The Effect And Mechanism Of MiR-192 And Its Target BLCAP In Acute Ischemia-Reperfusion Kidney Injury

Objective: Acute kidney injury(AKI)remains a common complication in hospitalized patients with prolonging hospital stay,cost increasing and poor outcome.Ischemia-reperfusion injury(IRI)is one of the most common causes of AKI.Micro RNAs(mi RNAs)are short,endogenous non-coding RNAs,with a length of 18-22 nts and regulate gene expression in post-transcriptional level by targeting messenger RNAs(m RNAs)and inhibiting translation of micro RNAs.Micro RNAs play important roles in various biological processes.There are increasing evidences that mi R-192 invovled in kidney fibrosis.However,the expression of mi R-192 and its mechanism in acute ischemia-reperfusion kidney injury is still unclear.Our study is to observe the mi RNA profiling,the dynamic change of mi R-192 in plasma and kidney tissue,as well as the role of its related target gene,so that to clarify the role and mechanism of mi R-192 in acute ischemia-reperfusion kidney injury.Methods: 1.18 male SD rats were randomly grouped into sham group and ischemia-reperfusioninjury(IRI)group(n=9 per group).Micro RNA PCR array was used to investigate plasma and kidney micro RNA profiling.Dynamic changes of plasma and kidney mi R-192 was tested by realtime PCR.HE stainning was used to observe histological injury.KIM-1 was tested by western blotting.2.Three databases including Targetscan,micro RNA.org and Mi RDB were used to predict target genes of mi R-192.Among the most conserved target genes which were simultaneously predicted by all databases,bladder cancer associated protein(BLCAP)which was closely related to cell cycle regulation,apoptosis and cell proliferation was chosen for verification.By co-transfection of dual-luciferase reporter plasmid containing BLCAP 3‘UTR region and mi R-192 expression vector to HEK293 T,the binding of mi R-192 with BLCAP 3'UTR was verified.Mi R-192 mimic and inhibitor was used to validate the role of mi R-192 on BLCAP regulation?BLCAP protein expression was detected by WB under IRI or hypoxia/reoxygenation(H/R)condition.3.Cell viability was tested by CCK-8 assay.Cell apoptosis was tested by Annexin V/PI stainning.Cell cycle was assessed by PI stainning.Cell cycle-related genes were examined by Realtime-PCR.RNA interference was used for BLCAP knockdown.Results: 1.Rat plasma global mi RNA PCR array showed a total of 42 differentially-expressed,among which 22 mi RNAs were aberrantly expressed with a more than 3-fold change between groups.The 15 elevated mi RNAs included mmu-mi R-409-3p,mmu-mi R-685,mmu-mi R-375,mmu-mi R-192,mmu-mi R-2182,mmu-mi R-429,mmu-mi R-1894-3p,rno-mi R-204#,rno-mi R-219-1-3p,mmu-mi R-1961,mmu-mi R-467 b,mmu-1954,mmu-mi R-1960,mmu-mi R-674,rno-mi R-504.The 7 down-regulated mi RNAs included mmu-mi R-126-5p,mmu-mi R-26 a,mmu-mi R-544,mmu-mi R-541,mmu-mi R-335-3p,hsa-mi R-338,and mmu-mi R-1930.Rat kidney global mi RNA PCR array showed 17 differentially-expressed mi RNAs,including 8 up-regulated mi RNAs and 9 down-regulated mi RNAs.The up-regulated mi RNAs included rno-mi R-384-3p ?rno-mi R-551b-5p?rno-mi R-125b-1-3p?rno-mi R-7b?rno-mi R-7a-5p?rno-mi R-708-3p?rno-mi R-494-5p and rno-mi R-92a-1-5p,while the down-regulated mi RNAs included rno-mi R-299b-3p?rno-mi R-3596a?rno-mi R-3596d?rno-mi R-143-5p?rno-mi R-329-5p?rno-let-7c-1-3p?rno-mi R-455-3p?rno-let-7e-3p and SNORD95?2.In dynamic study,plasma creatinine began to increase at IRI 3h and BUN level started to rise at IRI 6h,both reached their peak at IRI 12 h,began to decline at IRI 24 h,yet still significantly higher than the sham group.Plasma Cr and BUN were declined to baseline at IRI 3d.HE staining showed that significant injury of tubules at IRI 6h.At IRI 3d and 7d,although renal function has been completely back to normal,a few tubules were still not fully restored.Plasma mi R-192 levels began to rise at IRI 3h,peaked at IRI 12 h,declined slightly at IRI 12 h but still significantly higher than sham group and returned to baseline at IRI 3d;while kidney mi R-192 at IRI 6h were signifcantly lower than those in sham group and continued to remain at low levels till 7d after IRI.The trend of plasma and kidney mi R-192 levels was consistent with that of kidney structual and functional damage.3.Mi R-192 target genes were predicted by using three databases,including Target Scan,micro RNA.org and mi RDB.Six most conserved genes were simultaneously predicted by three databases.Wherein,BLCAP(Bladder cancer-associated protein)was one of them.The dual luciferase reporter system confirmed the combination of mi R-192 with 39-62 nt conserved sites of BLCAP 3'UTR.Mi R-192 mimic reduced the expression of BLCAP and mi R-192 inhibitor upregulated the expression of BLCAP.In the kidney tissues of IRI rats and NRK52 cells after H/R stimulation,BLCAP were increased.4.H/R induced NRK52 E cell viability decrease,increased apoptosis,cell cycle arrest and decreased expression of mi R-192.Under normoxic conditions,BLCAP knockdown significantly reduced the number of cell in G0/G1 phase compared to negative control group,while cells in S phase cells were signifcantly increased,,and the cell cycle-related genes,CDK2,CDK4,CCNA2,CCND1 and CCND3 were upregulated.While underhypoxic conditions,BLCAP knockdown can significantly improve cell viability,and cell cycle arrest and apoptosis induced by H/R.Conclusions: Plasma and kidney micro RNA profiling is dysregulated in rat acute ischemia-reperfusion kidney injury.The level of plasma mi R-192 was significantly increased while kidney mi R-192 was decreasd in kidney IRI rats in a time-dependent manner.Mi R-192 plays an important role in tubular epithelial cell injury through regulation of BLCAP.