The mechanism of Notch3 regulating Hes2 to reverse epithelial-mesenchymal transition in triple-negative breast cancer

doi:10.11958/20220306

Abstract

Abstract:

Objective To explore the molecular mechanism of Notch3 reversing epithelial-mesenchymal transition (EMT) and inhibiting tumor metastasis in triple negative breast cancer (TNBC). Methods The Notch3 overexpression plasmid (pcDNA3.1-Notch3-GFP) and its negative control plasmid (pcDNA3.1-NC-GFP), the small molecule interference plasmid for Hes2 knockdown (si-Hes2-GFP) and its negative control plasmid (si-NC-GFP) were transfected into TNBC MDA-MB-231 cells. Cells were divided into the control group (no transfection was performed), the pc-NC group (transfection pcDNA3.1-NC-GFP), the Notch3 overexpression group (pc-Notch3 group, transfection pcDNA3.1-Notch3-GFP), the Notch3 overexpression+si-NC group (pc-Notch3+si-NC group, co-transfected pcDNA3.1-Notch3-GFP and si-NC-GFP), and the Notch3 overexpression+Hes2 knockdown group (pc-Notch3+si-Hes2 group, co-transfected pcDNA3.1-Notch3-GFP and si-Hes2-GFP). Real-time quantitative PCR and Western blot assay were performed to measure mRNA and protein expression levels of Notch3 and Hes2 in cells to verify the transfection efficiency. The cell counting kit-8 assay was performed to measure cell proliferation activity. Scratch healing assay and Transwell chamber assay were performed to measure cell migration and invasion abilities. Western blot assay was performed to measure EMT-related proteins (E-cadherin and Vimentin) in cells. Results Compared with the control group and the pc-NC group, the Notch3, Hes2 mRNA and protein, E-cadherin protein levels were significantly increased in the pc-Notch3 group and the pc-Notch3+si-NC group, and the proliferation activity, mobility, number of transmembrane cells and Vimentin protein levels were significantly decreased (P<0.05). Compared with the pc-Notch3 group and the pc-Notch3+si-NC group, the Hes2 mRNA and protein levels and E-cadherin protein were significantly decreased in the pc-Notch3+si-Hes2 group, and the proliferation activity, mobility, number of transmembrane cells and Vimentin protein levels were significantly increased (P<0.05). Conclusion Notch3 may reverse the EMT of TNBC and inhibit tumor metastasis by up-regulating Hes2.

Key words: triple negative breast neoplasms, epithelial-mesenchymal transition, Notch3, Hes2, MDA-MB-231 cells, tumor metastasis

CLC Number:

R737.9

Figures/Tables 9

References 20

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基因名称	引物序列	产物大小（bp）
Notch3	上游：5'-TGGCGACCTCACTTACGACT-3'	195
Notch3	下游：5'-CACTGGCAGTTATAGGTGTTGAC-3'	195
Hes2	上游：5'-TCGGTTTCCCTTTGCGTG-3'	149
Hes2	下游：5'-AGGCTCTGGTTGATGCG-3'	149
β-actin	上游：5'-AGAGCTACGAGCTGCCTGAC-3'	175
β-actin	下游：5'-AGCACTGTGTTGGCGTACAG-3'	175

基因名称	引物序列	产物大小（bp）
Notch3	上游：5'-TGGCGACCTCACTTACGACT-3'	195
Notch3	下游：5'-CACTGGCAGTTATAGGTGTTGAC-3'	195
Hes2	上游：5'-TCGGTTTCCCTTTGCGTG-3'	149
Hes2	下游：5'-AGGCTCTGGTTGATGCG-3'	149
β-actin	上游：5'-AGAGCTACGAGCTGCCTGAC-3'	175
β-actin	下游：5'-AGCACTGTGTTGGCGTACAG-3'	175

组别	Notch3 mRNA	Notch3 蛋白	Hes2 mRNA	Hes2 蛋白
对照组	1.00±0.00	0.15±0.02	1.00±0.00	0.12±0.02
pc-NC组	1.04±0.13	0.17±0.03	1.01±0.12	0.13±0.02
pc-Notch3组	5.87±0.46^ab	0.56±0.07^ab	3.65±0.29^ab	0.43±0.05^ab
pc-Notch3+si-NC组	5.91±0.50^ab	0.58±0.06^ab	3.70±0.31^ab	0.45±0.06^ab
pc-Notch3+si-Hes2组	5.82±0.47^ab	0.54±0.07^ab	1.39±0.20^cd	0.17±0.03^cd
F	151.164^＊＊	49.235^＊＊	125.515^＊＊	52.692^＊＊

组别	Notch3 mRNA	Notch3 蛋白	Hes2 mRNA	Hes2 蛋白
对照组	1.00±0.00	0.15±0.02	1.00±0.00	0.12±0.02
pc-NC组	1.04±0.13	0.17±0.03	1.01±0.12	0.13±0.02
pc-Notch3组	5.87±0.46^ab	0.56±0.07^ab	3.65±0.29^ab	0.43±0.05^ab
pc-Notch3+si-NC组	5.91±0.50^ab	0.58±0.06^ab	3.70±0.31^ab	0.45±0.06^ab
pc-Notch3+si-Hes2组	5.82±0.47^ab	0.54±0.07^ab	1.39±0.20^cd	0.17±0.03^cd
F	151.164^＊＊	49.235^＊＊	125.515^＊＊	52.692^＊＊

组别	细胞增殖活性（A₄₅₀）
组别	0 h	24 h	48 h	72 h	F
对照组	0.22±0.03	0.40±0.05^A	0.77±0.09^AB	0.95±0.10^ABC	62.307^＊＊
pc-NC组	0.21±0.03	0.45±0.04^A	0.81±0.08^AB	0.97±0.09^ABC	83.577^＊＊
pc-Notch3组	0.23±0.04	0.36±0.05^A	0.50±0.06^abAB	0.65±0.08^abABC	27.830^＊＊
pc-Notch3+si-NC组	0.20±0.03	0.34±0.06^A	0.47±0.07^abAB	0.61±0.07^abABC	25.874^＊＊
pc-Notch3+si-Hes2组	0.23±0.02	0.42±0.07^A	0.72±0.08^cdAB	0.90±0.11^cdABC	45.290^＊＊
F	0.543	1.967	13.199^＊＊	10.728^＊＊