MicroRNA-27a Suppresses TLR4-induced Renal Ischemia-reperfusion Injury In Pregnant Rats

Objective:Ischemia reperfusion injury(IRI)is one of the primary causes of acute renal injury and even acute renal failure in pregnant rats.An accumulating amount of evidence has indicated that the aberrant expression of microRNAs(mi RNA)is tightly associated with the progression of IRI.In the process of renal IRI in pregnanct rats,inflammatory reactions,cell adhesion and the death of renal tubular epithelial cells serve important roles.The present study investigated the expression of renal miRNAs following renal IRI in an attempt to identify the miRNAs that exert pivotal functions in renal IRI in pregnant rats.Methods: Renal IRI in pregnant rats model were used in vivo and NRK52 E cells were used in vitro.MicroRNA microarrays and polymerase chain reaction(PCR)were used to screen differentially expressed microRNAs in renal after renal IRI in pregnant rats.miR-27 a control/agomir was injected into the tail vein of the rats for 48 h prior to ischemic surgery via a tail vein.And transfection of miR-27 a mimic and inhibitor into NRK52 E cells.Real-time quantitative PCR detection of miR-27 a expression,Western blot analysis of TLR4 protein level.The microRNA that can specially regulate TLR4 was determined by bioinformatic analysis and combined with dual luciferase reporter assay to confirm whether miR-27 a targets on the predicted 3'-UTR sequence of TLR4 mRNA,MTT colorimetric assay to test change in trophoblast cell proliferation capacity before and after transfection,and flow cytometry to assay cell apoptosis capacity before and after transfection.Results: The present study observed that miR-27 a,which was downregulated in IRI,and the 3'untranslated region(UTR)of Toll-like receptor 4(TLR4)have associated binding sites.The levels of TLR4,mir-27 a and specific associated proteins in the renal tissues of gestational rats were determined by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)analysis,immunohistochemistry and western blotting.The associations between miR-27 a and TLR4 were analyzed,and the regulatory effect of miR-27 a on TLR4 was detected by a luciferase reporter gene assay,western blotting and RT-qPCR in vivo and in vitro.In addition,the present study demonstrated that miR-27 a suppressed the expression of TLR4 by binding to the 3'UTR of TLR4 mRNA.The overexpression of mir-27 a downregulated the expression of TLR4,which in turn inhibited the inflammation,cell adhesion and death in IRI.mir-27 a inhibited inflammation in IRI via decreasing the expression of TLR4.Conclusions: The overexpression of mir-27 a downregulated the expression of TLR4,which in turn inhibited the inflammation,cell adhesion and death in renal IRI in pregnant rats.microRNA-27 a suppresses TLR4-induced renal ischemia-reperfusion injury in pregnant rats...