号通路下游的YAP蛋白磷酸化减少,从而促进EMT进程有关。

Background and Aims Triple negative breast cancer(TNBC)is more prone to invasion and metastasis,with a high degree of malignancy.Previous study of the authors’team found the low expression of Fat atypical cadherin 4(FAT4)in TNBC tissue and its prognostic relevance.Thus,this study was conducted to further investigate the effects of FAT4 on the biological behavior of TNBC cells and the associated mechanism.Methods The expression levels of FAT4 in normal mammary epithelial cell line(MCF-10A)and different TNBC cell lines(BT-549,MDA-MB-231,MDA-MB-468,and MDA-MB-436)were detected by qRT-PCR and Western blot,respectively.Then,the suitable TNBC cell lines were selected,and were transfected with FAT4-shRNA(FAT4 knockdown group)or scrambled sequences(negative control group),using untransfected TNBC cells as blank control group.In these cells,the changes in proliferation ability,apoptosis rate,and invasion/metastasis ability were determined by CCK-8 assay,flow cytometry and Transwell chamber assay,respectively.Meanwhile,the changes in YAP protein,a downstream target of the Hippo signaling pathway,as well as the epithelial mesenchymal transformation(EMT)-related proteins were also examined by Western blot analysis.Results Compared with normal mammary epithelial cell line,the mRNA and protein expression levels of FAT4 in all the studied TNBC cell lines were decreased with varying degrees(all P<0.05).The BT-549 and MDA-MB-436 cells were selected for functional experiments.In FAT4 knockdown group compared with blank control group of either the BT-549 or MDA-MB-436 cells,the proliferative abilities were significantly increased at 24,48,and 72 h after transfection,the apoptosis rate was significantly decreased,and the invasion/metastasis ability was significantly enhanced(all P<0.05);the expression level of YAP protein showed no significant change(P>0.05),but the level of phosphorylated YAP(p-YAP)was significantly decreased,and the level of epithelial marker E-cadherin was significantly decreased and mesenchymal marker N-cadherin was significantly increased(all P<0.05);the differences in all above parameters showed no statistical significance between negative control group and blank control group(all P>0.05).Conclusion The expression of FAT4 is generally down-regulated in TNBC cells.The FAT4 down-regulation can enhance the proliferation and invasion/migration abilities and weaken the apoptosis of TNBC cells,and the mechanism may be probably associated the reduced phosphorylation of YAP protein in downstream Hippo signaling pathway after FAT4 down-regulation,and thereby promoting the EMT process.