基于中性粒细胞活化核内小RNA宿主基因14在急性肺损伤中的调控作用机制分析

Analysis of Regulatory Mechanism of Small Nucleolar RNA Host Gene 14 in Acute Lung Injury Based on Polymorphonuclear Neutrophil Activation

目的:探讨基于中性粒细胞(PMN)活化核内小RNA宿主基因14(SNHG14)在急性肺损伤(ALI)中的调控作用机制。方法:生物信息学验证SNHG14、miR-331-3p和趋化因子受体1(CCR1)的靶向结合关系,合成相应的双荧光素酶报告基因。培养小鼠骨髓中性粒细胞,建立SNHG14过/抑制表达模型(siRNA-SNHG14),同时以miR-331-3p为核心行Rescue实验,检测细胞中miR-331-3p、SNHG14及CCR1的表达改变状况;脉波指示剂连续心排血量监测急性呼吸窘迫综合征(ARDS)患者血管外肺水指数和肺血管通透性指数等参数;qRT-PCR监测ARDS患者外周血SNHG14的表达,并分析SNHG14与ARDS的临床特征、肺通透指数和肺水含量的相关性;流式细胞检测PMN细胞转染siRNA-SNHG14后细胞凋亡率的变化。结果:SNHG14在脂多糖诱导的PMN细胞模型中表达显著升高;双荧光素酶证实SNHG14和miR-331-3p具有靶向关系,miR-331-3p与CCR1存在靶向关系,且SNHG14和miR-331-3p有结合位点,SNHG14干扰后miR-331-3p表达增加;ARDS患者外周血中SNHG14表达明显增加,且SNHG14与ARDS患者的肺通透指数和肺水含量呈正相关;siRNA-SNHG14可造成PMN细胞凋亡率明显降低。结论:在中性粒细胞中,SNHG14可能通过吸附miR-331-3p,调控CCR1的表达,进而可能参与ALI/ARDS的发生。

Objective:To investigate the regulatory mechanism of small nucleolar RNA host gene 14(SNHG14)in acute lung injury based on the polymorphonuclear neutrophil(PMN)activation.Methods:Bioinformatics was used to verify the targeted binding relationship between SNHG14,miR-331-3p and chemokine receptor 1(CCR1),and synthesize the corresponding dualluciferase reporter gene.Mouse bone marrow neutrophils were cultured to establish SNHG14 overexpression/inhibiting expression model(siRNA-SNHG14).Meanwhile,Rescue experiments were performed with miR-331-3p as the core to detect the expression changes of miR-331-3p,SNHG14 and CCR1 in cells.Pulse wave indicator continuous cardiac output was used to monitor the extravascular lung water index,pulmonary vascular permeability index(PVPI)and other parameters in patients with acute respiratory distress syndrome(ARDS).The expression of SNHG14 in peripheral blood of ARDS patients was monitored by qRTPCR,and the correlation between SNHG14 and the clinical features of ARDS,pulmonary permeability index and pulmonary water content was analyzed.Flow cytometry was used to detect the changes in apoptosis rate of PMN cells after transfection with siRNASNHG14.Results:The expression of SNHG14 was significantly increased in lipopolysaccharide-induced PMN cell models;Dualluciferase confirmed a targeting relationship between SNHG14 and miR-331-3p,between miR-331-3p and CCR1,and binding sites between SNHG14 and miR-331-3p,and the expression of miR-331-3p increased after SNHG14 interference;The expression of SNHG14 in peripheral blood of ARDS patients increased significantly,and SNHG14 was positively correlated with pulmonary permeability index and pulmonary water content of ARDS patients;siRNA-SNHG14 significantly reduced the apoptosis rate of PMN cells.Conclusion:In neutrophils,SNHG14 may regulate the expression of CCR1 through adsorption of miR-331-3p,and accordingly may participate in the occurrence of ALI/ARDS.