miR-139-5p调控Notch/RBP-J/Hes1轴促进支气管哮喘骨髓间充质干细胞“归巢”

doi:10.12122/j.issn.1673-4254.2024.12.03

摘要/Abstract

摘要：

目的通过将哮喘支气管上皮细胞与骨髓间充质干细胞共培养方式，观察miR-139-5p调控Notch1信号通路对哮喘骨髓间充质干细胞归巢的影响。方法将大鼠骨髓间充质干细胞和支气管上皮细胞共培养，并予miR-139-5p mimics进行干预。实验分为阴性对照组（NC组：正常大鼠骨髓间充质干细胞+支气管上皮细胞共培养）、模型对照组（MC组：正常大鼠骨髓间充质干细胞+哮喘大鼠支气管上皮细胞共培养）、miR-139-5p mimics组（miR-139-5p mimics+MC）、miR-139-5p mimics-NC组（miR-139-5p mimics-NC+MC）。检测细胞活力和细胞周期变化；免疫荧光染色检测CXCR4、SDF-1表达；经细胞转染后观察miR-139-5p表达量和BMSCs归巢水平；免疫印迹观察Notch1/RBP-J/Hes1蛋白表达量；ELISA检测Th1、Th2相关因子表达情况。结果与NC组比较，MC组miR-139-5p、IL-2、IL-12表达量降低（P<0.05），BMSCs归巢水平增加，CXCR4、SDF-1、IL-5、IL-9表达升高（P<0.05），Notch1、RBP-J、Hes1 mRNA、蛋白表达升高（P<0.05）。与MC组、miR-139-5p mimics-NC组比较，miR-139-5p mimics组miR-139-5p、BMSCs归巢水平升高，CXCR4、SDF-1、IL-2表达升高（P<0.05），Notch1、RBP-J、Hes1mRNA、蛋白表达降低，IL-5、IL-9表达降低（P<0.05）。相关性分析显示，BMSCs归巢水平与miR-139-5p、IL-12呈正相关，SDF-1与miR-139-5p呈正相关（P<0.05）；与IL-5表达呈负相关，CXCR4与Activated Notch1表达呈负相关，SDF-1与Notch1表达呈负相关（P<0.05）。结论 miR-139-5p可能通过靶向Notch1信号通路促进哮喘骨髓间充质干细胞归巢，作用于Th1、Th2细胞因子表达，改善哮喘气道炎症。

关键词: 支气管哮喘, 骨髓间充质干细胞, miR-139-5p, Notch1信号通路

Abstract:

Objective To observe the role of miR-139-5p and Notch1 signaling pathway in regulation of homing of bone mesenchymal stem cells (BMSCs) of asthmatic rats. Methods Normal rat BMSCs were co-cultured with bronchial epithelial cells from normal or asthmatic rats, followed by transfection with miR-139-5p mimics or a negative control sequence. The changes in cell viability and cell cycle were analyzed, and the cellular expressions of CXCR4 and SDF-1 were detected using immunofluorescence staining. The changes of BMSC homing after the transfection were observed, and the expressions of Notch1, RBP-J, and Hes1 mRNAs and proteins and Th1/Th2 cytokines were detected with RT-qPCR, Western blotting or ELISA. Results The co-cultures of BMSCs and asthmatic bronchial epithelial cells showed significantly decreased expressions of miR-139-5p, IL-2 and IL-12 and increased expressions of CXCR4, SDF-1, IL-5, IL-9, Notch1, RBP-J, and Hes1. Transfection with miR-139-5p mimics significantly increased the expressions of miR-139-5p, IL-2, CXCR4 and SDF-1 and lowered the expression levels of IL-5, IL-9, Notch1, activated Notch1, and Hes1 in the co-cultured cells. Correlation analysis showed that BMSC homing was positively correlated with miR-139-5p and IL-12 and negatively correlated with IL-5 expression. The expression of CXCR4 was negatively correlated with activated Notch1, and SDF-1 was positively correlated with miR-139-5p but negatively correlated with Notch1 expression. Conclusion High expression of miR-139-5p promotes homing of BMSCs in asthma by targeting the Notch1 signaling pathway to regulate the expressions of Th1/Th2 cytokines, thereby alleviating airway inflammation.

Key words: asthma, bone mesenchymal stem cells, miR-139-5p, Notch1 signaling pathway

王坤, 方昊翔, 曹晓梅, 朱子衡. miR-139-5p调控Notch/RBP-J/Hes1轴促进支气管哮喘骨髓间充质干细胞“归巢”[J]. 南方医科大学学报, 2024, 44(12): 2283-2290.

Kun WANG, Haoxiang FANG, Xiaomei CAO, Ziheng ZHU. MiR-139-5p regulates the Notch/RBP-J/Hes1 axis to promote homing of bone mesenchymal stem cells in bronchial asthma[J]. Journal of Southern Medical University, 2024, 44(12): 2283-2290.

图/表 11

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Primer	Sequence (5' to 3')	Length (bp)
Notch1-F	CCAGGAAAGAGGGCAGC	198
Notch1-R	GGAGCATCTCAAGCCTCTT	198
RBP-J-F	CAGGCATTCTACAGGAAGTTTG	99
RBP-J-R	TACTGTTTGATCCCCTCGTTC	99
Hes1-F	AAACCAAAGACAGCCTCTGA	184
Hes1-R	GGTGCTTCACTGTCATTTCC	184
miR139-5p-F	ACACTCCAGCTGGGTCTACAGTGCACGTGT	66
miR139-5p-R	TGGTGTCGTGGAGTCG	66
β-actin-F	CCCATCTATGAGGGTTACGC	150
β-actin-R	TTTAATGTCACGCACGATTTC	150

Primer	Sequence (5' to 3')	Length (bp)
Notch1-F	CCAGGAAAGAGGGCAGC	198
Notch1-R	GGAGCATCTCAAGCCTCTT	198
RBP-J-F	CAGGCATTCTACAGGAAGTTTG	99
RBP-J-R	TACTGTTTGATCCCCTCGTTC	99
Hes1-F	AAACCAAAGACAGCCTCTGA	184
Hes1-R	GGTGCTTCACTGTCATTTCC	184
miR139-5p-F	ACACTCCAGCTGGGTCTACAGTGCACGTGT	66
miR139-5p-R	TGGTGTCGTGGAGTCG	66
β-actin-F	CCCATCTATGAGGGTTACGC	150
β-actin-R	TTTAATGTCACGCACGATTTC	150

Group	Cell viability	G1(%)	S (%)	G2(%)
NC	1.000±0.014	56.74±2.15	8.80±0.72	34.46±0.77
MC	0.558±0.033^Δ	73.99±2.48 ^ΔΔ	4.24±0.63^ΔΔ	21.78±0.66^ΔΔ
miR-139-5p mimics	0.892±0.046*^▲	62.61±1.89*^▲	8.26±0.49*^▲	29.13±0.72*^▲
miR-139-5p mimics-NC	0.544±0.122^Δ	72.94±2.49	5.27±0.62	21.79±0.94

Group	Cell viability	G1(%)	S (%)	G2(%)
NC	1.000±0.014	56.74±2.15	8.80±0.72	34.46±0.77
MC	0.558±0.033^Δ	73.99±2.48 ^ΔΔ	4.24±0.63^ΔΔ	21.78±0.66^ΔΔ
miR-139-5p mimics	0.892±0.046*^▲	62.61±1.89*^▲	8.26±0.49*^▲	29.13±0.72*^▲
miR-139-5p mimics-NC	0.544±0.122^Δ	72.94±2.49	5.27±0.62	21.79±0.94

Group	Ave CT	2^-△△CT
NC	18.58	1.00±0.04
MC	19.08	0.54±0.05^Δ
miR-139-5p mimics	18.77	0.82±0.04*^▲
miR-139-5p mimics-NC	19.11	0.55±0.02^Δ