| Objective:Due to the limitation of creatinine and the rapid progress of AKI,the alteration of creatinine is too minimal to detect AKI in the early stage and alert clinical workers.Therefore,exploring early biomarkers for AKI diagnosis is constant goal of AKI clinicians.Micro RNAs(mi RNAs)are short-chain non-coding RNA molecules that are sensitive and remaining stable in the body fluid.So it is considered to be a good choice for disease diagnosis and prognostic evaluation.As a negative regulator of gene expression,mi RNA can target m RNA to reduce its stability or the effectiveness of translation,then participate in the pathophysiology of the disease.First aim of the current study is to explore the pathogenesis of mi RNA expression profile in AKI course,and whether mi RNA can be used as an early diagnostic marker of AKI.The second aim of this study is to explore the functional role of candidate mi RNA in the course of AKI.Methods:In the study,a rat animal model of AKI induced by ischemia-reperfusion was established.The serum creatinine level and kidney injury markers that reported previously were confirmed to ensure a successful model.Urine sample and renal cortex tissue were collected at different time points in rats.Mi RNAs were analyzed by high-throughput genechip for urine and renal cortex,respectively.The results of the genechip was verified by quantitative PCR.Patients with cardiac surgery were enrolled.The general information was collected in all patients.The creatinine levels was monitored in all the time points after surgery and urine was collected from patients.Also we analyzed the expression of NGAL and KIM-1 that reported as renal injury markers previously and analyzed the candidate mi RNAs that screened in the first part of this study.The hypoxia-reoxygenation model was established using human renal tubular epithelial cells(HK2)to simulate the ischemiareperfusion process in vivo.Mimics and inhibitor were used to regulate micro RNA-30c-5p expression in vitro.Apoptosis of hypoxia-reoxygenation model were observed after the oligo transfection by flowcytometry,TUNEL staining and Western Blot.Results : Results from the first part showed that compared with the sham group,the creatinine level of I/R group was significantly higher,and the pathological damage was significant.The infiltration of tubulointerstitial inflammation,tissue degeneration could be observed post operation.71 mi RNAs were screened out with the criteria of the mi RNA > 2 fold change upregulation and downregulation in three or more time points,when the mi RNAs were analyzed in the urine.The expression of mi RNAs was also analyzed in the renal cortex,and 19 mi RNAs were screened out with the same criteria above.In the second part of this study,the results showed that 38%(27 cases)of patients enrolled in the postoperative cardiothoracic surgery have developed AKI.Compared with Non-AKI group,the micro RNA-30c-5p,micro RNA-192-5p and micro RNA-378a-3p were increased in AKI group,and the first two was statistically significant.As mi RNA candidate marker,Micro RNA-30c-5p is more effective in the diagnosis than NGAL and KIM-1,and has the potential to become a new diagnostic marker.In the third part of this study,the results showed that the after up-regulate the expression of micro RNA-30c-5p,the apoptosis and apoptosis-related protein were also decreased.Upregulation of micro RNA-30c-5p could stabilize the expression of hypoxia-inducible factor-1?(HIF1?)exposed to hypoxiareoxygenation.The expression of micro RNA-30c-5p did not improve the apoptotic level of cells and did not improve the increase expression of apoptotic protein after HIF1? was knocked down.The cytokine signaling inhibitor-3(SOCS3),which may be a target gene of micro RNA-30c-5p predicted using database such as Target Scan,then the hypothesis was verified by the luciferase reporter assay.And the protein level of HIF1? was increased after knockdown of SOCS3 expression with si RNA,and the level of HIF1? did not change significantly after overexpression of SOCS3.Apoptosis were subsequently examined after regulation of SOCS3.It was found that the level of apoptosis was significantly reduced after lowering the level of SOCS3,and the level of apoptosis was also increased after overexpression of SOCS3.Apoptosis-related proteins also showed a corresponding trend.This suggests that the upregulated SOCS3 can inhibit the expression of HIF1?,and will increase the level of apoptosis.Conclusion: In the ischemia-reperfusion induced AKI animal model,the mi RNA in the urine and renal cortex have different expression profiles,and urine micro RNA-30c-5p has a good potential to be a novel diagnostic marker.The upregulation of the mi RNA can protect the cells from hypoxia-reoxygenation injury and reduce the apoptosis level.And the protective effect is achieved through the stabilization of HIF1?.Furthermore,the HIF1? stabilization is achieved by reducing the expression of SOCS3.At last,the SOCS3 reduction is achieved by the fact that micro RNA-30c-5p target gene SOCS3. |
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