Yao Liang, Tian Fuguo
Department of Breast Surgery, Shanxi Provincial Tumor Hospital and Affiliated Tumor Hospital of Shanxi Medical University, Taiyuan, Shanxi 030013, P.R. China.
Exp Ther Med. 2022 May 30;24(1):473. doi: 10.3892/etm.2022.11400. eCollection 2022 Jul.
Breast cancer is a highly heterogeneous tumor, among which triple negative breast cancer (TNBC) is the most invasive and prone to recurrence and metastasis. The present study aimed to investigate the regulatory mechanisms of glutamate-rich WD-repeat-containing protein 1 (GRWD1) in TNBC cells. The expression of GRWD1 in the normal human breast epithelial cells and human breast cancer cells was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The transfection effects of small interfering RNA (siRNA)-GRWD1 and overexpression (Ov)-Notch1 were also confirmed by RT-qPCR and western blotting. The proliferation, apoptosis, invasion and migration of transfected cells were in turn analyzed by Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine, Matrigel and wound healing assays. The expression of proteins related to proliferation, apoptosis, metastasis, epithelial-mesenchymal transition and the Notch signaling pathway was detected by western blotting. As a result, GRWD1 expression was upregulated in breast cancer cells and was revealed to be highest in MDA-MB-231 and HCC1937 cells. GRWD1 knockdown suppressed TNBC cell proliferation, invasion and migration and promoted TNBC cell apoptosis. Furthermore, the expression of Notch1 and Notch4 was inhibited by GRWD1 knockdown. The expression of downstream genes of the Notch signaling pathway Hes1, Hes5, Hey1, Hey2, p21, c-Myc, cyclin D1, human epidermal growth factor 2 receptor and NF-κB were all suppressed after siRNA-GRWD1 transfection. However, Notch1 overexpression reversed the effect of GRWD1 knockdown on biological behaviors of TNBC cells. In conclusion, GRWD1 knockdown could suppress the proliferation, invasion and migration and promoted apoptosis of TNBC cells through inhibiting the Notch signaling pathway.
乳腺癌是一种高度异质性肿瘤，其中三阴性乳腺癌（TNBC）侵袭性最强，最易复发和转移。本研究旨在探讨富含谷氨酸的WD重复结构域蛋白1（GRWD1）在TNBC细胞中的调控机制。通过逆转录定量聚合酶链反应（RT-qPCR）和蛋白质印迹分析检测GRWD1在正常人乳腺上皮细胞和人乳腺癌细胞中的表达。还通过RT-qPCR和蛋白质印迹法证实了小干扰RNA（siRNA）-GRWD1和过表达（Ov）-Notch1的转染效果。依次通过细胞计数试剂盒-8、5-乙炔基-2'-脱氧尿苷、基质胶和伤口愈合试验分析转染细胞的增殖、凋亡、侵袭和迁移情况。通过蛋白质印迹法检测与增殖、凋亡、转移、上皮-间质转化和Notch信号通路相关的蛋白质表达。结果显示，GRWD1在乳腺癌细胞中表达上调，且在MDA-MB-231和HCC1937细胞中表达最高。GRWD1基因敲低抑制了TNBC细胞的增殖、侵袭和迁移，并促进了TNBC细胞凋亡。此外，GRWD1基因敲低抑制了Notch1和Notch4的表达。siRNA-GRWD1转染后，Notch信号通路下游基因Hes1、Hes5、Hey1、Hey2、p21、c-Myc、细胞周期蛋白D1、人表皮生长因子2受体和核因子κB的表达均受到抑制。然而，Notch1过表达逆转了GRWD1基因敲低对TNBC细胞生物学行为的影响。总之，GRWD1基因敲低可通过抑制Notch信号通路来抑制TNBC细胞的增殖、侵袭和迁移，并促进其凋亡。
