| Part 1Microarray analysis of long noncoding RNA expression patterns in diabetic nephropathyBackground:Diabetic nephropathy(DN)is a common chronic complication of diabetes,which is characterized by increased urinary albumin excretion(microalbuminuria)and is the leading cause of end-stage renal disease(ESRD).Numerous pathologic changes occur in DN,such as thickening of the glomerular basement membrane,mesangial proliferation,arteriosclerosis,and glomerulotubular junction abnormalities,surviving podocytes show compensatory hypertrophy and foot process fusion.At present,the pathogenesis of diabetic nephropathy is still not entirely clear.Long noncoding RNAs(lncRNAs)are non-protein-coding transcripts longer than 200 nucleotides,involved in several biological processes such as chromatin modification,transcriptional regulation and post-transcriptional regulation.Increasing evidence indicates that lncRNAs are associated with DN.However,the role of lncRNAs in DN remains largely unknown.In this study,most of dysregulated lncRNAs were firstly reported and the potential functions of them explored in DN.Aims:To explore the potential functions of lncRNAs in DN.Methods:(1)6 female mice,5 weeks old,C57BL/KsJ db/db mice were randomly selected as db/db mice with diabetic nephropathy group and 6 female mice,5 weeks old,C57BL/KsJ db/m mice as normal control group.At the 9th week,urine samples from two groups were collected by metabolic cage to measure urine microalbumin.Then the mice were sacrificed and the body weight and blood glucose of two groups of mice were tested.The cortex of kidney from two groups were stainned by HE and PAS.(2)LncRNA microarray and co-expression analysis were performed in the cortex tissue of kidney.Some differential expressed IncRNAs were selected and validated by quantitative Real-time PCR.(3)LncRNA function was predicted by Gene Ontology enrichment and KEGG pathway analyses of lncRNAs-coexpressed mRNAs.Cis-and trans-regulation analyses were conducted to reveal potential relationships between IncRNAs and their target genes.Results:(1)Compared with db/m mice,the body weight,blood glucose,24-hour urine albumin levels of db/db mice with diabetic nephropathy were significantly increased.Renal HE and PAS staining results showed that the db/db mice with diabetic nephropathy were characterized by glomerular hypertrophy,capillary basement membrane thickening,and mesangial area widened.(2)The lncRNA microarray chip results showed that the expression of 311 IncRNAs in renal cortex of db/db mice with diabetic nephropathy was more than 2 times higher than that of db/m normal mice,of which 105 were significantly up-regulated and 206 were significantly down-regulated.Real-time quantitative RT-PCR analysis showed that the expression of 7 IncRNAs was different in the db/m normal group and db/db mice with diabetic nephropathy,and the results were significant(P<0.05).(3)GO and Pathway analysis showed that the expression of differentiated IncRNAs was associated with multiple signaling pathways related to diabetic nephropathy,such as MAPK,lysosome and glutathioneemia pathway.Cis-analysis and trans-analysis revealed that two IncRNAs may be involved in the development of diabetic nephropathy by cis-regulation of Map2kl and THBS1 genes,respectively.Transient analysis found that 272 1ncRNAs interacted with transcription factors.FOSB,JUNB,JUND,FOS,FOSL1 and HNF4A were closely related to the development of diabetic nephropathy.Conclusion:LncRNAs may be differentially expressed in early diabetic nephropathy and may be involved in the development of diabetic nephropathy through MAPK and other signaling pathways.Part 2MiR-205-5p induces apoptosis of podocyte in high glucose via activating wnt/β-catenin signaling pathwayBackground:The histopathological changes of renal biopsy in patients with early diabetic nephropathy show that the number of glomerular podocytes is reduced and the density is reduced.These changes are important causes of proteinuria formation in diabetic nephropathy.More and more studies have found that podocyte shedding,reduced and hyperglycemia-induced glomerular podocyte apoptosis.High glucose-induced podocyte apoptosis may be related to the formation of glycosylated terminal products,oxidative stress,mitochondrial damage,increased TGFβ-1,podocyte autophagy,but the current mechanism is still not entirely clear.MicroRNAs(MiRNAs)are non-coding RNAs of about 22 nucleotides and recognize specific sequences termed miRNA-response elements that are generally present in the 3‘-untranslated region(3’UTR)of target mRNAs.Interestingly,miRNAs have been shown to play a central role in DN regulation.Wnt signaling pathway is an evolutionary conservative signaling pathways involved in the development of multiple kidney diseases,including focal segmental glomerulosclerosis and diabetic nephropathy.In this study,we observed whether miR-205-5p could promote podocyte apoptosis in high glucose state for the first time and whether or not podocyte cell apoptosis was promoted by wnt/β-catenin signaling pathway in high glucose state.Aims:To investigate whether miR-205-5p could promote the apoptosis of podocytes in high glucose state and whether to promote podocyte apoptosis in high glucose state through wnt/β-catenin signaling pathway.Methods:(1)In vitro culture of human podocytes,37℃ differentiation and maturation,divided into normal group(CON)and high glucose group(HG).Annexin V-FITC and PI-dual-stained method were used to detect the change of apoptotic rate of podocytes.The expression of cleaved-caspase3 was detected by Western blot.QRT-PCR was used to detect the expression of miR-205-5p in both groups.Western blot was used to detect the expression of β-catenin protein.(2)MiR-205-5p mimics and negative control were transiently transfected into normal human podocytes for 48 hours.Annexin V-FITC and PI-dual-stained method were used to detect the changes of podocyte apoptosis rate.The expression of cleaved-caspase3 protein and β-catenin protein was detected by Western blot.(3)In vitro culture of human podocytes,small molecule selective inhibitor XAV-939 were added to inhibit Wnt pathway transcription factor β-catenin before high glucose stimulation.Annexin V-FITC and PI-dual-stained method were used to detect podocyte apoptosis rate.The expression of cleaved-caspase3 protein was detected by Western blot.Results:(1)Compared with the normal group,the expression of miR-205-5p was increased and the expression of β-catenin was increased in the high glucose group.(2)Compared with the no-load control,the podocyte apoptosis and(3-catenin expression increased in podocytes with miR-205-5p overexpression,cultured in normal glucose condition.(3)Compared with high-glucose-stimulated podocytes,the expression ofβ-catenin and the apoptosis of podocytes were significantly decreased in the high glucose group plus XAV-939 intervention.Conclusion:MiR-205-5p can induce apoptosis of podocytes under high glucose by activating wnt/β-catenin signaling pathway. |
