miR-155-5p介导PIK3R1负调控PI3K/AKT信号通路促进原发性干燥综合征人唾液腺上皮细胞增殖

doi:10.12122/j.issn.1673-4254.2025.01.09

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (1): 65-71.doi: 10.12122/j.issn.1673-4254.2025.01.09

miR-155-5p介导PIK3R1负调控PI3K/AKT信号通路促进原发性干燥综合征人唾液腺上皮细胞增殖

张玉如¹(), 万磊¹^,³^,⁴(), 方昊翔², 李方泽¹, 王丽文¹, 李柯霏¹, 闫佩文¹, 姜辉¹

^1.安徽中医药大学第一附属医院，安徽合肥 230031
^2.安徽中医药大学中医学院，安徽合肥 230031
^3.新安医学与中医药现代化研究所，安徽合肥 230012
^4.新安医学教育部重点实验室，安徽合肥 230012

收稿日期:2024-09-20 出版日期:2025-01-20 发布日期:2025-01-20
通讯作者: 万磊 E-mail:2693480130@qq.com;yxwanlei@163.com
作者简介:张玉如，在读硕士研究生，E-mail: 2693480130@qq.com
基金资助:
国家自然科学基金(82474482);安徽省自然科学基金(2208085MH276);新安医学与中医药现代化研究所揭榜挂帅项目(2023CXMMTCM020)

Inhibiting miR-155-5p promotes proliferation of human submandibular gland epithelial cells in primary Sjogren's syndrome by negatively regulating the PI3K/AKT signaling pathway via PIK3R1

Yuru ZHANG¹(), Lei WAN¹^,³^,⁴(), Haoxiang FANG², Fangze LI¹, Liwen WANG¹, Kefei LI¹, Peiwen YAN¹, Hui JIANG¹

^1.First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China
^2.College of Traditional Chinese Medicine, Anhui University of Traditional Chinese Medicine, Hefei 230031, China
^3.Xin'an Institute of Medicine and Traditional Chinese Medicine Modernization, Hefei 230012, China
^4.Xin'an Key Laboratory of Medicine, Ministry of Education, Hefei 230012, China

Received:2024-09-20 Online:2025-01-20 Published:2025-01-20
Contact: Lei WAN E-mail:2693480130@qq.com;yxwanlei@163.com
Supported by:
National Natural Science Foundation of China(82474482)

摘要/Abstract

摘要：

目的探讨miR-155-5p靶向作用于PIK3R1调控PI3K/AKT信号通路对原发性干燥综合征人唾液腺上皮细胞（pSS-HSGECs）的影响。方法通过双荧光素酶验证miR-155-5p与PI3K/AKT通路的靶向关系。利用TRAIL及INF-γ刺激细胞，模拟pSS-HSGECs细胞模型；空白组：HSGECs，模型组：TRAIL+INF-γ+HSGECs，空转组：TRAIL+INF-γ+HSGECs+miR-155-inhibitor-NC；miR-155抑制组：TRAIL+INF-γ+IHSGECs+miR-155-inhibitor；CKK-8法、流式细胞术、平板克隆形成实验分别检测细胞活力、细胞周期及凋亡率、细胞增殖能力。ELISA、RT-PCR方法分别检测相关细胞因子、miR-155-5p表达。蛋白质免疫印迹法检测PI3K/AKT信号通路蛋白表达。结果双荧光素酶检测结果表明，miR-155-5p与PI3K/AKT通路存在靶向关系，且PIK3R1mRNA为二者的结合位点。与空白组相比，模型组细胞活力、细胞克隆形成能力、IL-10、IL-4水平降低；细胞凋亡率、细胞周期G2期比例、TNF-α、IL-6、miR-155-5p、PIK3R1 mRNA表达、p-PI3K/PI3K、p-AKT/AKT比率、PIK3R1mRNA蛋白相对表达显著升高（P<0.01）。与模型组及空转组相比，miR-155抑制组细胞活力、G1期比例、克隆形成能力、IL-10和IL-4水平上升；同时细胞凋亡率、细胞周期G2期比例、TNF-α、IL-6、miR-155-5p、PIK3R1 mRNA表达、p-PI3K/PI3K、p-AKT/AKT比率及PIK3R1蛋白相对表达下降（P<0.05）。结论 miR-155-5p可靶向作用于PI3K1mRNA，负向调节PI3K/AKT信号通路的过表达，改善pSS-HSGECs增殖与凋亡异常，调节炎症反应。

关键词: 原发性干燥综合征, miR-155-5p, PIK3R1, PI3K/AKT信号通路, 炎症因子

Abstract:

Objective To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). Methods Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. Results Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. Conclusion In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.

Key words: primary Sjogren's syndrome, miR-155-5p, PIK3R1, PI3K/AKT signaling pathway, inflammatory factors

张玉如, 万磊, 方昊翔, 李方泽, 王丽文, 李柯霏, 闫佩文, 姜辉. miR-155-5p介导PIK3R1负调控PI3K/AKT信号通路促进原发性干燥综合征人唾液腺上皮细胞增殖[J]. 南方医科大学学报, 2025, 45(1): 65-71.

Yuru ZHANG, Lei WAN, Haoxiang FANG, Fangze LI, Liwen WANG, Kefei LI, Peiwen YAN, Hui JIANG. Inhibiting miR-155-5p promotes proliferation of human submandibular gland epithelial cells in primary Sjogren's syndrome by negatively regulating the PI3K/AKT signaling pathway via PIK3R1[J]. Journal of Southern Medical University, 2025, 45(1): 65-71.

图/表 11

表1 A、B液成分

Tab.1 Ingredients of Liquid A and B

246 μL serum-free

medium+4 μg plasmid

240 μLserum-free

medium+10 μL

DNA

Lipofectamine 2000

图1 双荧光素酶检测结果

Fig.1 Double luciferase detection results and binding sites. **P<0.01.

图2 miR-155对pSS-HSGECs细胞活力的影响

Fig.2 Effect of miR-155 inhibitor on viability of HSGEC model of pSS (pSS-HSGECs). **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

图3 miR-155-5p对pSS-HSGECs细胞凋亡的影响

Fig.3 Effect of miR-155 inhibitor on apoptosis of pSS-HSGECs. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

图4 miR-155p对pSS-HSGECs细胞周期的影响

Fig.4 Effect of miR-155p inhibitor on cell cycle distribution in pSS-HSGECs. **P<0.01 vs the blank group; ##P<0.01 vs the Model group; ΔΔP<0.01 vs the Idling group.

图5 细胞周期流式图

Fig.5 Flow cytometric analysis of cell cycle distribution in the cells.

图6 miR-155p对pSS-HSGECs增殖能力的影响

Fig.6 Effect of miR-155p inhibitor on proliferation of pSS-HSGECs. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

图7 各组细胞因子水平

Fig.7 Cytokine levels in pSS-HSGECs in each group. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

图8 各组细胞miR-155-5p、PIK3R1mRNA表达情况

Fig.8 Expression of miR-155-5p and PIK3R1 mRNA in pSS-HSGECs in each group. **P<0.01 vs Blank group; ##P<0.01 vs Model group; ΔΔP<0.01 vs Idling group.

图9 各组细胞PI3K/AKT通路蛋白表达

Fig.9 Protein expressions of the PI3K/AKT pathway in pSS-HSGECs in each group. A: Blank group. B: Model group. C: Idling group. D: miR-155 inhibition group.

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miR-155-5p介导PIK3R1负调控PI3K/AKT信号通路促进原发性干燥综合征人唾液腺上皮细胞增殖

Inhibiting miR-155-5p promotes proliferation of human submandibular gland epithelial cells in primary Sjogren's syndrome by negatively regulating the PI3K/AKT signaling pathway via PIK3R1

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