CircRNA1860/hsa-miR-125b-5p Regulates NDRG1 Gene And Inhibition Effect On Epithelial-Mesenchymal Transition In Renal Epithelial Cell Induced By High Glucose

Objective:In recent years,with the increasing incidence of diabetes mellitus(DM)all over the world,heavy burden has been loaded to patients caused by DM and related complications.Diabetic kidney disease(DKD)is one of the most serious complications of diabetes.In present,it has been the the second cause of end stage renal failure and the first cause of hospital cause of renal disease in China.DKD is a kidney disease characterized by progressive renal fibrosis.The pathogenesis of DKD is not clear.There may be a number of factors involved.At present,the classic pathogenesis of DKD is based on the center of glomerulus theory,but recent studies have found that the pathological changes of tubular cells are separately earlier than glomerular changes at the early statge of DKD,and the proportion of severe tubular lesions was higher than that of typical glomerular structural changes in microalbuminuria DKD,suggesting that tubular injury is also an important pathological change of DKD.Therefore,it has been suggested that although the glomerulus is greatly affected in DKD,the tubulointerstitium is the main initiation of progression.Among mechanisms of tubular changes,epithelial-mesenchymal transformation(EMT)of renal epithelial cells is one of the hot spots.Recent studies have shown blocking EMT of renal epithelial cells can effectively alleviate renal fibrosis and improve renal function,indicating that EMT plays an important role in the process of renal fibrosis.Therefore,it is important to study the mechanism of EMT in diabetic kidney disease.N-MYC downstream regulated gene(NDRG)is a new discovered gene family in recent years.Studies on NDRG family have demonstrated that they are involved in EMT process in tumor disease,and overexpression of NDRG family significantly inhibited EMT process.NDRG1,an important member of NDRG family,is expressed highly in the kidney organ.But,there is no research on NDRG1 against EMT in DKD.Therefore,this study is desiged to explore the effect of NDRG1 on DKD to provide new theory mechanism of DKD.Methods: Part Ⅰ: Transcriptome sequencing analysis We used 30 mmol/L glucose DMEM medium as high glucose group and 5.6 mmol/L glucose DMEM medium as control.72 hours later,cells were collected and subjected to whole transcriptome sequencing(transcriptome sequencing,RNA-seq),the differentially expressed m RNA was analyzed by GO and KEGG enrichment analysis.According to the results,we combined the results of m RNA,miRNA and circRNA sequencing to further analyze the endogenous competition inhibition(Ce RNA)analysis and get the candidate Ce RNA regulatory networks.Part II: Study on inhibition effect of NDRG1 on epithelial-to-mesenchymal transition(EMT)of renal epithelial cells induced by hyperglycemia.Firstly,the renal tubular epithelial cells HK2 were cultured in DMEM medium with different gradients of glucose concentrations,and the cells were collected after 72 hours.The expression of NDRG1,STAT3,Snai1,E-cadherin and α-SMA were analyzed by q RT-PCR,Western-blot and immunofluorescence.The lentiviral plasmids p SLenti-SFH-NDRG1,p SLenti-U6-sh RNA-NDRG1 were constructed and transfected into HK2 cells,respectively.Cells were collected after 72 hours of 30mmol/L high glucose DMEM culture,the m RNA and protein expressions changes of NDRG1,STAT3,Snai1,E-cadherin and α-SMA were examined.Then,STAT3 inhibitor JSI-124 were used to inhibit STAT3 in HK2 cells,the expression levels of NDRG1,STAT3,Snai1,E-cadherin and α-SMA were detected.Db/db mice were adopted to establish diabetic kidney disease model.Body weight and blood glucose were measured continuely in each two-week period.At 20-week-age,24-hour urine protein was examined.The mice were sacrificed,and the renal tissues were obtained for observation under light microscopy(HE and Masson staining)and electron microscopy(EM).Immunofluorescence stain assay was adopted for detecting of NDRG1,STAT3,Snai1,E-cadherin and α-SMA.The m RNA expressions level of NDRG1,STAT3,Snai1,E-cadherin and α-SMA in renal tissues were analyzed by q RT-PCR method.Db/m and C57BL/6 mice were selected as controls.A standard diet and free water were given to all mice in the experiment process.Part III.circRNA 1860/hsa-miR-125b-5p/NDRG1 Ce RNA regulatory network for EMT Firstly,we tested candidate circRNAs and miRNAs by transcriptome gene sequencing by q RT-PCR in HK2 cells cultured in high glucose.The final target Ce RNA regulatory network was identified as circRNA1860/hsa-miR-125b-5p/NDRG1.Circular structure of circRNA1860 was identified by RT-PCR using divergent primer and convergent primer.Subcellular localization of circRNA1860 and hsa-miR-125b-5p was tested under high glucose.FISH suggested that circRNA1860/hsa-miR-125b-5p regulated the expression of NDRG1 by Ce RNA mechanism.The overexpression and knockout plasmids of circRNA1860 were constructed and transfected into HK2 cells.q RT-PCR was adopted to analyze the expression changes of hsa-miR-125b-5p,NDRG1,E-cadherin and α-SMA.Western-blot analysis assay was used to detected protein expressions of NDRG1,E-cadherin and α-SMA.The hsa-miR-125b-5p mimic and hsa-miR-125b-5p inhibitor were transfected into HK2 cells.72 hours later,cells were collected,and the m RNA expressions of hsa-miR-125b-5p,NDRG1,E-cadherin and α-SMA were detected by q RT-PCR.Western-blot method was used to examine the protein exression of NDRG1,E-cadherin and α-SMA.The binding of circRNA 1860 with hsa-miR-125b-5p were detected by dual luciferase method.Results: Part Ⅰ: Transcriptome sequencing analysis A total of 66072 m RNA expressions were obtained in the transcriptome sequencing.Among them,when compared with the HK2 cells cultured in control DMEM medium,194 genes were differentially expressed in high glucose DMEM medium,among which 131 genes were up-regulated and 63 genes were down-regulated.24 miRNAs were differentially expressed in the high glucose group,including 10 up-regulated and 14 down-regulated.39 circRNAs were differentially expressed cultured in the high glucose medium,including 22 up-regulated and 17 down-regulated circRNAs.The top five significant up-and down-regulated m RNAs were selected for preliminary verification.The five up-regulation m RNAs were EIF2S3 B,SMIM15,SLC28A3,BASP1 and C12ORF45;and the five down-regulated m RNAs were RIMBP3 b,HSPA6,HID1,LAMP3 and NDRG1.q RT-PCR results showed that EIF2S3 B,SMIM15,BASP1 were up-regulated genes and NDRG1 was down-regulated genes.Functions and pathways were searched for the above proteins in public databases,and NDRG1 was found to be associated with EMT.Therefore,the role of NDRG1 for EMT in renal tubular epithelial cells was further explored in the follow-up studies.According to Targetscan and Miranda databases,the predicted candidate circRNA-miRNA-m RNA Ce RNA network had 26 circRNA nodes,24 miRNA nodes,and 677 m RNAs,constituting 5870 possible Ce RNA interaction networks.After screening the NDRG1-related nodes,5 circRNA nodes and 5 miRNA nodes were obtained,forming 6 kinds of interaction networks.Therefore,the candidate Ce RNA network will be explored for further studies.Part II: NDRG1 participated in the process of epithelial-mesenchymal transition of renal epithelial cells under high glucose.Under the condition of high glucose DMEM medium,in which the concentration of glucose was 30mmol/L,the expression of E-cadherin m RNA decreased and α-SMA m RNA increased in HK2 cells.Meanwhile,the protein expression of E-cadherin decreased and α-SMA increased in HK2 cells.HK2 cells underwent wedge-shaped deformation,and the migration ability of HK2 cells was enhanced by wound-healing and transwell test.All the above results demonstrated that HK2 cells underwent EMT under high glucose.The results of q RT-PCR and Western-blot analysis showed that the m RNA and protein expression of NDRG1 decreased under high glucose.These results suggest that NDRG1 may be involved in the EMT process of HK2 cells under high glucose condition.NDRG1 overexpression was designed in HK2 cells transfected with p SLenti-SFH-NDRG1 lentivirus.After overexpression of NDRG1,the expression of NDRG1 and E-cadherin increased signigicantly,STAT3,Snai1 and α-SMA decreased by examined by q RT-PCR and Western-blot assays.These results suggested that NDRG1 overexpression can inhibit EMT and may be related to STAT3/Snai1 pathway.NDRG1 was knocked down by transfected with p SLenti-U6-sh RNA-NDRG1 vector.The expression of STAT3,Snai1,E-cadherin and α-SMA in HK2 cells was detecterd by q RT-PCR and Western-blot method.Results showed that the expression of NDRG1 and E-cadherin decreased signigicantly.However,the expression of STAT3,Snai1 and α-SMA increased after NDRG1 knocked out.Then,JSI-124,a STAT3 inhibitor was used to inhibit STAT3.Results showed the expression of Snai1 and α-SMA decreased and the expression of E-cadherin increased.Thus,NDRG1 inhibited EMT in HK2 cells under high glucose concentration via STAT3/Snai1 pathway.Spontaneous DKD was constructed by db/db mice model.The db/db mice were in obese,with polydipsia,polyphagia and polyuria.In H-E staining,enlarged glomerular,diminished renal capsule,and thickened glomerular basement membrane were observed.In tubular cells,extensive edema,granular and vacuolar degeneration,and thickened basement membrane were observed.Masson’s staining showed increased basement membrane stroma in glomerulus and increased fibrinogen in tubulobasement membrane with thickened basement membrane.Under the electron microscope,db/db kidney glomerular cells had homogeneous thickened glomerular basement membrane and increased mesangial matrix.The number and morphology of mitochondria in renal tubular epithelial cells were mainly decreased.In db/db mice,the expression of NDRG1 and E-cadherin decreased,while the expression of α-SMA,STAT3 and Snai1 increased in kidney.The immunofluorescence results showed that NDRG1 gene was expressed specifically in renal tubules,but not in glomeruli.And the NDRG1 expression level decreased significantly in db/db mice kidney.Part III.The circRNA 1860/hsa-miR-125b-5p/NDRG1 Ce RNA regulatory network for EMT In the study,five circRNAs and five miRNAs were screened by q RT-PCR,which might be related to NDRG1 gene expression.Ultimately,we selected circRNA1860 and hsa-miR-125b-5p for follow-up studies.The convergent primers and divergent primers were designed and amplified by RT-PCR.After detection,the c DNA could be amplified by divergent primer,while not g DNA.After RNase treatment,agarose gel electrophoresis showed that circRNA1860 bands still existed.Sequencing results showed that circRNA 1860 was formed by reverse splicing cyclization of exon 2-10 of FBXL5.Therefore,circRNA1860 is a ring structure.After overexpression of circRNA 1860,the expression of NDRG1 m RNA was increased.However,the expression of NDRG1 was decreased after knockdown of circRNA 1860.The results of Western-blot were in accordance with those of q RT-PCR.The m RNA expression of hsa-miR-125b-5p was significantly increased by hsa-miR-125b-5p mimic,and NDRG1 m RNA and NDRG1 protein was significantly decreased afterward when testing by q RT-PCR and Western-blot analysis.Conversely,both NDRG1 m RNA and protein expression levels increased after inhibition of hsa-miR-125b-5p by hsa-miR-125b-5p inhibitor.Results of in situ hybridization of circRNA 1860 and hsa-miR-125b-5p showed that circRNA 1860 and hsa-miR-125b-5p were mainly located within the cytoplasm.Under high glucose condition,the expression of circRNA 1860 decreased and hsa-miR-125b-5p increased significantly,suggesting that they may regulate expression of NDRG1 by competitive inhibition.Dual-luciferase assay showed that circRNA 1860 was able to bind specifically to hsa-miR-125b-5p,and hsa-miR-125b-5p could bind with NDRG1 m RNA.Therefore,circRNA 1860/hsa-miR-125b-5p regulated the expression of NDRG1 gene through Ce RNA mechanism.Conclusion: NDRG1 gene can inhibit EMT in tubular cells induced by high glucose.Under high glucose,the expression of NDRG1 decreased,EMT was actived via STAT3/SNAI1 pathway by decreased NDRG1.The reduction of NDRG1 was related to decreasing of circRNA 1860.circRNA 1860 regulated the transduction of NDRG1 by competing combining with hsa-miR-125b-5p,thus causing the occurrence of EMT.This is the first study to demonstrate that NDRG1 is involved in the occurance of EMT in renal tubular epithelial cells under high glucose,which provides a new potential direction for the study of DKD.