The Study On The Expression And Clinical Significance Of LncRNA DLX6-AS1 In DKD

BackgroundThe percentage with chronic kidney disease related to diabetes has exceeded the percentage with chronic kidney disease related to glomerulonephritis in China.Diabetic kidney disease(DKD)is the leading cause of chronic kidney disease.The development and progression of DKD is multifactorial,involving complex molecular networks and processes.Advances in the understanding of the pathogenetic mechanism that these factors mediate in the context of diabetic nephropathy will facilitate the discovery of reliable biomarkers and new therapeutic targets.Long noncoding RNAs(lncRNAs)are longer than 200 nucleotides,which are involved in the regulation of several human diseases,including cancer and kidney disease.Their contributions to development and progression of DKD are emerging.Increasing experimental evidence supports the hypothesis that lncRNAs may act as competing endogenous RNAs(ce RNAs)or natural microRNA sponges.Expression profiles from array experiments show that a significant increase in the expression of lncRNA DLX6-AS1 in diabetic nephropathy patients,while miR-346 is downregulated.lncRNA DLX6-AS1 may contain miR-346-binding sites through bioinformation analysis.The lncRNA–miRNA competitive interactions may be responsible for triggering the pathophysiological events in diabetic nephropathy.In this study,we will examine the role of lncRNA DLX6-AS1 in DKD-induced podocytes dysfunction,and the correlation of the relative expression of lncRNA DLX6-AS1 between albuminuria and other clinical variables in patients with DKD.Methods(1)Tissue specimens from Human Subjects:A total of 15 patients with diabetic nephropathy confirmed by kidney biopsy were included in the study.15 samples of kidney tissue were taken adjacent to kidney tumors in patients with kidney cancer and were used as diabetes group and controls.All kidney tissues were collected from patients at the First Affiliated Hospital of Zhengzhou University.(2)Serum samples from Human Subjects:A total of 65 diabetes mellitus patients with or without nephropathy and 32 control participants were included in the study.Supernatants were collected as serum by centrifuging for analyses.All blood samples were collected from patients at the First Affiliated Hospital of Zhengzhou University.(3)Real-time qPCR:Real-time qRT-PCR for lncRNA DLX6-AS1 and miR-346expression analysis was performed following protocols.GAPDH and U6 were used as internal control.(4)Fluorescence in situ hybridization(FISH):lncRNA DLX6-AS1 FISH was performed with locked nucleic acid detection probes,followed by immunofluorescence staining of podocin,which is podocytes marker protein.(5)Morphometric analysis:FFPE kidney tissue sections were processed for PAS staining.Transmission electron microscopy was used to quantify GBM thickness,the lesions of podocyte foot and so on.(6)Statistical Analyses:Differences among multiple groups were evaluated by One Way Anova for continuous variables or Chi square test for categorical variables.Two groups were compared by t test for normally distributed data and Mann–Whitney U test for non-normally distributed data.The correlation between relative levels of lncRNA DLX6-AS1 and clinical variables was analyzed by Spearman correlation coefficient after determining the non-normal distribution of data.Linear regression models were performed with albumin/creatinine ratio as the dependent variable.Results(1)The expression of lncRNA DLX6-AS1 in human kidney tissue was significantly up-regulated and the expression of miR-346 was significantly down-regulated in diabetes mellitus and diabetic nephropathy patients,comparing with the normal control group.These expression patterns were similar to those observed in human serum sample,the circulating DLX6-AS1 shows the tendency of increasing with albuminuria.(2)PAS staining and EM showed effacement of podocyte foot processes,thickening of glomerular basement membrane,mesangial expansion with increased matrix.(3)Albumin/creatinine ratio(r_s=0.626,P<0.001)and eGFR(r_s=-0.289,P=0.020)were correlated with lnc RNA DLX6-AS1 expression in patients serum with diabetes both with and without nephropathy.(4)FISH results indicate the elevated expression of lncRNA DLX6-AS1 in the glomeruli of DKD patients.With the podocyte injuries,DLX6-AS1 shows the increasing tendency in the podocytes.ConclusionlncRNA DLX6-AS1 is up-regulated in DKD circumstance,which may reversly regulate the expression of miR-346.There is a positive correlation between DLX6-AS1 and albuminuria.lncRNA DLX6-AS1 mediates kidney dysfunction in DKD that might be relevant to podocyte injuries.Understanding this novel RNA crosstalk will lead to significant insight into DKD regulatory networks and have implications in diagnostic biomarker.