| BackgroundIn our study we use angiotensin Ⅱ(Ang Ⅱ)to simulate the internal environment of hypertension,then we detected the expression of long non-coding RNA(lnc RNA)RNA methyltransferase SUN domain family member NSUN5 pseudogene 1(NSUN5P1)in human renal tubular epithelial cell line HK-2.In addition,bioinformatics analysis was used to explore the endogenous competitive RNAs(ce RNA)associated with NSUN5P1,and identify the possible key signal molecules regulated by ce RNA mechanism in hypertensive kidney damage.Micro RNA(miRNA)has-miR-940 and thrombospondin-1(THBS1).The role of NSUN5P1/ miR-940/THBS1 axis in the pathogenesis of hypertensive renal fibrosis was verified in the cell model.Methods1.The expression level of genes was detected by high-throughput sequencing in HK-2 cells with Ang Ⅱ intervention in vitro,and the differentially expressed genes were screened out by bioinformatics analysis.2.Based on the ce RNA mechanism,bioinformatics software was used for prediction and analysis of target genes in all differential genes,Pearson correlation coefficient was calculated,ce RNA network that met the conditions was found,and key lncrnas and candidate target genes were identified.Gene Ontology database,Kyoto Encyclopedia of Genes and Genomes database,Disease Ontology database,Gene enrichment analysis were used to analyze the signal pathway of the above genes.The protein interaction network encoded by target genes was validated using String online database.3.Reverse transcription polymerase chain reaction(RT-PCR)was used to verify the sequencing results in HK-2 interfered by Ang Ⅱ,and lentivirus transfection technology was used to construct cell models of NSUN5P1overexpressed/knockdown stable strain.Cell fluorescence in situ hybridization and double luciferase reporter gene assay were used to verify the localization and specific binding of target gene in cells.4.The expression levels of transforming growth factor β1(TGF-β1),SMAD2,SKP1 and THBS1 in HK-2 cells overexpressing NSUN5P1 were detected by Western blot.The protein expressions of α smooth muscle actin,type Ⅰ,type Ⅱ,type Ⅳcollagen,fibronectin and cadherin E were detected by ELISA.5.In HK-2 cells with NSUN5P1 knockdown,the expression levels of TGF-β1,SMAD2,SKP1 and THBS1 were determined by Western blot,and the protein expressions of α actin,type Ⅰ,type Ⅱ and type Ⅳ collagen,fibronectin and cadherin E were detected by ELISA.6.It was verified that NSUN5P1 plays a regulatory role by binding with miR-940 and activating TGFβ1 signaling pathway in HK-2 cells interfered by Ang Ⅱ.The expression levels of TGF-β1,SMAD2,SKP1 and THBS1 in HK-2 cells were determined by Western blot.The protein expressions of α actin,t type Ⅰ,type Ⅱ,typeⅣ collagen,fibronectin and cadherin E were detected by ELISA.7.Ten patients with hypertensive nephropathy and 10 healthy controls in our hospital were collected,and peripheral blood mononuclear cells were extracted from human peripheral venous blood,respectively,and the expression of target genes in ce RNA was detected by RT-PCR.In an in vitro model of hypertensive renal injury cells,namely HK-2 cells with Ang Ⅱ intervention,gene expression levels were detected by high-throughput sequencing,and differential genes were screened out by bioinformatics analysis.Results1.There were 76333 lnc RNAs and 15,446 m RNAs screened by high-throughput sequencing,including 2657 differentially expressed lnc RNAs.Among them 757 lnc RNAs were up-regulated and 1900 lnc RNAs were down-regulated,and lnc RNA NSUN5P1 was most significantly up-regulated.The decrease of IMT2 C was most significant.Meanwhile,676 differentially expressed m RNAs were found,among which 153 m RNAs were significantly up-regulated and 523 m RNAs were significantly down-regulated.THBS1 was the most up-regulated and SLC7A2 was the most down-regulated(P < 0.05).A total of 80 differentially expressed miRNAs were screened,of which 35 miRNAs were up-regulated and 45 were down-regulated(P < 0.05).On this basis,a ce RNA network of NSUN5P1/ miR-940 /THBS1 was finally screened out.2.Bioinformatics analysis showed that differential genes were mainly enriched in TGF-β signaling pathway,Wnt signaling pathway,m TOR signaling pathway and other signaling pathways,and functionally closely related to TGF-β signaling pathway,ECM receptor interaction,p13K-Akt and so on.There were differences in TGF-β signaling pathways,complement and coagulation cascade,proximal tubule bicarbonate recovery and other signaling pathways between the hypertensive model group and the normal control group,and the differentially expressed m RNA was mainly closely related to cardiovascular diseases,vascular diseases,renal insufficiency and other diseases(P < 0.05).3.Confocal fluorescence microscopy showed that NSUN5P1 and miR-940 were mainly located in the cytoplasm of HK-2 cells,and the expression of NSUN5P1 in the cytoplasm was several times higher than that in the nucleus(P < 0.05).In the double luciferase reporter gene test results,compared with the negative control group,the co-transfer group of wild-type gene and miR-940 significantly reduced the luciferase activity(P < 0.05),suggesting the targeted binding of miR-940 to NSUN5P1 and THBS1,respectively.4.In HK-2 treated with Ang II,the expression levels of TGF-β1,THBS1 and related proteins were increased in Ang Ⅱ group,and the expression levels of extracellular matrix were increased(P < 0.05).After knockdown of NSUN5P1 expression,the levels of related proteins and extracellular matrix were significantly decreased,and on this basis,the levels of TGF-β1-related proteins and extracellular matrix were significantly increased by knockdown of miR-940(P < 0.05).The expression levels of TGF-β1-related proteins and extracellular matrix were further increased after overexpression of NSUN5P1,while the expression levels of TGF-β1-related proteins were significantly decreased while the overexpression of miR-940,while the expression levels of extracellular matrix were decreased(P <0.05).5.In HK-2 treated with Ang II,the expression levels of TGF-β1,THBS1 and related proteins in Ang II group were increased,while the extracellular matrix level was up-regulated(P < 0.05).TGF-β1,THBS1 and related proteins were further up-regulated in extracellular matrix after knockdown of miR-940 expression.The levels of TGF-β1,THBS1,related proteins and extracellular matrix were significantly decreased when miR-940 was overexpressed(P < 0.05).6.In this study,the levels of NSUN5P1 and THBS1 in peripheral blood of 10 patients with hypertensive nephropathy were significantly higher than those in the non-hypertensive nephropathy group,and the level of miR-940 was significantly decreased(P < 0.05),which had the same trend as the results of in vitro validation experiment.Conclusion1.The NSUN5P1/ miR-940/THBS1 axis based on ce RNA regulation mechanism exists in the simulated hypertensive renal injury cells in vitro,and miR-940 can specifically bind to the other two gene.In Ang Ⅱ induced HK-2 cell injury model,NSUN5P1 can up-regulate the expression of THBS1 and TGF-β1-related proteins,and the pro-fibrosis effect of NSUN5P1 can only be exerted through miR-940.2.The function of NSUN5P1/ miR-940 /THBS1 axis in regulating renal fibrosis may be related to the regulation of TGF-β1 signaling pathway,thereby increasing extracellular matrix production and promoting collagen deposition. |
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