Notch1信号通过调控细胞增殖、迁移、侵袭和糖酵解参与子宫腺肌病的发生及发展

doi:10.12122/j.issn.1673-4254.2024.08.19

摘要/Abstract

摘要：

目的基于Notch1信号观察子宫腺肌病（AM）在位内膜及Ishikawa细胞系糖酵解相关因子和蛋白的表达，并探讨子宫腺肌病的发生机制。方法收集AM（AM组，n=8）及子宫肌瘤（CON组，n=8）在位子宫内膜组织，通过临床特征评估两组子宫内膜的可比性， qRT-PCR和Western blot检测其Notch1信号及糖酵解相关因子和蛋白的表达，通过葡萄糖和乳酸试验检测其葡萄糖和乳酸的含量；建立过表达Notch1的Ishikawa细胞慢病毒稳转株， CCK-8检测其细胞存活率， Transwell迁移和侵袭实验检测其迁移和侵袭能力，细胞外酸化速率检测其糖酵解储备。结果与CON组子宫内膜组织比较，AM组糖类抗原125（CA125）水平明显高于CON组（P<0.01），AM患者在位子宫内膜组织Notch1、HK2和PDHA的mRNA相对表达量增加（P<0.01或P<0.05），Notch1、GLUT1、HK2、PKM和PDHA蛋白相对表达量均显著升高（P<0.01或P<0.05），葡萄糖含量显著下降、乳酸水平显著升高（P<0.01或P<0.05）。与对照病毒空载体（ov-NC）比较，过表达Notch1的Ishikawa细胞慢病毒稳转株（ov-Notch1）的细胞存活率显著升高（P<0.05），细胞迁移和侵袭细胞数量显著增加（P<0.05），酵解容量和酵解储备显著升高（P<0.01或P<0.05）。结论 Notch1信号可能通过调控细胞的增殖、迁移、侵袭和糖酵解过程，进而参与AM的发生发展。

关键词: 子宫腺肌病, 子宫内膜, 糖酵解, Ishikawa细胞系, 慢病毒

Abstract:

Objective To investigate the expressions of glycolysis-related factors and changes in Notch1 signaling in endometrial tissues of adenomyosis (AM) and Ishikawa cells to explore the pathogenesis of AM. Methods Eutopic endometrial tissues were collected from 8 patients with AM and 8 patients with uterine fibroids matched for clinical characteristics (control group). The expressions of Notch1 signaling proteins and glycolysis-related factors in the collected tissues were detected using qRT-PCR and Western blotting, and the levels of glucose and lactic acid were determined. An Ishikawa cell model with lentivirus-mediated stable Notch1 overexpression was established for assessing cell survival rate with CCK-8 assay, cell migration and invasion abilities with Transwell migration and invasion assays, and glycolytic capacity by determining the extracellular acidification rate. Results Compared with those in the control group, the endometrial tissues in AM group showed significantly increased expression level of carbohydrate antigen 125 (CA125), increased mRNA expression levels of Notch1, HK2 and PDHA and protein expressions of Notch1, GLUT1, HK2, PKM and PDHA, lowered glucose level and increased lactate level. The Ishikawa cell models with stable Notch1 overexpression exhibited significantly increased cell survival rate with attenuated cell migration and invasion abilities and decreased glycolysis capacity and reserve. Conclusion The Notch1 signaling pathway participates in the pathogenesis of AM possibly by regulating the proliferation, migration, invasion and glycolysis of endometrial cells.

Key words: adenomyosis, endometrium, glycolysis, Ishikawa, lentivirus

温小慧, 黄诗雅, 刘学红, 李坤寅, 关永格. Notch1信号通过调控细胞增殖、迁移、侵袭和糖酵解参与子宫腺肌病的发生及发展[J]. 南方医科大学学报, 2024, 44(8): 1599-1604.

Xiaohui WEN, Shiya HUANG, Xuehong LIU, Kunyin LI, Yongge GUAN. Role of Notch 1 signaling and glycolysis in the pathogenic mechanism of adenomyosis[J]. Journal of Southern Medical University, 2024, 44(8): 1599-1604.

图/表 9

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Primer	Sequence (5' to 3')
Notch1	TGGACCAGATTGGGGAGTTC
Notch1	GCACACTCGTCTGTGTTGAC
GLUT1	TGGCATCAACGCTGTCTTCT
GLUT1	AGCCAATGGTGGCATACACA
HK2	GTGAATCGGAGAGGTCCCAC
HK2	GCTAACTTCGGCCACAGGAT
PKM	ATGTCGAAGCCCCATAGTGAA
PKM	TGGGTGGTGAATCAATGTCCA
PDHA	ATGGAATGGGAACGTCTGTTG
PDHA	CCTCTCGGACGCACAGGATA
GAPDH	CAGGAGGCATTGCTGATGAT
GAPDH	GAAGGCTGGGGCTCATTT

表2 子宫内膜来源患者的基线特征

Tab.2 Baseline characteristics of the patients with adenomyosis and uterine fibroids ［n=8, Mean±SD or M (P25-P75)］

Group	Age (year)	Uterine volume (cm³)	Number of pregnancies	Number of abortions	CA125 (U/mL)
CON	46.63±2.78	280.38 (128.75-1009.46)	3.00 (2.00-3.00)	0.00 (0.00-1.75)	14.44 (12.16-23.82)
AM	44.88±2.95	636.08 (396.14-1020.82)	3.50 (2.25-5.00)	1.50 (0.25-3.50)	45.01 (28.51-73.14)**

表3 在位子宫内膜组织Notch1信号及糖酵解相关因子mRNA的表达

Tab.3 Relative mRNA expressions of Notch1 signaling pathway and glycolysis-related factors in the endometrial tissue ［n=8, Mean±SD or M (P25-P75)］

Group	Notch1	GLUT1	HK2	PKM	PDHA
CON	1.31±0.78	0.71 (0.65-1.13)	1.01 (0.50-5.26)	1.01 (0.29-3.63)	1.78±1.68
AM	5.12±3.46*	3.04 (1.21-3.85)	3.31 (1.58-12.38)*	3.10 (1.40-8.02)	6.92±5.05*

图1 在位子宫内膜组织Notch1信号及糖酵解相关蛋白的表达

Fig.1 Expression of Notch1 signal and glycolysis-related proteins in the endometrial tissues detected by Western blotting.

表4 在位子宫内膜组织Notch1信号及糖酵解相关蛋白的表达

Tab.4 Protein expressions of Notch1 signaling pathway and glycolysis-related factors in the endometrial tissue (n=7, Mean±SD)

Group	Notch1	GLUT1	HK2	PKM	PDHA
CON	0.21±0.11	0.15±0.09	0.85±0.07	0.16±0.05	0.21±003
AM	0.60±0.28**	0.36±0.22*	0.24±0.13*	0.31±0.14*	0.40±0.14*

表5 在位子宫内膜组织葡萄糖、乳酸含量

Tab.5 Glucose and lactic acid levels in the endometrial tissue (n=5, Mean±SD)

Group	Glucose	Lactate
CON	88.8±24.82	50.16±4.09
AM	50.51±23.50*	74.34±8.00**

图2 过表达Notch1信号对Ishikawa细胞系迁移、侵袭的影响

Fig.2 Effects of overexpression of Notch1 signaling on migration and invasion of Ishikawa cells (Original magnification: ×200).

表6 过表达Notch1信号对Ishikawa细胞系迁移、侵袭细胞数的影响

Tab.6 Effects of overexpression of Notch1 signaling on migration and invasion of Ishikawa cells (n=3, Mean±SD)

Group	Migration cells	Invasion cells
ov-NC	4.78±1.68	9.89±4.00
ov-Notch1	64.56±17.67*	140.89±48.49*

表7 过表达Notch1信号对Ishikawa细胞系糖酵解能力的影响

Tab.7 Effect of overexpression of Notch1 signaling on glycolysis capacity of Ishikawa cells (n=3, Mean±SD)

Group	Fermentation capacity	Fermentation reserve
ov-NC	10.43±1.27	9.61±1.88
ov-Notch1	17.13±1.68**	16.20±1.62*

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