miR⁃143⁃3p通过调控CX3CL1/CX3CR1信号通路对脂多糖诱导肺泡上皮细胞损伤的影响

The effect of miR⁃143⁃3p on lipopolysaccharide⁃induced alveolar epithelial cell injury by regulating the CX3CL1/CX3CR1 signaling pathway

目的探讨miR⁃143⁃3p通过调控趋化因子(C⁃X3⁃C基元)配体1/趋化因子(C⁃X3⁃C基元)受体1(CX3CL1/CX3CR1)信号通路对脂多糖(LPS)诱导肺泡上皮细胞损伤的影响。方法收集急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)患者及健康体检者外周血血清,标记为ALI/ARDS组和Normal组。双荧光素酶实验证实miR⁃143⁃3p与CX3CL1的靶向关系。体外培养人肺泡上皮细胞A549,并将细胞分成Con⁃trol组、LPS组、LPS+NC mimic组(转染NC mimic)、LPS+miR⁃143⁃3p mimic组(转染miR⁃143⁃3p mimic)、LPS+miR⁃143⁃3p mimic+pcDNA组(共转染miR⁃143⁃3p mimic和pcDNA)、LPS+miR⁃143⁃3p mimic+pc⁃CX3CL1组(共转染miR⁃143⁃3p mimic和pc⁃CX3CL1)。qRT⁃PCR检测miR⁃143⁃3p和CX3CL1、CX3CR1 mRNA表达水平;Western blot检测CX3CL1、CX3CR1蛋白表达;MTT和流式细胞术检测细胞活力和凋亡;ELISA分析肿瘤坏死因子⁃α(TNF⁃α)、白介素⁃1β(IL⁃1β)、白介素⁃6(IL⁃6)、血管内皮生长因子(VEGF)和降钙素原(PCT)水平。结果与Normal组相比,ALI/ARDS组患者外周血中miR⁃143⁃3p表达(0.52±0.05)降低,CX3CL1、CX3CR1的mRNA(1.79±0.08)、(1.65±0.06)和蛋白(1.03±0.15)、(0.98±0.12)表达升高(P<0.05)。与Control组比较,LPS组A549细胞中miR⁃143⁃3p表达减小,细胞活力降低,凋亡率[(23.56±0.85)%]及TNF⁃α、IL⁃1β、IL⁃6、VEGF、PCT含量增加(P<0.05)。miR⁃143⁃3p过表达后,细胞活力升高,凋亡率[(13.74±0.69)%]及TNF⁃α、IL⁃1β、IL⁃6、VEGF、PCT含量降低(P<0.05)。双荧光素酶实验结果显示,miR⁃143⁃3p直接负调控CX3CL1表达。CX3CL1过表达可明显逆转miR⁃143⁃3p过表达对LPS诱导的A549细胞损伤的影响和对CX3CR1 mRNA和蛋白表达的抑制作用(P<0.05)。结论miR⁃143⁃3p通过抑制CX3CL1/CX3CR1信号通路,减轻LPS诱导的肺泡上皮细胞损伤。

Objective To investigate the effect of miR⁃143⁃3p on lipopolysaccharide(LPS)⁃induced alveolar epithelial cell injury by regulating the Chemokine(C⁃x3⁃C unit)ligand 1/Chemokine(C⁃x3⁃C unit)receptor 1(CX3CL1/CX3CR1)signaling pathway.Methods Peripheral blood serum were collected from patients with acute lung injury/acute respiratory distress syndrome(ALI/ARDS)and healthy individuals.The samples were classified into ALI/ARDS group and normal group.Dual luciferase experiment was performed to confirm the targeting relationship between miR⁃143⁃3p and CX3CL1.Human alveolar epithelial cells A549 were cultured in vitro,and the cells were separated into normal control group,LPS group,LPS+NC mimic group(transfected with NC mimic),LPS+miR⁃143⁃3p mimic group(transfected with miR⁃143⁃3p mimic),LPS+miR⁃143⁃3p mimic+pcDNA group(co⁃transfected with miR⁃143⁃3p mimic and pcDNA),and LPS+miR⁃143⁃3p mimic+pc⁃CX3CL1 group(co⁃trans⁃fected with miR⁃143⁃3p mimic and pc⁃CX3CL1).QRT⁃PCR was performed to measure the expression levels of miR⁃143⁃3p,CX3CL1 and CX3CR1 mRNAs.Western blot was performed to measure CX3CL1 and CX3CR1 protein expression.The viability and apoptosis of alveolar epithelial cells were detected by MTT and flow cytometry.ELISA was performed to analyze the levels of tumor necrosis factor⁃α(TNF⁃α),interleukin⁃1β(IL⁃1β),interleukin⁃6(IL⁃6),vascular endothelial growth factor(VEGF)and procalcitonin(PCT).Results Compared with the Normal group,the expression of miR⁃143⁃3p(0.52±0.05)in the peripheral blood of the ALI/ARDS group decreased,and the mRNA(1.79±0.08),(1.65±0.06)and protein(1.03±0.15),(0.98±0.12)expression of CX3CL1 and CX3CR1 increased(P<0.05).Compared with the Control group,the expression of miR⁃143⁃3p in the A549 cells of the LPS group decreased,the cell viability decreased,and the apoptosis rate[(23.56±0.85)%]and the contents of TNF⁃α,IL⁃1β,IL⁃6,VEGF,and PCT increased(P<0.05).After miR⁃143⁃3p was overexpressed,cell viability was obviously increased,and the apoptosis rate[(13.74±0.69)%]and the contents of TNF⁃α,IL⁃1β,IL⁃6,VEGF,and PCT were obviously reduced(P<0.05).The results of dual luciferase assay showed that miR⁃143⁃3p directly negatively regulated the expression of CX3CL1.CX3CL1 overexpression significantly reversed the effect of miR⁃143⁃3p overexpression on LPS⁃induced A549 cell damage and the inhibitory effect on CX3CR1 mRNA and protein expression(P<0.05).Conclusion MiR⁃143⁃3p can reduce the injury of alveolar epithelial cells induced by LPS by inhibiting the CX3CL1/CX3CR1 signaling pathway.