| BackgroundDiabetic nephropathy(diabetic kidney disease,DKD)is one of the most common and devastating complications of diabetes caused by diabetic nephropathy.It is also the most common cause of end-stage renal disease(ESRD)and significantly increases the risk of diabetes Patient's mortality rate.In recent years,the incidence of diabetes has been increasing.According to the International Diabetes Federation,the global number of people with diabetes will increase from 382 million in 2013 to 592 million in 2035.Diabetes has become a global health problem,in order to prevent complications,attention should be paid to effectively delay the occurrence and development of diabetic nephropathy.Despite the control of blood glucose,blood pressure and blood lipids,such as proteinuria and other comprehensive treatment,but the current clinical efficacy is still poor.Because of the complex metabolic disorder in patients,it is difficult for DKD to progress to end-stage treatment,which can easily lead to death.Therefore,looking for an earlier and more effective prevention and treatment of diabetic nephropathy new entry point,to prevent or delay the progress of DKD is the medical profession has so far failed to solve the problem of exploring the pathogenesis of DKD in order to find a new and effective method of prevention has important medical And social research value.Diabetic nephropathy pathological features of early renal hypertrophy,glomerular and tubular basement membrane thickening,with the course of the disease,can gradually develop into the glomerular extracellular matrix progressive accumulation of tubulointerstitial fibrosis,and eventually Development of irreversible renal tissue damage.In the past that diabetic nephropathy is a glomerular disease-based disease,recent studies found that:tubular cell damage in the pathogenesis of DKD also plays an important role.In the early stage of DKD,there are different degrees of renal tubular injury,which is characteristic of early DKD and may not depend on glomerular lesions,and has a close relationship with renal function.Dallas et al.Found that renal pathology was found in DKD patients:only 30%of patients with diabetic microalbuminuria had typical glomerular structural damage;40%patients had no or slight glomerular injury,but renal tubular injury Serious,manifested as tubular basement membrane thickening,tubulointerstitial lesions,tubular atrophy,increased apoptosis,interstitial fibrosis,peritubular capillaries and other diabetic renal tubular sparse lesions;the other 30%of patients with normal renal structure.In addition,urinary NAG enzymes,al-MG,?2-MG and RBP,which are indicators of renal tubular function,have been significantly elevated in diabetic patients before microalbuminuria,whereas erythropoietin-1,25-(OH)2VitD3 significantly decreased;prompted changes in the renal tubules may precede the development of DKD may play a key role.Therefore,renal tubular injury has become an important area of DKD research in recent yearsmiRNA is a single-stranded,non-coding RNA consisting of about 22-24 nucleotides in length.It can specifically degrade mRNA or inhibit protein translation through specific binding to the 3 'noncoding region of the target gene mRNA.This binding mechanism is very flexible and complex,it can act on a biological function of the protein mRNA,but also can act on a variety of different biological function of the mRNA,fully involved in cell growth,proliferation,differentiation,death Each link,plays an important role,and therefore has a very close relationship with the process of life,whether it is normal development or pathological disease.The abnormal expression of miRNA is closely related to many diseases[5,6].Studies have shown that miRNA can also participate in the various stages of autophagy,and play a corresponding direct or indirect regulatory role.Sirt1,a homologous protein of yeast sirtuin,is a type ? deacetylase dependent on niacinamide adenine dinucleotide(NAD +),an analogue of Sirt2 in humans.It was first discovered in human body in 1999 that it is a kind of important molecule for tissue cells to self-protect and plays a very important role in the biological processes of regulating cell cycle,anti-oxidant stress,anti-inflammatory reaction and regulating glycolipid metabolism Important corner role.Studies have shown that the expression and activity of Sirtl are found in many diseases and are closely related to the occurrence of autophagy.It plays an important regulatory role in a large number of energy metabolism,gene transcription and cell senescence,not only can promote lipid mobilization and liver gluconeogenesis,but also can regulate the insulin secretion function of pancreatic ?-cells.Sirtl has the ability to deacetylate histone proteins and also has the effect of deacetylating a part of non-histone proteins,such as the p53 tumor suppressor gene.The existing studies have shown that Sirtl can play a regulatory role in regulating the acetylation status of its target genes in multiple signal transduction pathways,in particular through regulation of lysine acetylation and deacetylation In order to regulate the activity of its corresponding target protein,Sirtl also plays a regulatory role in stabilizing target protein and subcellular localization.Many studies have found that Sirtl plays an important role in maintaining the stability of the genome,regulating the energy metabolism of cells,prolonging cell life,delaying cellular senescence,regulating the development of malignant tumors,reducing the production of inflammatory cytokines and inhibiting apoptosis.Sirtl is also a key gene regulating many kinds of cell autophagy.Autophagy or autophagy is defined as the process by which a cell self-digests a cellular component or an original cellular component under the action of a lysosomal system.When mammalian cells are under stress,such as starvation,energy deficiency,and disease,autophagy is the main way to maintain intracellular environment stability and ensure macromolecular protein,organelle recycling,and organelle quality.It is a kind of ubiquitin-The more powerful and specific proteolytic pathways of the protease system are specific and powerful for the elimination of intracellular invaders,misfolded proteins,peroxidation and aging proteins.Autophagy can prevent tissue damage in the long-term,and it can also maintain the homeostasis of the internal environment under short-term stress conditions.Method1.Cell cultureHuman renal proximal tubule(HK-2)cells were cultured in MEM(Gibco,Invitrogen)supplemented with 10%FBS(Gibco,NZL)at 37? and 5%C02.The culture medium was changed every two days,waiting for the growth of HK-2 cells to 60%,and the medium was supplemented with MEM(Gibco,Invitrogen)supplemented with 2%FBS(Gibco,NZL)and cultured for 24 hours,and the cells were divided into two groups.Group 1:Medium with normal glucose concentration(5.5 mmol/L)served as a control.Group 2:High glucose stimulation group supplemented with high glucose(30 mM)medium.All cells were incubated with the medium for 72 hours.2.RNA extraction,reverse transcription and quantitative real-time RT-PCRThe Trizol method extracts total cellular RNA and quantifies nucleic acids using a spectrophotometer.RNA was reverse transcribed to cDNA using M-MLV reverse transcriptase reagent.The real-time fluorescence quantitative PCR reaction was performed using the SYBR method.Gene expression was calculated by 2_??Ct method.B-actin serves as an internal reference gene.3.Protein extraction and Western blotThe RIPA method was used to extract the total protein of the tissue or cell.The protein concentration of the sample was determined by the BCA method and the protein was denatured at 100?.Prepare 8%-15%SDS-PAGE gel,electrophoretically separate the sample protein,and wet the target protein onto the PVDF membrane.The PVDF membrane was blocked with TBST containing 5%nonfat dry milk or 5%BSA.The primary antibody of the desired protein was diluted and incubated with a PVDF membrane on a 4? shaker overnight.On the next day,the PVDF membrane was placed in TBST and then incubated with fluorescent secondary antibody(1:15000)in the dark for 1 h.After being placed in TBST and washed again,it was visualized using the Tanone 5500 automated chemiluminescence imaging system and condensing with condensate.Results of the analysis of the Quantity One system.4.Transfection of cells1)In order to achieve a change in expression of miR-155-5p,HK-2 cells were treated with miR-155-5p mimic/inhibitor using LipofectamineTM 3000 as a transfection reagent.2)In order to achieve changes in Sirt1 expression,HK-2 cells were treated with Sirt1 plasmid or Sirt1 si while using LipofectamineTM 3000 as a transfection reagent.3)In order to achieve inhibition of P53 expression,HK-2 cells were treated with the P53 inhibitor Pifithrin-a while the non-inhibitory group was used as a negative control(NC).5.Determination of luciferase reporter genePCR amplification of miR-155-5p and Sirtl 3'UTR binding sites from genomic DNA of HK-2 cells.The amplified Sirtl 3' UTR fragment was inserted into the psi-Check2 vector(Ribobio,Guangzhou,China)using the Xhol and Notl restriction sites,respectively.HK-2 cells were placed at a density of 60%in 24 well plates and cultured in MEM medium containing 2%Gibico fetal bovine serum for 24 hours.Dual-luciferase reporter vector psi-Check2(1 jig)and miR-155-5p mimic/inhibit constructed with Sirtl and miR-155-5p binding sites were co-transfected with 30 nm D-glucose and 10%Gibico The fetal bovine serum was cultured in a MEM medium for 48 hours,cells were collected,and luminescence was analyzed using a dual luciferase assay system.6.Chip analysisThe software predicts the presence of a p53 binding site in the miR-155-5p promoter region.Cells were treated with 1%formalin and sonicated to collect soluble chromatin supernatants.With anti-p53 antibody overnight,mouse IgG immunoprecipitation served as a negative control.The immune complex is washed and a DNA sample is obtained.The recovered DNA was analyzed by using primer qRT-PCR containing the miR-155-5p promoter region and the P53 binding site.7.Statistical AnalysisAll test data are represented by mean standard error(X-ray SEM).One-way ANOVA was used to compare multiple groups of means.The normal distribution of the two samples was compared using two independent sample t-tests.Statistical results were considered statistically significant when the P-value was<0.05.Result1.Determination of general expression levels of high glucose stimulated HK-2 cellsThe test was divided into two groups,normal glucose(5.5 mM)and high glucose(30 mM),synchronized with HK-2 cells and stimulated for 72 hours to extract total RNA and protein for analysis.After high glucose stimulation,miR-155-5p expression increased,Sirtl expression was inhibited,P53 expression was elevated,autophagy activity was inhibited,and fibrosis related indicators were enhanced.2.MiR-155-5p directly adjusts SirtlTransfection of HK-2 cells with miR-155-5p mimic or inhibitor was successful.After qRT-PCR and WB detection,expression of Sirtl was decreased,autophagic activity was inhibited,and expression of fibrosis markers was enhanced after miR-155-5p overexpression.The corresponding change after-155-5p expression is suppressed will be reversed.Subsequently,we constructed a dual luciferase reporter plasmid vector,psi-Check2,containing the Sirtl 3' UTR region and the miR-155-5p binding site,and co-transfected with miR-155-5p mimic/inhibitor.The results showed that the psi-Check2 fluorescence ratio co-transfected with miR-155-5p mimic was significantly reduced,and conversely increased,indicating that the miR-155-5p binds to the Sirtl 3'UTR region.3.Sirtl directly regulates the expression of P53 in high-glucose cultured HK 2 cellsAfter Sirtl gene or si-Sirtl was transfected with LipofectamineTM 3000,and after successful overexpression or suppression was verified,it was observed by qRT-PCR and WB that P53 expression was significantly reduced after Sirtl overexpression under high glucose conditions,and after Sirtl was inhibited This effect will be reversed.It was demonstrated that sirtl can inhibit the expression of P53 in high glucose-treated HK-2 cells.4.Regulation of miR-155-5p by P53We used Chip experiments to verify whether p53 in high glucose cultured HK-2 cells can regulate miR-155-5p gene expression.It was found that high glucose stimulation can up-regulate the binding of P53 and miR-155-5p promoter regions.After inhibiting P53 expression with inhibitor Pifithrin-?,a decrease in miR-155-5p expression was observed.Conclusion1.The expression of miR-155-5p was increased under high glucose status,and protective factors such as Sirtl and autophagy were inhibited,and the expression of P53 and fibrosis markers were enhanced.2.miR-155-5p can specifically bind to Sirtl 3'UTR region and inhibit its expression.3.Sirtl can inhibit the expression of P53,promote autophagy activity and inhibit the expression of fibrosis markers.4.P53 specifically binds to the miR-155-5p promoter region and promotes miR-155-5p expression.The presence of high glucose-induced tubular cell injury signaling loop p53/miR-155-5p/Sirtl was demonstrated. |
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