| BackgroundDiabetic kidney disease(DKD)is the most important microvascular complication of diabetes.Diabetes has replaced glomerulonephritis as the leading cause of chronic kidney disease(CKD).Diabetic patients with renal impairment will significantly increase the risk of cardiovascular disease and increase patient mortality.Long non-coding RNA(LncRNA)refers to non-coding RNAs greater than 200 nucleotide(nt)in length.Although non-coding RNA does not encode a protein,it can act as a regulatory element in different functions in the cytoplasm and in the nucleus.LncRNA forms a fine,complex regulatory network by interacting with DNA,RNA and proteins.In the interaction between LncRNA and RNA,some LncRNAs can competitively bind with microRNA(miRNA),resulting in the loss of regulation of target genes by miRNA.These LncRNAs are called competitive endogenous RNA(ceRNA).The ceRNA mechanism is a unique mechanism of LncRNA.We used LncRNA microarray and microRNA microarray to analyze LncRNA expression profiles and miRNA expression profiles in diabetic kidney disease patients and normal controls,respectively,and found that LncRNA DLX6-AS1 expression increased,while miR-346 expression decreased,and we know miR-346 can inhibit the activity of GSK-3? in a high glucose environment and reduce kidney damage in a high sugar environment.We also predicted that LncRNA DLX6-AS1 and miR-346 have a binding site through bioinformatics,so it is speculated that Dlx6-AS1 may competitively bind to miR-346,thereby causing miR-346 to lose its regulation of GSK-3?.Then,promoting the development of diabetic kidney disease.The homologous sequence of DLX6-AS1 in mouse is homeobox transcription factor 6,anti-sense RNA 1(Dlx6-os1).This study is to analyze the expression of DLX6-AS1/Dlx6-os1 and its significance in diabetic kidney disease patients and diabetic nephropathy mice model,explore the possible mechanism of DLX6-AS1/Dlx6-os1 in the progression of diabetic kidney disease.Method1.A total of 12 renal biopsy specimens from patients with diabetic kidney disease were collected and biochemical data were recorded.The expression of DLX6-AS1 in kidney tissue was detected by qRT-PCR.2.Type 1 and type 2 diabetic nephropathy mice model were constructed,and the expression of Dlx6-os1 in mouse kidney tissue was detected by qRT-PCR.3.Fluorescence in situ hybridization assay was used to locate the expression of Dlx6-os1 in mouse immortalized podocytes.4.Dual luciferase reporter gene detection technology detects whether there is a direct effect between Dlx6-os1 and miR-346.5.Type 1 diabetic nephropathy mice model were constructed and grouped as follows: no intervention group: CON 7w,CON 15 w,STZ 7w,STZ 15 w.Dlx6-os1 overexpression group: con+saline,con+con-LV,con+Dlx6-os1 4w,con+Dlx6-os1 12 w.Dlx6-os1 expression inhibition group: STZ+saline,STZ+con-shRNA,STZ+Dlx6-os1 shRNA 4w,STZ+Dlx6-os1 shRNA 12 w.Type 2 diabetic nephropathy mice model were grouped as follows: no intervention group: db/m 14 w,db/m 22 w,db/db 14 w,db/db22 w.Dlx6-os1 overexpression group: db/m+saline,db/m +con-LV,db/m+Dlx6-os1 4w,db/m+Dlx6-os1 12 w.Dlx6-os1 expression inhibition group: db/db+saline,db/db+con-LV,db/db+Dlx6-os1 shRNA 4w,db/db+Dlx6-os1 shRNA 12 w.6.qRT-PCR was used to detect the expression of Dlx6-os1,miR-346 and GSK-3? mRNA in each group of mice,and the biochemical indicators of each group were monitored.7.Statistical analysis of the data: The Spearman correlation coefficient was used for the single factor correlation analysis,and the unpaired t test was used for the difference analysis of the normal data between the two groups.Result1.The relative expression of DLX6-AS1 in kidney tissues of patients with diabetic kidney disease was significantly increased,about 3.1 times of that in the control group,and there was a significant positive correlation with uACR(r=0.601,P<0.05).2.The relative expression of Dlx6-os1 in kidney tissues of STZ mice was significantly increased,about 2.3 times of that in the control group,and there was a significant positive correlation with uACR(r=0.57,P<0.01).3.The relative expression of Dlx6-os1 in kidney tissues of db/db mice was significantly increased,about 2.0 times of that in the control group,and there was a significant positive correlation with uACR(r=0.534,P<0.05).4.The FISH showed that Dlx6-os1 was expressed in the cytoplasm and nucleus of podocytes.The expression level of Dlx6-os1 was higher in the high glucose concentration group(HG)than in the normal glucose concentration group(NG).5.Dual luciferase reporter assay showed that the relative activity of the Dlsx6-os1 group was reduced by approximately 38% compared to the MUTA group.6.Type 1 diabetic nephropathy mice model,no intervention group: Compared with CON 7w group,the expression level of Dlx6-os1 in kidney tissues of STZ 7w and STZ 15 w mice was significantly increased,which was about 1.73 times and 2.30 times of that in CON 7w group,respectively;The expression level of miR-346 was significantly decreased,which was about 0.40 times and 0.36 times of that in CON 7w group,respectively;the expression level of GSK-3? mRNA was significantly increased,which was about 1.50 times and 1.75 times of that in CON 7w group,respectively.Dlx6-os1 overexpression group: Compared with the con+saline group,the expression level of Dlx6-os1 in the kidney tissues of con+Dlx6-os1 4w and con+Dlx6-os1 12 w mice were significantly increased,which was about 5.88 times and 11.21 times of that in con+saline group,respectively;The expression level of miR-346 was significantly decreased,which was about 0.44 times and 0.36 times of that in con+saline group,respectively;The expression level of GSK-3? mRNA was significantly increased,which was about 1.68 times and 3.23 times of that in con+saline group,respectively.Dlx6-os1 expression inhibition group: Compared with the STZ+saline group,the expression level of Dlx6-os1 in the kidney tissues of STZ+Dlx6-os1 shRNA 4w and STZ+ Dlx6-os1 shRNA 12 w mice was significantly decreased,which was about 0.48 times and 0.31 times of that in STZ+saline group,respectively;The expression level of miR-346 was significantly increased,which was about 2.95 times and 3.36 times of that in STZ+saline group,respectively;The expression level of GSK-3? mRNA was significantly decreased,which was about 0.58 times and 0.43 times of that in STZ+saline group,respectively.7.Type 2 diabetic nephropathy mice model,no intervention group: Compared with db/m 14 w group,the expression level of Dlx6-os1 in kidney tissues of db/db 14 w and db/db 22 w mice was significantly increased,which was about 1.62 times and 2.08 times of that in db/m 14 w group,respectively;The expression level of miR-346 was significantly decreased,which was about 0.56 times and 0.48 times of that in db/m 14 w group,respectively;the expression level of GSK-3? mRNA was significantly increased,which was about 1.30 times and 1.47 times of that in db/m 14 w group,respectively.Dlx6-os1 overexpression group: Compared with the db/m+saline group,the expression levels of Dlx6-os1 in the kidney tissues of db/m+Dlx6-os1 4w and db/m+Dlx6-os1 12 w mice were significantly increased,which was about 5.87 times and 8.06 times of that in db/m+saline group,respectively;The expression level of miR-346 was significantly decreased,which was about 0.58 times and 0.38 times of that in db/m+saline group,respectively;The expression level of GSK-3? mRNA was significantly increased,which was about 1.69 times and 2.27 times of that in db/m+saline group,respectively.Dlx6-os1 expression inhibition group: Compared with the db/db+saline group,the expression levels of Dlx6-os1 in the kidney tissue of db/db+Dlx6-os1 shRNA 4w and db/db+Dlx6-os1 shRNA 12 w mice was significantly decreased,which was about 0.55 times and 0.39 times of that in db/db+saline group,respectively;The expression level of miR-346 was significantly increased,which was about 1.72 times and 1.90 times of that in db/db+saline group,respectively;The expression of GSK-3? mRNA was significantly decreased,which was about 0.66 times and 0.52 times of that in db/db+saline group,respectively.ConclusionDLX6-AS1/Dlx6-os1 is significantly increased in kidney tissue of diabetic nephropathy patients and diabetic nephropathy mice model.One of the mechanisms may be to regulate the expression of GSK-3? by negatively regulating the expression of miR-346. |
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