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

作　　者：张玉如 万磊[1,3,4] 方昊翔 李方泽 王丽文 李柯霏 闫佩文 姜辉 ZHANG Yuru;WAN Lei;FANG Haoxiang;LI Fangze;WANG Liwen;LI Kefei;YAN Peiwen;JIANG Hui(First Affiliated Hospital of Anhui University of Traditional Chinese Medicine,Hefei 230031,China;College of Traditional Chinese Medicine,Anhui University of Traditional Chinese Medicine,Hefei 230031,China;Xin'an Institute of Medicine and Traditional Chinese Medicine Modernization,Hefei 230012,China;Xin'an Key Laboratory of Medicine,Ministry of Education,Hefei 230012,China)

机构地区：[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

出　　处：《南方医科大学学报》2025年第1期65-71,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(82474482,82274501);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)。

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(pSSHSGECs)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pSSHSGECs增殖与凋亡异常,调节炎症反应。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.

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 

分 类 号：R593.2[医药卫生—内科学]