Notch3调控Hes2逆转三阴性乳腺癌上皮-间质转化的机制研究

doi:10.11958/20220306

天津医药 ›› 2022, Vol. 50 ›› Issue (8): 785-790.doi: 10.11958/20220306

• 细胞与分子生物学 • 下一篇

Notch3调控Hes2逆转三阴性乳腺癌上皮-间质转化的机制研究

田园¹^,²(), 牟雅君¹^,², 杨文秀¹, 豆晓伟²^,^Δ()

1贵州医科大学临床医学院病理教研室（邮编550004）
2贵州医科大学附属医院临床研究中心

收稿日期:2022-03-03 修回日期:2022-04-18 出版日期:2022-08-15 发布日期:2022-08-12
通讯作者: 豆晓伟 E-mail:tianyuan967@163.com;douxw@gmc.edu.cn
作者简介:田园（1996）,女,硕士在读,主要从事乳腺癌转移及机制方面研究。E-mail： tianyuan967@163.com
基金资助:
国家自然科学基金资助项目(81860516)

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

TIAN Yuan¹^,²(), MOU Yajun¹^,², YANG Wenxiu¹, DOU Xiaowei²^,^Δ()

1 Department of Pathology, School of Clinical Medicine, Guizhou Medical University, Guiyang 550004, China
2 Clinical Research Center, the Affiliated Hospital of Guizhou Medical University

Received:2022-03-03 Revised:2022-04-18 Published:2022-08-15 Online:2022-08-12
Contact: DOU Xiaowei E-mail:tianyuan967@163.com;douxw@gmc.edu.cn

田园, 牟雅君, 杨文秀, 豆晓伟. Notch3调控Hes2逆转三阴性乳腺癌上皮-间质转化的机制研究[J]. 天津医药, 2022, 50(8): 785-790.

TIAN Yuan, MOU Yajun, YANG Wenxiu, DOU Xiaowei. The mechanism of Notch3 regulating Hes2 to reverse epithelial-mesenchymal transition in triple-negative breast cancer[J]. Tianjin Medical Journal, 2022, 50(8): 785-790.

摘要/Abstract

摘要：

目的探究Notch3逆转三阴性乳腺癌（TNBC）上皮-间质转化（EMT）及抑制肿瘤转移的分子机制。方法将Notch3过表达质粒（pcDNA3.1-Notch3-GFP）及其阴性对照质粒（pcDNA3.1-NC-GFP）、Hes2敲低的小分子干扰质粒（si-Hes2-GFP）及其阴性对照质粒（si-NC-GFP）转染TNBC MDA-MB-231细胞,分为对照组（不进行任何转染）、pc-NC组（转染pcDNA3.1-NC-GFP）、Notch3过表达组（pc-Notch3组,转染pcDNA3.1-Notch3-GFP）、Notch3过表达+si-NC组（pc-Notch3+si-NC组,共转染pcDNA3.1-Notch3-GFP和si-NC-GFP）、Notch3过表达+Hes2敲低组（pc-Notch3+si-Hes2组,共转染pcDNA3.1-Notch3-GFP和si-Hes2-GFP）。采用实时荧光定量PCR和Western blot分别检测细胞Notch3、Hes2 mRNA和蛋白表达;细胞计数试剂盒-8检测细胞增殖活性;划痕愈合实验和Transwell小室实验检测细胞迁移、侵袭能力;Western blot检测细胞EMT相关蛋白[E-钙黏蛋白（E-cadherin）和波形蛋白（Vimentin）]表达。结果与对照组和pc-NC组比较,pc-Notch3组和pc-Notch3+si-NC组Notch3、Hes2 mRNA和蛋白、E-cadherin蛋白水平显著升高,细胞增殖活性、迁移率、穿膜细胞数、Vimentin蛋白水平显著降低（P<0.05）;与pc-Notch3组和pc-Notch3+si-NC组比较,pc-Notch3+si-Hes2组Hes2 mRNA和蛋白、E-cadherin蛋白水平显著降低,细胞增殖活性、迁移率、穿膜细胞数、Vimentin蛋白水平显著升高（P<0.05）。结论 Notch3可能通过上调Hes2来逆转TNBC的EMT,抑制肿瘤转移。

关键词: 三阴性乳腺癌, 上皮-间质转化, Notch3, Hes2, MDA-MB-231细胞, 肿瘤转移

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

中图分类号:

R737.9

图/表 9

参考文献 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^＊＊

Notch3调控Hes2逆转三阴性乳腺癌上皮-间质转化的机制研究

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

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组别	细胞迁移率（%）	穿膜细胞数（个/视野）
对照组	60.49±7.15	355.05±46.47
pc-NC组	57.30±6.82	347.61±45.34
pc-Notch3组	36.28±5.45^ab	152.73±27.28^ab
pc-Notch3+si-NC组	34.61±5.70^ab	149.56±23.31^ab
pc-Notch3+si-Hes2组	54.02±7.26^cd	336.21±38.59^cd
F	10.479^＊＊	25.240^＊＊

组别	E-cadherin蛋白	Vimentin蛋白
对照组	0.20±0.03	0.36±0.04
pc-NC组	0.22±0.04	0.39±0.05
pc-Notch3组	0.57±0.06^ab	0.15±0.03^ab
pc-Notch3+si-NC组	0.61±0.07^ab	0.13±0.02^ab
pc-Notch3+si-Hes2组	0.25±0.04^cd	0.30±0.05^cd
F	48.631^＊＊	27.209^＊＊

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