| Background Diabetic Nephropathy(DN)is one of the most dangerous complications of Diabetes Mellitus(DM),which has become one of the main causes of chronic kidney disease(CKD).Until now,there is still a lack of effective treatment for DN.Its pathogenesis and treatment have been the focus of scholars at home and abroad.The shedding and apoptosis of podocytes,compensatory hypertrophy of surviving podocytes and fusion of podocytes are one of the most important pathological changes in the early stage of DN,and proteinuria induced by injury of podocytes can lead to continuous progression of DN.Due to the limited repair and regeneration ability of podocytes,the degree of injury is an important factor in determining the prognosis of DN.Therefore,it is of great significance to further explore the molecular mechanisms and prevention strategies of DN podocytic injury.Circular RNAs(CircRNAs)are members of A new family of non-coding RNAs(ncRNA).They are ubiquitous in eukaryotes and do not have a 5’-end cap or 3’-end poly(A)tail.Instead,circRNA form circular structures by covalent bonds via reverse splicing of exons or introns.The closed loop structure of circRNA makes it difficult to be degraded by exonuclease and has a more stable state than linear RNA.It is sequentially conserved,mainly located in the cytoplasm or stored in exosomes,and plays a regulatory role at the transcriptional or post-transcriptional level.CircRNAs have a variety of biological functions,including the role of miRNA sponges or ce RNAs,regulating the expression of parental genes,binding directly to proteins to affect their function,and a small number of circRNAs that can be translated into proteins.In recent years,more and more evidence has shown that circRNA plays a key regulatory role in the progress of various diseases(including cancer,diabetes,cardiovascular diseases,etc.),while its role in the pathogenesis of kidney disease remains to be clarified.Recent studies have shown that circ HLA-C plays an important role in lupus nephritis by binding to miR-150.Circ Nr1h4 regulates the pathogenesis of hypertensive nephropathy by targeting miR-155-5p.In patients with kidney cancer,circRNAZNF609 affects FOXP4 expression by binding to miR-138-5p.It is well known that miRNAs are short non-coding RNAs that are involved in various biological processes by directly binding to the 3’ untranslated region to degrade targeted m RNAs.Currently,studies have shown that miRNA is closely related to the pathological progression of DN,including miR-146 a affecting the occurrence of DN through its anti-inflammatory effect and miR-23 b inhibiting DN fibrosis and albuminuria by directly affecting the G3BP2 pathway.Therefore,whether circRNA can also play a role through the "CircRNA-miRNA-m RNA" axis in the process of DN podocide injury has not been reported.In recent years,authoritative studies have found that circRNAs have more abundant N6-methyladenosine(m6A)modification.From the production mechanism,m6 A modification of circRNAs is mainly added by m6 A modification enzymes METTL3 and METTL14,and reversibly removed by demodified enzymes FTO and ALKBH5.m6 A modification plays an important role in maintaining the biological activity of circRNA.In addition to the classical function of regulating the stability of circRNA,m6 A modification can also affect the miRNA sponge effect of circRNA by regulating the interaction between circRNA and miRNA.However,whether m6 A modification regulates circRNA expression in DN remains to be discussed.Our previous study found that MEETL3-mediated m6 A methylation in podocytes regulates the progression of DN.Therefore,this study will further explore the role and mechanism of circ-0000953 regulating DN podocyte injury and m6 A methylase METTL3 regulating the expression level of circ-0000953,which is helpful to provide new theories and new drug targets for the prevention and treatment of DN.Method:Part Ⅰ: To construct a mouse model of type 1 diabetes induced by intraperitoneal injection of STZ.Whole transcriptome high-throughput sequencing was used to screen circRNA with differential changes.The changes of circRNA in mouse kidney were verified by RT-PCR.Then we simulated DN environment in vitro by high glucose stimulation of renal tubular epithelial cells(m TEC),podocytes(MPC5),endothelial cells(VEC),and mesangial cells(SV40-MS-13).The expression levels of circRNA in different cells were detected by Real-time PCR,and the results showed significant changes in the expression of podocytes.At the same time,fluorescence in situ hybridization(FISH)was used to detect circRNA location in the glomeruli of renal puncture specimens of diabetic nephropathy patients and paracancer kidney specimens.The ring structure of circ-0000953 was verified by Sanger sequencing and bioinformatics prediction,and the stability of circ-0000953 structure was investigated using Actinomycin D and RNase.Part Ⅱ: A stable circ-0000953 overexpressed podocyte line was constructed by sh RNA.The expression levels of Nephrin and WT-1 in high-glucose stimulated podocytes were detected by western blot(WB).The expression levels of inflammatory factors TNF-α,IL-1β and MCP-1 were detected by RT-PCR.And the activation levels of classical inflammatory transcription factor P65 were detected by WB.In order to further elucidate the function of circ-0000953,transcriptomic sequencing was performed on high-glucose-stimulated overexpressed circ-0000953 podocytes to screen out the most significant autophagy pathway.WB,immunofluorescence and transmission electron microscopy were used to detect the high-glucose-induced autophagy changes of podocytes in each group.In vivo experiments,we used AAV9 to overexpress circ-0000953 in type 1 and type 2diabetic mice,respectively.At the same time,we constructed mouse kidney podocytes to conditionally knock-in circ-0000953,and detected the general indexes and 24-hour urinary albumin levels of mice.PAS staining was used to observe the degree of renal tissue damage,and the changes in glomerular ultrastructure were observed by electron microscopy.IHC and WB were used to detect the expression changes of podocyte marker proteins,and WB,immunofluorescence double staining and RT-PCR were used to detect the levels of renal tissue inflammation and autophagy.We then reverse-validated its function by silencing circ-0000953 in type 1 diabetic mice.The degree of renal tissue damage was observed by PAS staining and the changes of glomerular ultrastructure were observed by electron microscopy.The expression changes of podocyte marker proteins were detected by IHC and WB.The levels of renal tissue inflammation and autophagy were detected by WB,immunofluorescence double staining and RT-PCR.Part Ⅲ: We screened circRNA-bound miRNAs using RNA-pulldown combined with RT-PCR.The binding of circ-0000953 to miR-665-3p was confirmed by bioinformatics prediction and luciferase reporting assay.miR-665-3p was silenced in vivo and in vitro experiments to verify its function.The degree of renal tissue damage was observed by PAS staining and the changes of glomerular ultrastructure were observed by electron microscopy.The expression changes of podocyte marker proteins were detected by IHC and WB.The levels of renal tissue inflammation and autophagy were detected by WB,immunofluorescence double staining and RT-PCR.Rescue experiments were used to further elucidate the protective role of circ-0000953 through miR-665-3p in high-glucose-stimulated podocytes.The target protein of miR-665-3p was analyzed as the autophagy related protein Atg4 b by targetscan prediction website,and the binding of miR-665-3p to Atg4 b was verified by luciferase reporting assay and bioinformatics prediction.In vitro and in vivo,overexpression of Atg4 b was used to verify its function.The degree of renal tissue damage was observed by PAS staining and the changes of glomerular ultrastructure were observed by electron microscopy.The expression changes of podocyte marker proteins were detected by IHC and WB.The levels of renal tissue inflammation and autophagy were detected by WB,immunofluorescence double staining and RT-PCR.Part Ⅳ: Predicting the m6 A methylation sites of circRNA using biological information prediction websites.The expression of circ-0000953 in kidney tissue of mice with METTL3 conditional-knockout in podocytes was detected by RT-PCR.The interaction between circ-0000953 and METTL3 was investigated using luciferase reports.The m6 A methylation level of circ-0000953 in METTL3 conditional-knockout mice was determined by Me RIP-q PCR.si RNA silencing m6 A reading protein YTHDF2 was used to investigate its effect on circ-0000953 expression.Results:Part Ⅰ.Expression and difference analysis of circRNA in vitro and in vivo in diabetic kidneyIn order to explore the expression changes of circRNA in DN,we used full transcriptome high-throughput sequencing to screen circRNA with differential changes in the kidney of diabetic mice.We found the most significant change in circ-0000953 in the kidney of diabetic mice at 12 weeks after STZ induction.Then,we verified the changes of circ-0000953 in renal proper cells stimulated by high glucose: renal tubular epithelial cells,podocytes,endothelial cells,and mesangial cells.RT-q PCR results confirmed that circ-0000953 was mainly enriched in podocytes and located in the cytoplasm of podocytes.Bioinformatics predicted that circ-0000953 was formed by the head-tail clipping cyclization of exon 2 of parent gene D19 ertd.The cyclic structure of circ-0000953 was demonstrated by sanger sequencing,and circ-0000953 also showed a more stable state than linear RNA when treated with actinomycin D and endonucrenase.In order to investigate the correlation between circ-0000953 and clinical diabetic patients,we found that the expression of circ-0000953 was significantly decreased in the glomerulus of diabetic nephropathy patients by fluorescence in situ hybridization,and the expression of circ-0000953 was negatively correlated with the 24-hour urinary albumin and creatinine of diabetic nephropathy.There was a positive correlation with estimate glomerular filtration rate.Part Ⅱ: Functional study of circRNA in diabetic kidney podocytes injury Overexpression of circ-0000953 in podocytes mitigated the loss of high-glucose-induced Nephrin and WT-1 of podocytes marker proteins,inhibited the expression of inflammatory cytokines TNF-α,IL-1β,and MCP-1,and also inhibited the phosphorylation of the classical inflammatory transcription factor P65.In order to further elucidate the function of circ-0000953,transcriptomic sequencing was performed on the high-glucose-stimulated overexpressed circ-0000953 podocytes to screen out the autophagy pathway with the most significant changes.The overexpression of circ-0000953 reduced the disorder of autophagy level in high-glucose-stimulated podocytes and increased the key autophagy protein Atg7,Atg4 b and LC3.In vivo,circ-0000953 was overexpressed in mice with type 1 and type 2 diabetes,and we constructed podocyte conditional knock-in circ-0000953 mice.We found that circ-0000953 overexpression reduced the level of 24-hour urinary albumin and inhibited the degree of kidney injury induced by diabetes in mice.It also reduced the loss of podocyte marker proteins.In addition,we found that circ-0000953 overexpression inhibited inflammation and autophagy levels in renal tissue of diabetic mice.We then reverse-validated its function by silencing circ-0000953 in type 1 diabetic mice.We found that circ-0000953 silencing increased the 24-hour urinary albumin level of mice,increased the degree of diabetic kidney injury,and promoted the loss of podocytes marker protein.In addition,we found that circ-0000953 silencing further aggravated renal tissue inflammation and autophagy levels in diabetic mice.Part Ⅲ: Study on the mechanism of circ-0000953 mediated podocytes injury in diabetic kidneyWe used RNA-pulldown combined with RT-PCR to screen out circRNA-bound miRNAs,among which miR-665-3p had the most significant changes.The binding of circ-0000953 to miR-665-3p was verified by luciferase reporting assay and bioinformatics prediction.In vitro and in vivo experiments,we found that silencing miR-665-3p reduced the level of 24-hour urinary albumin in mice,inhibited the degree of kidney injury induced by diabetes in mice,and alleviated the loss of podocytes marker protein.Silencing miR-665-3p also inhibited renal tissue inflammation and autophagy levels.Using Rescue experiments,we found that circ-0000953 in high-glucose-stimulated podocytes played a protective role mainly through miR-665-3p,while further overexpressed miR-665-3p in circ-0000953 overexpressed podocytes,and circRNA could not further play its protective role.The target protein of miR-665-3p was analyzed as the autophagy related protein Atg4 b by targetscan prediction website,and the binding of miR-665-3p to Atg4 b was verified by luciferase reporting experiment and bioinformatics prediction.In vivo and in vitro experiments,overexpression of Atg4 b reduced the level of 24-hour urinary albumin in mice,inhibited the degree of kidney injury induced by diabetes,and alleviated the loss of podocyte marker protein.Meanwhile,overexpression of Atg4 b inhibited the level of renal tissue inflammation and autophagy.Part Ⅳ: The effect and mechanism of m6 A methylation mediated circ-0000953We used the bioinformatics prediction website to predict that circ-0000953 has a highly credible m6 A methylation site.Then,we used RT-PCR to detect the expression level of circ-0000953 in the kidney tissue of mice with METTL3 conditional-knockout in podocytes,and the results showed that the knockout of METTL3 reversed the expression of circ-0000953.The interaction between circ-0000953 and METTL3 was investigated using luciferase reports.Me RIP-q PCR found that m6 A methylation levels in circ-0000953 decreased in METTL3 conditional-knockout podocytes.Luciferase assay also confirmed the interaction between METTL3 and circ-0000953.We silenced the m6 A reading protein YTHDF2,and found that the silencing of YTHDF2 reduced the degradation of circ-0000953 and restored the expression of circ-0000953.Conclusion1.In this study,overexpression of circ-0000953 in vitro significantly reversed inflammation and impaired autophagy in high-glucose stimulated podocytes,and overexpression of circ-0000953 in vivo significantly reduced pathological injury,inflammation and impaired autophagy in type 1 and type 2 diabetes kidneys.2.This study found that circ-0000953 regulates autophagy of diabetic renal podocytes by targeting the miR-665-3p/Atg4 b axis.3.This study further found that m6 A methylase METTL3 regulates the expression level of circ-0000953 in a YTHDF2-dependent manner. |
PDF Full Text Request