Screening And Analysis Of Differential LncRNA In Diabetes Mice With And Without Nephropathy

Background Diabetes mellitus?DM?is the leading cause of end stage renal disease?ESRD?.In 2015,the global prevalence of DM was as high as 8.8%,and the number of people with DM will exceed about 642 million by 2040.As the country with the largest number of DM patients in the world,the prevalence rate of DM among adults in China reached 10.9% in 2013,with the number of DM patients reaching more than 100 million.It is expected that the total number of DM patients will reach 150 million by 2030.Although only 20%-40% DM patients complicated with diabetic kidney disease?DKD?,the large base DM patients make the DKD to be extensive and serious hazard.According to the data statistics of inpatients from 2011 to 2016 of the national health and family planning commission of China,DKD has become the primary cause of chronic kidney disease?CKD?in China and one of the most urgent problems in the prevention and treatment of CKD.Therefore,it is of great scientific significance and clinical application value to further explore the pathogenesis of DKD and propose effective new methods to prevent and treat DKD from the root.Generally considered renal microvascular damage,namely the diabetic glomerular nephropathy?DN?is typical pathological changes in DKD.Scientists have conducted systematic studies on DN from gene,environment,proteomics,metabolomics and other aspects,but still cannot explain "why some DM patients do not have DN".Long non-coding RNA?lncRNA?refers to a class of functional RNA that is longer than 200 nucleotides?nt?,lacks open reading framework?ORF?and does not encode proteins.Lnc RNA is not only widely distributed,but also regulates genes at epigenetic level,transcriptional level and post-transcriptional level.Lnc RNA can also tightly regulate its own expression.Different Lnc RNA molecules may interact with each other and with other genes,eventually forming a complex regulatory network.Specific role of lnc RNA now still not entirely clear,but with the deepening of the research on lnc RNA,More and more studies have shown that lnc RNA is significantly expressed in renal tissues of normal people and DN patients induced by fibrosis and inflammation,autophagy.And can be used as the new biomarkers and therapeutic targets of DN.But for age,gender and duration matching T2 DM patients,there is no related research of differentially expressed lnc RNA in the kidney tissues of patients with DN and without DN.Objective To find the differentially expression long non-coding RNA?lnc RNA?between db/db mouse that with nephropathy?DN?or not?DM?.Methods RNA of kidney samples from 3 db/m controls?CON?,3 db/db mouse without nephropathy?DM?and 3 db/db mouse with nephropathy?DN?prove by renal biopsy.Then,expression of lnc RNA was detected by high-throughput next generation sequencing technology.Sequencing data were analyzed to filter out the differentially ex pressed lnc RNA,and their function was preliminary investigated by bioinformatics analysis and functional enrichment analysis to their target genes.Then we extracted total RNA of kidney from these 9 mice to run RT-q PCR to verify the outcomes of the high-throughput sequencing.Results The urinary microalbumin/creatinine ratio?UACR?,blood glucose and serum creatinine of diabetic nephropathy db/db mice?DN?were higher than those of diabetic db/db mice?DM??p<0.05?.Totally 160 lnc RNA were upregulated and 99 lnc RNA were downregulated in kidney of DN mice compared with those of DM mice.And 119 lnc RNA were upregulated and 239 lnc RNA were downregulated in kidney of DN mice compared with those of CON mice.Choose lnc RNA that has more higher fold changes and FPKM values to run RT-q PCR and filtrated whose variation trend consistent with the outcome of the highthroughput sequencing.RT-q PCR confirmed that lnc RNA linc279227 was significantly upregulated in cultured renal innate cells stimulated by high glucose and the expression in podocytes was both abundant and earlier.Fluorescence in situ hybridization?FISH?experiments indicated its expression both in glomeruli and renal tubules.And the linc279227 were mostly expressed in cytoplasm.The cis target gene TMEM72 and co-expressed target gene of linc279227 were predicted and the enrichment analysis results showed that linc279227 may be involved in the regulation of the progression of diabetes to diabetic nephropathy through podocyte migration,mitochondrial autophagy.Conclusions There was a significantly different expression pattern of lnc RNA between the kidneys of DN and DM mice,which may involve in the progress of DN.Lnc RNA TCONS₀0279227 may play a significant role in this course.This project deeply revealed the mechanism of lnc RNA in DN from the new perspective of the development of lnc RNAmediated DM into DN,which is expected to obtain new targets for early diagnosis and intervention in DN at the level of lnc RNA.