Exosomes Regulate Epithelial Mesenchymal Transition of Endometrial Cells in Mice with Recurrent Spontaneous Abortion

doi:10.12114/j.issn.1007-9572.2023.0768

Abstract

Abstract:

Background

Recurrent spontaneous abortion (RSA) is a common reproductive disorder closely linked with the weakened epithelial-mesenchymal transition (EMT) in the endometrium during embryo implantation. Endometrium-derived exosomes and miRNAs they transport are linked with RSA via the involvement in cellular communication and EMT, although the specific mechanisms have not yet fully revealed.

Objective

Focusing on the function of exosomes in EMT, this study aims to validate the regulatory role of endometrium-derived exosomes and miRNA-221 they transport in EMT of endometrial cells in RSA mice.

Methods

From October 2021 to May 2022, a total of 24 eight-week-old female mice in the specific pathogen-free (SPF) level were randomly assigned into the control group (n=12) and RSA group (n=12) for modeling. After RSA modeling, male and female mice were housed together. On the 4th day of pregnancy, female mice were sacrificed for isolating endometrial epithelial cells. They were cultured for extracting exosomes, which were identified by transmission electron microscopy and Western blotting. Cell proliferation, invasion, and migration abilities were evaluated using CCK-8 assay, Transwell assay, and fluorescence-activated cell sorting (FACS) before and after exosome interventions. Expression levels of EMT-related genes [E-cadherin, N-cadherin, Vimentin, matrix metalloproteinase 7 (MMP-7), matrix metalloproteinase 9 (MMP-9) ] and exosome marker proteins [CD₆₃, CD₈₁, tumor susceptibility gene 101 (TSG101) ] were measured. Expression levels of EMT marker proteins (CD₂₄^-APC, CD₄₄^- FITC) in exosome-treated cells were analyzed. Transfection of miRNA-221 mimics and inhibitors was performed to analyze the effects of exosome-derived miRNA-221 on EMT of endometrial epithelial cells in RSA mice. Relevant analyses were conducted using Western blot and dual-luciferase reporter assay.

Results

Abnormal EMT was observed in the endometrial epithelial cells of mice in RSA group, and the mouse endometrial epithelial cells were able to secrete exosomes. The proliferation rate, migration ability, invasion ability, and CD₄₄⁺/CD₂₄⁺ ratio of cells in the RSA group treated with exosomes were significantly higher than those measured in cell supernatant of RSA group without exosome intervention (P<0.05). Compared with those in the cell supernatant of RSA group, induction of exosomes significantly downregulated EMT marker protein E-cadherin, but upregulated stromal marker proteins N-cadherin and Vimentin, as well as matrix marker proteins MMP-7 and MMP-9 (P<0.05). The expression level of miRNA-221 in the RSA group was significantly lower than that of the control group (P<0.05). Compared with those transfected with miRNA negative control, transfection of miRNA-221 mimic significantly activated the phosphatidylinositol 3-kinase (PI3K) -AKT pathway, nuclear factor-kappa B (NF-κB) pathway, and p38 pathway (P<0.05), but downregulated mRNA level of PTEN (P<0.05). Transfection of miRNA-221 inhibitor significantly upregulated mRNA level of PTEN (P<0.05) .

Conclusion

Endometrium-derived exosomal miRNA-221 negatively regulates the PTEN gene, activates the PI3K-AKT pathway, promotes EMT in endometrial epithelial cells, and influences the pregnant outcome of RSA mice.

Key words: Recurrent spontaneous abortion, Epithelial-mesenchymal transition, Exosomes, miRNA-221, PTEN gene, PI3K-AKT pathway

摘要：

背景

复发性流产（RSA）是常见的生殖障碍性疾病，在胚胎植入过程中，子宫内膜发生上皮间充质转化（EMT），RSA的发生与该过程减弱密切相关。子宫内膜来源外泌体及其携带的miRNA参与细胞间通讯，影响细胞EMT，与RSA关系密切，但具体作用机制尚不明确。

目的

本研究聚焦于外泌体在细胞EMT中的功能，通过实验验证子宫内膜来源外泌体及其分泌的miRNA-221对RSA小鼠子宫内膜细胞EMT的调控作用。

方法

2021年10月—2022年5月，选取SPF级、健康8周龄雌性小鼠24只，随机选取12只作为正常对照组，剩余12只小鼠制作RSA模型，造模结束后雌雄合笼，妊娠第4日处死雌性小鼠，分离和培养子宫内膜上皮细胞并收集外泌体，通过透射电镜和免疫印迹法（WB）进行外泌体鉴定；通过CCK-8、Transwell和流式细胞术检测外泌体干预前后两组细胞增殖、侵袭、迁移能力；细胞EMT相关蛋白[E-cadherin、N-cadherin、Vimentin、基质金属蛋白酶7（MMP-7）、MMP-9]及外泌体标志蛋白（CD₆₃、CD₈₁、TSG101）的表达；外泌体处理后的细胞EMT标志蛋白（CD₂₄^- APC、CD₄₄^- FITC）的表达；转染miRNA-221模拟物和抑制物分析外泌体源性miRNA-221对小鼠子宫内膜上皮细胞EMT的作用，并通过WB和双荧光素酶报告基因检测进行分析。

结果

RSA组小鼠的子宫内膜上皮细胞具有异常的EMT，小鼠子宫内膜上皮细胞能够分泌外泌体。添加外泌体的RSA组细胞增殖率、迁移能力、侵袭能力、CD₄₄⁺/CD₂₄⁺表达水平高于细胞上清液RSA组（P<0.05）。与细胞上清液RSA组相比，添加外泌体的RSA组细胞的EMT标志蛋白E-cadherin水平降低，间质样标志蛋白N-cadherin和Vimentin以及基质标志蛋白MMP-7、MMP-9蛋白水平升高（P<0.05）。RSA组miRNA-221表达水平低于正常对照组（P<0.05）。与对照随机miRNA相比，miRNA-221模拟物激活了PI3K-AKT通路、NF-κB通路和p38通路（P<0.05）。与对照随机miRNA相比，miRNA-221模拟物降低了PTEN mRNA的表达水平（P<0.05），而miRNA-221抑制剂转染则增加了PTEN mRNA的表达水平（P<0.05）。

结论

子宫内膜来源外泌体miRNA-221可通过负向调控Pten基因，激活PI3K-AKT通路，促进子宫内膜上皮细胞EMT，影响RSA妊娠结局。

关键词: 复发性流产, 上皮间充质转化, 外泌体, miRNA-221, Pten基因, PI3K-AKT通路

CLC Number:

R 714.21

LI Mengyuan,LI Guanshan,HAN Qian, et al. Exosomes Regulate Epithelial Mesenchymal Transition of Endometrial Cells in Mice with Recurrent Spontaneous Abortion[J]. Chinese General Practice, 2025, 28(08): 962-972. DOI: 10.12114/j.issn.1007-9572.2023.0768.
李梦元,李冠杉,韩倩等. 外泌体调节复发性流产小鼠子宫内膜细胞上皮间充质转化的作用机制研究[J]. 中国全科医学, 2025, 28(08): 962-972. DOI: 10.12114/j.issn.1007-9572.2023.0768.

Figures/Tables 14

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组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
正常对照组	12	26.33±0.15	43.17±0.70	81.07±0.51	92.87±1.53	126.83±3.68	18.48±0.26	47.10±0.51	85.78±2.80
RSA组	12	26.57±0.55	42.07±0.25	70.77±0.51	81.27±1.50	92.00±2.55	16.34±0.34	37.07±0.45	71.70±1.31
t值		0.707	2.554	24.580	9.381	13.460	8.571	25.550	7.885
P值		0.518	0.063	<0.001	<0.001	<0.001	0.001	<0.001	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
正常对照组	63.10±2.74	29.62±1.06	1.01±0.08	2.74±0.15	2.14±0.31	2.39±0.05	2.60±0.07
RSA组	39.03±2.63	13.11±1.65	1.89±0.05	2.16±0.15	0.79±0.12	1.78±0.14	2.12±0.07
t值	11.110	14.600	16.600	4.626	7.112	7.303	6.785
P值	<0.001	<0.001	<0.001	0.010	0.002	0.002	0.002

组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
正常对照组	12	26.33±0.15	43.17±0.70	81.07±0.51	92.87±1.53	126.83±3.68	18.48±0.26	47.10±0.51	85.78±2.80
RSA组	12	26.57±0.55	42.07±0.25	70.77±0.51	81.27±1.50	92.00±2.55	16.34±0.34	37.07±0.45	71.70±1.31
t值		0.707	2.554	24.580	9.381	13.460	8.571	25.550	7.885
P值		0.518	0.063	<0.001	<0.001	<0.001	0.001	<0.001	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
正常对照组	63.10±2.74	29.62±1.06	1.01±0.08	2.74±0.15	2.14±0.31	2.39±0.05	2.60±0.07
RSA组	39.03±2.63	13.11±1.65	1.89±0.05	2.16±0.15	0.79±0.12	1.78±0.14	2.12±0.07
t值	11.110	14.600	16.600	4.626	7.112	7.303	6.785
P值	<0.001	<0.001	<0.001	0.010	0.002	0.002	0.002

组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
RSA	12	27.27±0.21	42.57±0.64	70.47±1.42	80.97±1.42	95.57±1.58	17.15±0.05	35.11±6.56	54.85±0.55
RSA+Ex	12	26.59±0.65	42.50±0.60	80.97±1.94	93.30±3.16	129.47±5.23	17.24±0.13	48.89±1.15	64.17±0.43
t值		0.707	0.132	7.560	6.171	10.740	1.103	3.583	23.600
P值		1.721	0.901	0.002	0.004	<0.001	0.332	0.023	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
RSA	26.27±1.91	11.82±1.53	3.47±0.47	2.44±0.15	2.32±0.25	1.92±0.15	1.69±0.10
RSA+Ex	54.33±2.52	30.88±1.46	1.21±0.33	4.45±0.51	3.20±0.08	2.36±0.10	1.96±0.10
t值	15.380	15.610	16.600	6.535	5.855	4.133	3.432
P值	<0.001	<0.001	<0.001	0.003	0.004	0.0145	0.026

组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
RSA	12	27.27±0.21	42.57±0.64	70.47±1.42	80.97±1.42	95.57±1.58	17.15±0.05	35.11±6.56	54.85±0.55
RSA+Ex	12	26.59±0.65	42.50±0.60	80.97±1.94	93.30±3.16	129.47±5.23	17.24±0.13	48.89±1.15	64.17±0.43
t值		0.707	0.132	7.560	6.171	10.740	1.103	3.583	23.600
P值		1.721	0.901	0.002	0.004	<0.001	0.332	0.023	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
RSA	26.27±1.91	11.82±1.53	3.47±0.47	2.44±0.15	2.32±0.25	1.92±0.15	1.69±0.10
RSA+Ex	54.33±2.52	30.88±1.46	1.21±0.33	4.45±0.51	3.20±0.08	2.36±0.10	1.96±0.10
t值	15.380	15.610	16.600	6.535	5.855	4.133	3.432
P值	<0.001	<0.001	<0.001	0.003	0.004	0.0145	0.026

组别	例数	miRNA-133a	miRNA-158-5P	miRNA-221	miRNA-720	miRNA-365	miRNA-215	miRNA-34a
正常对照组	12	0.92±0.06	0.93±0.07	5.07±0.10	0.92±0.06	0.92±0.05	0.97±0.12	0.92±0.06
RSA组	12	1.18±0.04	2.10±0.12	1.05±0.14	0.49±0.03	1.13±0.07	5.65±0.46	0.42±0.03
t值		6.390	14.000	40.550	11.970	4.209	17.190	13.690
P值		0.003	<0.001	<0.001	<0.001	0.014	<0.001	<0.001