A Novel Long Non-coding RNA ENSMUST00000147869 Regulates Proliferation And Fibrosis In Diabetic Nephropathy

BackgroundDiabetic nephropathy is a common microvascular complications of diabetes mellitus and it is one of the main cause of the death of diabetic patients.Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix,glomerular and tubular basement membrane thickening,mesangial cell proliferation and mesangial matrix increased,finally can appear glomerular sclerosis and renal tubular interstitial fibrosis.And the pathogenesis of diabetic nephropathy is extremely complicated,and has not yet fully understood,so the study of the pathogenesis of diabetic nephropathy is critical for the treatment of diabetic nephropathy.Long noncoding RNA is a kind of RNA that more than 200nt,and can control gene expression levels of RNA in epigenetic regulation,transcriptional regulation and transcription regulation such as level control gene expression levels of RNA,which is not involved in or rarely participate in protein coding.LncRNA is closely related to many diseases’ occurrence and development,such as breast cancer,liver cancer,gastric cancer,but its research is relatively small with diabetic nephropathy,therefore,IncRNA related to the development of diabetic nephropathy needs to explore.This study through the lncRNA microarrays technology in diabetic nephropathy abnormal expression of lncRNA,ENSMUST00000147869 is significant lower in the normal group.Further research of ENSMUST00000147869 found that its high in sugar glomerular mesangial cells in mice after expressing can significantly affect the glomerular mesangial cells proliferation and fibrosis.Method1.lncRNA microarrays was used to detected 3 pairs of db/db mice and db/m mice kidney tissues,screening analysis in diabetic nephropathy in the organization has the difference expression of mRNA and the lncRNA.2.Choose differentially expressed in such aspects of lncRNA which has 2 times from the expression level and above,the enrichment of IncRNA length,GO and KEGG pathway analysis associated with diabetic nephropathy,using real-time quantitative PCR(RT-qPCR)with nest in 8 of diabetic nephropathy and normal control mice kidney tissues to express its verification,select predict potential critical IncRNA as the research object.3.High glucose in glomerular mesangial cells build express selection potential critical IncRNA stable cell line model of infection,the experimental cell can be divided into four groups:low glucose,high glucose,Lv-Mock,Lv-lncRNA.Western blot detecting various proliferation and fibrosis expression differences between experimental group;used RT-qPCR to detect proliferation between the experimental group and fibrosis corresponding mRNA expression level.Used CCK8 to detect the experimental cell proliferation.Result1.According to increase or reduce 2 times and 2 times above,P value is less than 0.05 standard,filter has been expressed in diabetic nephropathy group differences of 690 mRNAs,including 268 upregulated mRNAs and 422 downregulated mRNAs,1018 lncRNAs,including the rise of 221 upregulated lncRNAs and 797 downregulated lncRNAs.2.RT-qPCR was used to detected 8 diabetic nephropathy in mice to verify the ENSMUST00000147869 was the same with microarrays,Finally confirmed ENSMUST00000147869 as the ultimate research target.3.Construct plasmid pCDH-ENSMUST00000147869 by cloning sequencing,and successfully infected glomerular mesangial cells under high glucose,RT-qPCR showed that ENSMUST00000147869 level increased obviously after infected.4.Western blot showed glomerular mesangial cells under high glucose decreased the levels of PCNA,Cyclin D1 and Collagen I,and overexpression of ENSMUST00000147869 can significantly reduce the expression of PCNA,Cyclin D1,Collagen I,Fibronectin after overexpression of ENSMUST00000147869.5.RT-qPCR showed the level of PCNA,Cyclin D1 and Collagen I,Fibronectin mRNA under high glucose was significantly higher than that of low glucose condition;and overexpression of ENSMUST00000147869 could obviously decrease hyperplasia and fibrosis markers of mRNA level.6.CCK8 showed that high glucose condition increased the growth rate of mesangial cell,overexpression of ENSMUST00000147869 can significantly reduce the cell proliferation ability.Conclusions1.LncRNA microarrays was high flux and high sensitivity,it can filter the lncRNA in diabetic nephropathy effectively,lncRNA expression provided a new thought for the pathogenesis of diabetic nephropathy research.2.The level of ENSMUST00000147869 mRNA was significantly lower in diabetic nephropathy tissues than in normal tissue,long noncoding RNA ENSMUST00000147869 can be used as a treatment for diabetic nephropathy.3.ENSMUST00000147869 can regulate the proliferation and fibrosis of mesangial cells,and play a role in the process of diabetic kidney disease,so ENSMUST00000147869 may become new targets for clinical diagnosis and treatment of diabetic nephropathy.