| Part I Screening of miRNAs Expression profiles in rats after kidney ischemia-reperfusion injuryObjectiveTo Screen the change of miRNAs expression profiles in rats after renal IRI through microarray chip and analysis.Methods1.Building kidney ischemia-reperfusion injury model SD rat:rats adaptive feed after 1 week and fasted about 12 h, anesthesiaed through abdominal cavity and disinfection, incisioned the skin and peritoneum, then exposed bilateral renal pedicle, using noninvasive micro vascular clamp clip bilateral renal pedicle;loosen the vascular clamp at the same time after 45 min and sutured peritoneum and skin.After 12 h, anesthesiaed rats again and collected specimens.2.the experimental group:10 weeks of 12 rats were randomly divided into two groups,6 rats in each group:1) the experimental group:cliped bilateral renal vascular clamp for 45 minutes and collected specimens after 12 hours’reperfusion;(2) control:exposed bilateral renal pedicle by the same way and waited 45 min, layered suture incision after 12 h. 3.specimen collection and detection:Serum creatinine and urea nitrogen were detected o verify the success of modeling. For miRNA screening with microarray, kidney was harvested 12 h after reperfusion. At last, Verified the chip results by RT-PCR.Results1.Establishing the kidney ischemia-reperfusion injury model successfully, the levels of serum creatinine and urea increased significantly after IRI.2.There are 36 miRNAs’expressions changed significantly in microRNAs chip after kidney IRI.15 miRNAs of them changed more than two times. The results of RT-PCR consistent with the results of miRNAs chip. The expression of 10 miRNAs is increased. The expression of 5 miRNAs is decreased.Conclusion miRNAs expression profiles of rat is changed obviously after kidney IRI. MicroRNAs is involved in the occurrence and development of the kidney IRI through inflammatory activation, cell apoptosis and proliferation, angiogenesis, and interstitial fibrosis. The specific mechanism needs further study.Part II Expression of miR-98 after hypoxia reoxygenation injury and its influence on apoptosis through targeted Caspase3 in HUVECObjectiveExplore the change of miR-98 expression level and its influence on apoptosis after hypoxia reoxygenation injury in microvascular endothelial cells, and analyze the possible mechanism.Methods1. Constructed a model of hypoxia reoxygenation injury in vitro by cultivating human umbilical vein endothelial cell. Ischemia is simulated through light mineral oil cover cell surface for 60 min, and join DMEM medium without serum and sugar for 24 h. miR-98 were further detected at lh/4h, lh/12h and lh/24h to observe the MiR-98 expression in different time points after hypoxia reoxygenation injury in microvascular endothelial cells.2. Constructed a model of hypoxia reoxygenation injury after transfected miR-98 inhibitor and mimics and the corresponding control. Then we detect the expression of caspase 3 mRNA and protein level by RT-PCR and western-blot, t the cell apoptosis is detected by flow cytometry and TUNEL.3.We find miR-98 and caspase-3 complementary sequence in the 3’UTR region through bioinformatics retrieval, and build 3’UTR(Casp3) reporter gene carrier and 3’UTR (Casp3) mut report gene carrier in accordance with the complementary sequence,then join the 3 ’UTR region of purpose gene to the back of the luciferase gene. comparing the expression of microRNA, the change of report gene expression, by monitoring luciferase enzyme activity change, reflect the inhibition by miRNA quantitatively.Results1.the expression of miR-98 rose at 4h after A/R injury in HUVEC, and achieved about five times to NC group at 24h.2.we observed the negative feedback regulation between miR-98 and caspase-3 after transfection interferring the expression of miR-98. The cell apoptosis decreased significantly when the expression of miR-98 increased.The cell apoptosis increased significantly when the expression of miR-98 reduced.3.We build 3’UTR(Casp3) reporter gene carrier and 3’UTR (Casp3) mut report gene carrier successfully. the model of mutation is CTACCTCA mutatie to AACAUCGA. miR-98 had a regulation to the expression of CASP3 3’UTR luciferase gene(p<0.0001),but not to the expression of CASP3 mut 3’ UTR luciferase gene.Conclusion1. the expression of miR-98 increased after hypoxia reoxygenation injury in HUVEC.2. MiR-98 has a negative regulatory role on the levels of caspase-3 protein and cell apoptosis after A/R injury in HUVEC which shows miR-98 could exert certain protective effect to microvascular endothelial cells after hypoxia reoxygenation injury.3. Caspase-3 is the target genes of miR-98. |
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