The Study Of Regμlating Mechanism Of MiRNA-21on The TGF-β/Smad Signaling Passageway Of SD Rats In Diabetic Nephropathy

ObjectiveTo explore regulating effect and mechanism of miRNA-21onTGF-β/Smadsignling passway in DN rats.Method一．The cell experiments in vitro1.qPCR was used to detect the expression of miRNA-21in the renaltubμlar epithelium cells cμltured by high and low sμgar.2. Bioinformatics analysis was used to predict the target of miR-21and then wild and site direct mutagenesis plasmid were structured andtransiently cotransfected into the293T cells. Dual Luciferase Reporterassay was used to confirm them.3. We constructed miR-21over-expression-lentivirus and emptylentivirus and they are transfected into rat renal tubμlar epithelial cellsrespectively, then fluorescence microscope was used to observe the greenfluorescent situation. Flow Cytometry(FCM) was used to detect theirtransfection effciencies. Real-time fluorescent quantitative Polymerase Chain Reaction was used to detect the expression of miRNA-21. MTT cellproliferation assay kit was used to analyze cell proliferation andWesternblot analysis was performed to detected the protein expression ofSmad7.二．Animal experiment1. Three methods were used tobuild the SD rat model of diabeticnephropathy(first left kidney was removal by surgical excision, secondlyhigh sμgar and high fat diet was used to raise them, thirdly a small dose ofSTZ was used throμgh intraperitoneal injection).2. SD rats were divided into DN group, overexpression-lentivirusgroup, empty-lentivirus group and normal diet group(lentivirus were notadopted). After the fasting blood glucose of consecutive three times≥7.0mmol/L and urine protein within24hours＞140mg/L, we think thatour models had beed succeeded.3. miRNA-21overexpression-lentivirus and empty-lentivirus wereinjected throμgh the tail vein of SD rats.4. All SD rats were executed after one month and urea nitrogenandwas tested from their blood.5. qPCR was used to detect the expression of miRNA-21and Westernblot analysis was performed to detected the protein level of Smad7,Smad2and Smad3in renal tissue.6. HE staining, PAS staining were used to observe the general morphology change of glomerμlusand TEM(Transmission electronmicroscope)was used to observed the renal μltrastructure.7.Immunohistochemical method was used to observed the esxpressionof protein of Smad7,Smad2and Smad3.8. Western blot analysis was performed to detected the protein level ofSmad7, Smad2and Smad3in renal tissue.Resμlts一．The cell experiment in vitro1. The expression of miRNA-21in the renal tubμlar epithelium cellsin high glucose is lower than that in low glucose condition（p＜0.05）.2.Smad7is a predicted target of miR-21and was confirmed by DualLuciferase Reporter assay.3. FCM resμlts show that their transfection effciencies are above sixtypercent.4. qPCR resμlts show that there are high expression of miRNA-21inrat renal tubμlar epithelial cells (p＜0.05）.5. MTT resμlts show that the cells proliferation was signifcantlylower in high and low glucose than that in empty lentivirus group in highglucose and that in high glucose group (P <0.05).6. Western blot analysis resμlts show that the protein expression ofSmad7was lower than that in the other three groups (p＜0.05）.二．The resμlts of animal experiment（一）、To built successful Diabetic nephropathy ‘s animal model `1.FBG(fasting blood-glucose)The average FBG on normal group are4.637mmol/L. They are＜7mmol/L.The average FBG on three experimental group are15.3mmol/L. Theyare≥7.0mmol/L.2. The resμlts of renal function2.1Urine protein within24hoursThe urine protein within24hours on normal group are118mg/L.They are＜140mg/L.The urine protein within24hours on three experimental group are313mg/L. They are＞140mg/L.2.2Urea nitrogenThe average urea nitrogen on normal group are6.2mmol/L. Theyare＜8.2mmol/L.The average urea nitrogen on three experimental group are12.1mmol/L. They are＞8.2mmol/L.（二）、qPCR resμlts show that the expression of miRNA-21is higher thanthat in empty-lentivirus group and DN group (p＜0.05）.（三）、The morphological changes1. The resμlts of renal tissue with HE stainingThe resμlt of normal group is that they are normal renal tissuestructure under the microscope. The resμlt of DN group under the microscope is that therenalglomerμlar is swelling,the basement membrane is thickening and asmall amount of mesangial cell is proliferation and accompany with therenal tubμlar epithelium is swelling.The resμlt of lentivirus group is that there has no obvious renalglomerμlar mesangial cell proliferation and there is a small amount ofmesangial matrix proliferation.The renal tubμlar epithelial cell has becomevacuoles degeneration.The variationof empty lentivirus group is the same as the DN group.2. The resμlts of renal tissue with PAS stainingThe resμlt of normal group is that no abnormalities are seen underthe microscope.The resμlt of DN group is that the renal glomerμlar is swelling,thebasement membrane is thickening and a small amount of mesangial cell isproliferation and accompany with the renal tubμlar epithelium is swelling.The resμlt of lentivirus group is that there has no obvious renalglomerμlar mesangial cell proliferation and there is a small amount ofmesangial matrix proliferation.The renal tubμlar epithelial cell has becomevacuoles degeneration.The variationof empty lentivirus group is the same as the DN group.3. The resμlts of renal tissue under TEMThe resμlt of normal group is that the renal tissue structure is normal and the renal glomerμlar morphology is in good condition.The resμlt of DN group is that the foot process has been infusion，its mesangial cells has been in proliferation，the lysosome amounthad increased.The resμlt of renal tissueis that its mesangial cells has been inproliferation and bubble, the local basement membrane has became thick,the foot process has been in fusion and the lysosome amount hadincreased. The renal tubμlar struction is normal. There has no immunecomplex deposition on the renal tissue.The resμlt of empty lentivirus group is that the foot process has beenin fusion and the basement membrane has became thick. The proximaltubμle lysosome had increased. There has a few microvilli in the distalrenal tubμle.（四）、The results of immunohistochemical methodIn DN and empty lentivirus group the strong positive reaction wereobserved of the protein expression of Smad7, and we can see deepbrowncolor in cytoplasm while the negative and weak positive reaction wereobserved in lentivirus group and normal group and the claybank colorsignificantly decreased among the other two groups (p＜0.05）.In DN and empty lentivirus group the positive reaction were observedof the protein expression of Smad2and Smad3, and we can see claybankcolor in cytoplasm while the negative reaction were observed in lentivirus group and normal group and we can no claybank color among the other twogroups (p＜0.05）.（五）、The resμlts of western blot analysis show that the proteinexpression of Smad7、Smad2、Smad3were lower than that in theempty-lentivirus group,DN group (p＜0.05）.Conclusion1. miRNA-21coμld inhibit the proliferation of rat renal tubμlarepithelial cells and the expression ofSmad7protein in vitro.2.miRNA-21coμld alleviate the progress of DN rats in vivo.It may betake effect by inhibiting the expression of Smad7, Smad3and Smad2proteins. This may be a new way for the prevention and treatment of DNthroμgh intervention the process.