| Objective:Congenital hydronephrosis caused by ureteropelvic junction obstruction(UPJO)is a common urinary system malformation in children.It is the main cause of congenital urinary tract obstruction in fetuses,infants and children.Congenital obstructive nephropathy is one of the most common causes of chronic kidney disease(CDK)and end-stage renal disease(ESRD)in children and infants.It manifests as progressive irreversible structural damage and/or renal damage,and is increasingly recognized as an important public health issue.Renal function damage caused by urinary tract obstruction is a complex process of multi-gene,multi-factor and multi-mechanism.It involves multiple pathological processes such as oxidative stress,apoptosis,inflammation,epithelial-mesenchymal transition and fibrosis.In the past decade,many studies have been devoted to exploring the molecular mechanism of CKD progression,and it has been proved that a variety of classical signaling pathways play an irreplaceable role in the progression of the disease.However,the mechanism of gene-related upstream molecular expression and regulation and renal fibrosis and functional damage is still unclear.MicroRNAs(miRNAs or miRs)are a class of endogenous,small,non-coding RNAs that play an important role in regulating gene expression at the post-transcriptional level.Previous clinical and experimental animal studies have shown that miRNAs play an important role in the pathogenesis of various kidney diseases,and miRNAs may be used as biological markers or used to intervene in disease progression to diagnose or treat diseases.But these studies are either based on animal models or limited to one miRNA of interest.High-throughput sequencing technology was used to obtain differentially expressed miRNAs in the kidney tissues of children with congenital hydronephrosis with normal renal function and decreased renal function.To the miRNAs that have a key role,we launched the first part of the experiment in response to this idea.The effects of differential miRNAs on cell epithelial-mesenchymal transition were observed by transfection of human kidney epithelial cell line(HK-2).Use database prediction software to select m RNAs that may be targeted to bind to differential miRNAs,amplify and inhibit differential miRNAs through cell transfection experiments to screen out reverse-change m RNAs,and through literature search,screen out the previous dual-luciferase reporter gene detection confirmation.And differential miRNAs can be used to regulate the epithelial-mesenchymal transition function of HK-2 cells through targeted binding m RNA.For this part of the idea,we launched the second part of the experiment.A model of complete unilateral ureteral obstruction in neonatal rats was made to simulate human congenital hydronephrosis,and kidney tissues were collected to verify the differential expression of differential miRNAs and targeted binding m RNA in the ureteral obstruction group and the normal group.Differential miRNAs agomir were transfected into a hydronephrotic animal model by intravenous injection of the mouse tail,and the regulatory relationship between the two was verified in vivo.It was found that the differential miRNAs can regulate the unilateral ureter of newborn rats through targeted binding of m RNA.The epithelial-mesenchymal transition function of renal tissue after complete obstruction.For this part of the idea,we launched the third part of the experiment.To better explore the role of differential miRNAs in the renal function damage of congenital hydronephrosis,provide a scientific basis for the future realization of miRNAs as diagnostic biological markers or molecular treatment of renal damage in children with congenital hydronephrosis An important foundation.Methods:1.We screened children with congenital hydronephrosis caused by obstruction at the junction of the renal pelvis and ureter,and divided them into the normal renal function group according to the renal function shown by preoperative diuretic nephronuclide imaging(different renal function>45%),Renal function impairment group(different renal function<30%),take the material during surgery,obtain the thinnest part of the renal parenchyma,immediately freeze it in liquid nitrogen,and store it in a refrigerator at-80°C for high throughput Sequencing,qRT-PCR.2.High-throughput sequencing technology was used to screen the differentially expressed miRNAs in the kidney tissues of the renal impairment group and the normal group,and predict the main functions and signal pathways of the parental gene enrichment.qRT-PCR was used to analyze the differentially expressed miRNAs in the kidney tissues of children.The unilateral ureteral obstruction model of newborn rats and HK-2 cell model were validated,and the temporal and spatial expression of differential miRNAs in animal models and cell models were obtained.3.Predict the m RNA bound to the differential miRNA through the database.Use HK-2 cell transfection experiment to amplify and inhibit miRNA187-3p to screen out reversed m RNA.Through literature search,the previous dual-luciferase reporter gene detection confirmed that miR-187-3p has a direct target regulatory relationship with PTRF and NT5 E.Verified that miR-187-3p can regulate the function of HK-2 cell epithelial-mesenchymal transition,fibrosis and TGFβ/Smad2 pathway through targeted binding PTRF.4.Making a unilateral ureteral obstruction model in newborn rats.The expression position of PTRF and NT5 E in renal tissue after obstruction was located by immunohistochemical staining.The expression differences of mir-187-3p,PTRF and nt5 e were verified in renal tissues of ureteral obstruction and normal newborn rats.Detect the effect of up-regulation or down-regulation of miRNA187-3p on renal epithelial-mesenchymal transition,fibrosis and TGFβ/Smad2 pathway after ureteral obstruction.5.Analysis: If the stripe difference is not obvious in Western blot,the relative density of the strip is detected by image-j software.Real-time PCR data were expressed by 2-△△Ct value,and spss21.0 statistical software was used to analyze the statistics,and the graph was drawn by graphpad prism 8.All the experimental data were expressed by mean ± standard deviation,P<0.05 is considered to be statistically different.Results:1.All children with congenital hydronephrosis had fetal SFU 4 degree hydronephrosis.Among the 6 children used for high-throughput sequencing,the age of the normal renal function group was 6-14 weeks(median age 10 weeks),and the age of the abnormal renal function group was 7-18 weeks(median age 10 weeks).Among the other 6 children used for result verification,the age distribution of the normal renal function group was 12-20 weeks(median age 13 weeks),and the age distribution of the abnormal renal function group was 6-22 weeks(median age 12weeks).The results of high-throughput sequencing indicated that there were 22 differentially expressed miRNAs in the kidney tissues of children with congenital hydronephrosis with decreased renal function and normal renal function.Compared with the normal renal function group,the expression of 5 miRNAs in the decreased renal function group Up-regulated and down-regulated expression of 17 miRNAs.Twelve human and mouse homologous miRNAs were selected for RT-q PCR verification in children’s kidney tissues,animal model kidney tissues,and cell models.It was found that the expression of miR-21-5p and miR-187-3p differed between the groups,which was consistent with the sequencing results.Basically the same,but the difference multiples are different.Among them,miR-187-3p has not been studied in the miRNA-target gene-signaling pathway network of obstructive renal disease,so we choose miR-187-3p as the object of further research.2.Using qRT-PCR technology in unilateral ureteral obstruction animal models and HK-2 cell models to verify the temporal and spatial expression trend of miR-187-3p.Compared with the normal group,the expression of miR-187-3p in the ureteral obstruction group and the TGFβ induction group increased in the early stage,and gradually decreased with the extension of the modeling time.On the 5th day of ureteral obstruction and the 6th day of TGFβ induction,it fell below the normal group level.It is consistent with our previous study found that the neonatal mouse ureteral obstruction appeared after 5 days of renal tubular epithelial-mesenchymal transition.3.A total of 23 m RNAs that bind to miR-187-3p are predicted from the database.It was verified in HK-2 cells that miR-187-3p negatively regulates the expression of PTRF and NT5 E.After over-expression of miR-187-3p,the expression of PTRF and NT5 E decreased;after inhibiting the expression of miR-187-3p,the expression of PTRF and NT5 E increased.Retrieving the results of dual luciferase reporter gene detection in published literature indicated that miR-187-3p has binding sites with PTRF and NT5 E,the latter two are the direct downstream target genes of miR-187-3p.Transfection of miR-187-3p mimics into cell models inhibited the epithelial-mesenchymal transition and fibrosis induced by TGFβ,and at the same time inhibited the phosphorylation of Smad2 in the TGFβ/Smad2 pathway.From this we obtained that miR-187-3p negatively regulates the expression of PTRF and NT5 E,and regulates the epithelial-mesenchymal transition function of HK-2 cells and the TGFβ/Smad2 pathway.4.We transfected miR-187-3p agomir into animal models.Experimental verification suggests that miR-187-3p can negatively regulate the expression of PTRF and NT5 E.miR-187-3p also inhibit the epithelial-mesenchymal transition and fibrosis of the kidney tissue in the unilateral ureteral obstruction model,and inhibit the phosphorylation of Smad2 in the TGF/Smad2 pathway.Therefore,we believe that miR-187-3p targeted regulation of PTRF plays a regulatory role in the renal dysfunction of congenital hydronephrosis,and the abnormal expression or regulation of any molecule may affect the progression of obstructive nephropathy.Conclusions:1.The above results indicate that miRNAs are differentially expressed in kidney tissues with normal renal function and decreased renal function in children with congenital hydronephrosis.2.miR-187-3p is related to renal damage in congenital hydronephrosis.miR-187-3p increases in the early stage of obstructive renal injury and decreases in the late stage.miR-187-3p plays an important role in the progression of renal dysfunction in congenital hydronephrosis through negative regulation of PTRF and NT5 E.3.Increased expression of miR-187-3p in kidney tissue inhibits the progression of kidney tissue epithelial-mesenchymal transition and fibrosis,and at the same time inhibits the phosphorylation of Smad2 in the TGFβ/Smad2 pathway.Increased expression of miR-187-3p in HK-2 cells inhibits cell epithelial-mesenchymal transition and fibrosis,and at the same time regulates the phosphorylation of Smad2 in the TGFβ/Smad2 pathway. |
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