The Role And Mechanism Of MicroRNA-129-5p In LPS-induced Hpmecs Injury

Objective:Sepsis is one of the most common causes of death in ICU patients,with a fatality rate of up to 30%～50%.Sepsis often affects the lungs,causing acute respiratory distress syndrome(ARDS),which can be life-threatening.Controlling of inflammatory cascade-like reaction is the key to prevent and treat ARDS.MicroRNAs(miRNAs)are important regulatory factors in a variety of pathophysiological processes,involved in the regulation of cell cycle,differentiation,apoptosis and inflammatory response,etc.Recent studies have found that miRNAs play an important regulatory role in ARDS.MicroRNA-129-5p(miR-129-5p),a member of the miR-129 family,has recently been shown to inhibit inflammation,but its role in ARDS has not yet been studied.In this study,we investigated the role and mechanism of miR-129-5p in LPS-induced human lung microvascular endothelial cells(HPMECs)injury.Methods:1.Blood samples of healthy volunteers and clinical sepsis patients were collected,serum was separated,and the level of miR-129-5p in serum was detected by RT-qPCR.Clinical data of the patients were collected for statistical analysis.2.In this study,human pulmonary microvascular endothelial cells(HPMECs)were stimulated with LPS to simulate the damage effect of HPMECs cells in ARDS.HPMECs cells were stimulated with 10 μg/ m L LPS for 0h,6h,12 h,24h and 48 h,respectively.RNA was extracted from the cells and the expression level of miR-129-5p in the cells was detected by RT-qPCR.3.The cells were divided into Control group and LPS group.The LPS group was treated with 10 μg/m L LPS,the Control group was treated with the same amount of PBS,and then DMEM high glucose medium containing 10% fetal bovine serum(FBS)was added for culture.RNA was extracted to detect the level of miR-129-5p,scratch assay and Transwell assay were used to evaluate the migration ability of cells,and RTCA assay was used to detect the proliferation ability of HPMECs.4.It was found that PIK3R1 may be one of the target genes of miR-129-5p through Target Scan database analysis,and PIK3R1 is the regulatory subunit of PI3K,which can activate the PI3K/Akt pathway.The presence of binding sites for miR-129-5p and PIK3R1 was verified by dual luciferase reports.5.HPMECs were transfected with miR-129-5p mimic and corresponding negative control(NC).The expressions of miR-129-5p and PIK3R1 mRNA were detected by RT-qPCR,and the levels of inflammatory factors were detected by ELISA.Western blot assay was used to detect the expression of PIK3R1 protein level.Transwell and scratch assay were used to analyze the migration ability of cells,RTCA assay was used to analyze the proliferation ability of cells,Flow cytometry was used to detect apoptosis,and the expression of PI3K/Akt pathway proteins PI3K,Akt and p-Akt were detected.6.HPMECs was transfected with PIK3R1 small interfering RNA(siRNA)and NC.The expression of PIK3R1 mRNA was detected by RT-qPCR,and the expression of PIK3R1 protein and PI3K/Akt signaling pathway proteins PI3K,Akt and p-Akt were detected by Western blot.Cell migration ability was evaluated by Transwell assay wound healing,and cell proliferation was detected by RTCA assay.Results:1.Expression of miR-129-5p in serum of subjectsCompared with normal volunteers,the expression level of miR-129-5p in serum of sepsis patients was significantly decreased(P < 0.05).The expression level of miR-129-5p in serum of patients with sepsis complicated with ARDS was lower(P < 0.05).2.Effect of LPS stimulation on HPMECs cellsWhen LPS was used to stimulate HPMECs to induce cell injury,the expression level of miR-129-5p was decreased(P < 0.05),and the expression level of miR-129-5p was the lowest at 24 h.The scratch experiment showed that the scratch healing rate of LPS was lower than that of Control group(P < 0.05).Transwell results showed that the number of cells migrated in the LPS group was lower than that in the Control group(P< 0.05).3.PIK3R1 is a target gene of miR-129-5pDual-luciferase reports showed that miR-129-5p and PIK3R1 had aseven base pairs binding site.4.Effects of transfection with miR-129-5p mimic on HPMEC cellsCompared with the miR-NC group,the expression of miR-129-5p mimic was up-regulated(P < 0.01),the wound healing rate was higher,the number of cell migration was increased,the proliferation ability was stronger and the apoptosis was reduced.After LPS treatment,the levels of inflammatory cytokines such as IL-1β,IL-6,IL-10 and TNF-α were increased,and the overexpression of miR-129-5p could reduce the expression level of inflammatory cytokines.RT-qPCR results showed that the expression of PIK3R1 mRNA was up-regulated in the LPS group.Western blot and immunofluorescence results showed that the expression of PIK3R1 protein was increased in the LPS group,while the expression of PI3K and p-Akt protein was decreased.After transfection with miR-129-5p mimic.5.Influence of interference with PIK3R1 expression on HPMECs.PIK3R1 mRNA levels were decreased in the PIK3R1 siRNA group when compared with the siRNA NC group(P < 0.001).Interference with PIK3R1 expression increased cell migration and proliferation,decreased PIK3R1 protein expression,and up-regulated PI3K and p-Akt protein expression.Conclusion:Mi R-129-5p targeted PIK3R1 gene and regulated the PI3K/Akt pathway to reduce LPS-induced HPMECs cell damage and inflammatory response.Upregulation of miR-129-5p could inhibit the production of inflammatory factors in LPS-induced sepsis model,promote cell migration and proliferation,and reduce endothelial cell damage.The expression of miR-129-5p in sepsis patients serum and HPMECs cells treated by LPS were decreased,this study found that miR-129-5p can regulate PI3K/Akt pathway by targeting PIK3R1 gene,to alleviate the HPMECs cell damage and inflammation induced by LPS.Overexpression of miR-129-5p can inhibit the production of inflammatory cytokines in ARDS model induced by LPS,and promote cell proliferation,migration and reduce the endothelial cell injury.