| Objective:Through the establishment of Sepsis-AKI animal model and cell model,we studied the expression of miR-30 a,miR-30 e and related matures(miR-30a-3p,miR-30e-3p)in Sepsis-AKI rats and human proximal tubule epithelial cells(HK-2)induced by LPS at different time points.To explore the relationship between miR-30 s expression and inflammatory response;predict and verify the target gene of miR-30a-3p,explore the role of miR-30a-3p in Sepsis-AKI and its mechanism.Methods:SD rats with healthy body and normal activities were used for animal experiments.They were randomly divided into normal control group(0h),CLP 3h,6h,12 h,24h four subgroups,each with 6 rats.Thirty valid samples were included in the experiment;LPS was used to stimulate HK-2 cells to establish a cell model.The cell experiments were divided into 7 groups: blank control group(0h group)and LPS(10ug/ml)for 0.5h,1h,3h,6h,12 h,24h group,the sample size of each group is n=3.Quantitative analysis of miRNA-30 a and miRNA-30 e and their related mature bodies in CLP-induced Sepsis-AKI rat model and LPS-induced HK-2 Sepsis-AKI cell model and their corresponding control group by RT-qPCR method.ELISA and qPCR were used to detect and analyze the inflammatory factors of TNF-?,IL-1? and IL6,and to investigate whether there is a correlation between miR-30 a and miR-30 e and related matures and inflammatory factors.Bioinformatics and western blot were used to predict and validate miR-30a-3p target genes and target sites,and to explore the role and mechanism of miR-30a-3p in Sepsis-AKI.Searching for possible target genes of hsa-miR-30a-3p by miRWalk,TargetScan,miRDB,mirDIP four miRNA target analysis databases and interpreting the predicted results of multiple databases as the final candidate target genes;Prediction of possible binding site information of miRNA-30a-3p and TEAD1 3' UTR by RNA hybrid database and Targetscan database;HK-2 cell transfection experiments were divided into four groups: LPS+NCm group,LPS+NCi group,LPS+miR-30a-3p mimics group and LPS+miR-30a-3p inhibitor group.The final concentration of l0ug/ml LPS was given 36 h after transfection.After 12 hours' stimulation,the qPCR was used to verify the transfection efficiency.After another 12 h,the cell pellet was collected and Western Blot was used to detect the expression of TEAD1.Result:1.General manifestations of Sepsis-AKI rat model,abdominal infection,pathological changes in renal tissue and expression of markers of renal injury.In the model group,postoperative mental dysfunction,fever,shortness of breath,chills,trembling,erect hair,slow activity,not drinking water,more eye secretions.At 24 hours after operation,intestinal edema was significantly aggravated,mucosal congestion,ascites exudation increased significantly,and infection characteristics were obvious.With the prolongation of postoperative time,the abdominal infection of the rats increased,the intestinal edema and ascites exuded more and more.HE staining of paraffin sections of renal tissue revealed that the tubular epithelial cells in the CLP group were swollen,the tubules were dilated,the tubular structure was destroyed,the epithelial cells were interstitial edema,the glomerular capillaries were dilated,and the blood was congested.And as time goes on,the pathological changes are more obvious.After rat modeling,serum creatinine levels of each group increased at different time points compared with that of the control group,with statistical differences at 6h 12 h 24h(P=0.0054,P=0.0001,P=0.0001).The serum urea value increased gradually from 3h after model establishment and increased significantly after 24 h.Group comparisons were statistically significant(P = 0.0343).2.General manifestations after establishment of the rat model,abdominal infection,pathological changes in renal tissue and expression of markers of renal injury.The expressions of inflammatory factors TNF-a and IL-1? were significantly increased at 3h after surgery(P<0.05)(TNF-a: P=0.0157,IL-1?: P=0.0224),and the inflammatory factor bursts rapidly after 6h.The expression of miR-30e-3p and miR-30a-3p was down-regulated at 12h(P<0.05)and then decreased.The expression of miR-30e-5p and miR-30a-5p began to decrease significantly at 3h postoperatively(P<0.05)and continued to decrease with prolonged postoperative time.There was a positive correlation between the expression of miR-30e-3p and IL-1? in the Sepsis-AKI rat model(r=0.44,P<0.05).The results of the other groups were not statistically significant,and there was no significant correlation between the two groups.3.The expression of miRNA-30a-3p,miRNA-30a-5p,miRNA-30e-3p,miRNA-30a-5p and inflammatory factors were correlated with LPS after stimulation of HK-2 cells.miR-30e-3p and miR-30a-3p,miR-30e-5p and miR-30a-5p had been down-regulated 3h after LPS treatment of HK-2 cells(P<0.05),and gradually decreased since the treatment time was prolonged.The expression of inflammatory cytokines TNF-a and IL-1? was up-regulated 1h after LPS treatment,and it was down-regulated 3 h later,that is,the expression level gradually recovered after inflammation burst.IL-6 expression in cells increased after LPS treatment,and reaches its peak after 6h and falls to its initial level within a few hours.The expression levels of miR-30e-3p,miR-30e-5p and miR-30a-5p in the Sepsis-AKI cell model were positively correlated with the expression of inflammatory factor IL-1?(correlation coefficients r value for 0.5187,0.6326,0.5893,respectively),with statistically significant(P<0.05).There was no statistically significant correlation between the expression of miR-30a-3p and IL-1? expression(P>0.05).As for miRNAs of each group(including miR-30e-3p,miR-30a-3p,miR-30e-5p,miR-30a-5p),there was no statistically significant correlation between miRNAs and inflammatory factors TNF-a and IL-6(P>0.05).4.Prediction and validation of miR-30a-3p target genes.Through bioinformatics prediction studies such as multiple database searches and screening,we find the TEAD1 gene may be a target gene regulated by miRNA-30a-3p.miRNA-30a-3p mimics and miRNA-30a-3p inhibitor were introduced into HK-2 cells by liposome lipo-3000.The transfected miRNA-30a-3p was highly expressed in the mimics group;the expression of Inhibitor group has no significant changes statistically.The unique transcript mRNA of the target gene TEAD1 was significantly up-regulated in the transfected miRNA-30a-3p inhibitor group(P=0.0008),while the expression change in the transfected miRNA-30a-3p mimics group was not statistically significant.The expression of TEAD1 in the Inhibitor group was significantly increased after transfection(P<0.0001),while the expression in the mimics group was significantly decreased(P<0.01).Conclusions:1.miR-30e-3p and miR-30a-3p,miR-30e-5p,and miR-30a-5p in the miR-30 family were down-regulated during the pathological process of Sepsis-AKI,and the expression is gradually reduced within 24 hours.They have the potential to be biomarkers for the diagnosis of Sepsis-AKI.(innovation)2.In the process of Sepsis-AKI,the inflammatory factors TNF-? and IL-1? rapidly increased in the inflammatory reaction and then decreased rapidly.IL-6 had the same upward and downward trend,but the time was expressed after TNF-?,IL-1?.(verification)3.The expression levels of miR-30e-3p,miR-30e-5p and miR-30a-5p were positively correlated with the expression of pro-inflammatory factor IL-1? in Sepsis-AKI in vitro,especially miR-30e-3p,in vivo and in vitro.A positive correlation with IL-1? expression was observed in the experiments.(innovation)4.TEAD1 is a direct target gene of miR-30a-3p.(innovation)5.Hypothesis: During the development of Sepsis-AKI,LPS down-regulates the expression of miR-30a-3p,and up-regulates the target gene TEAD1 to influence its Hippo signaling pathway to exert some regulation and influence on cell proliferation and apoptosis. |
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