| Objective : Diabetes is one of the most prevalent chronic diseases in the world,which is a serious threat to human health.Diabetic kidney diseases(DKD)is one of the most common microvascular complications of diabetes,whether it is type1 or type2 diabetes,about30%~40% of the patients have renal involvement,which becomes the main cause of end-stage renal disease(ESRD).Recent epidemiological studies indicate that since 2011,the proportion of diabetic chronic kidney disease in China has exceeded that of glomerulonephritis.The exact pathogenesis of diabetic nephropathy is still unclear.Blood glucose and its metabolites can activate protein kinase C,polyol pathway and non-enzymatic glycosylation,which together lead to changes in renal structure and function.For a long time,we all use urine trace albumin to diagnose and monitor progress DKD severity,however Shimizu,found that they had limited effects in some aspects,In some patients,diffuse glomerular fibrosis,interstitial fibrosis and tubular atrophy predate the presence of microalbuminuria,a recent study found that noncodingRNA and genetic susceptibility was also involved in DKD disease process.The main pathological feature of DKD is the accumulation of extracellular matrix(ECM)in the glomeruli.However,DNA methylation,lncRNA and miRNA all affect the accumulation of ECM,leading to the occurrence of diseases.Studies have confirmed that lncrna-pvt1 and TGF-1 are up-regulated in mesangial cells.Under the action of high glucose and TGF-1,the expression of mir-1207-5 p derived from PVT1 was significantly increased in renal cells,but TGF1 was independent of PVT1.These evidences suggest that lncrna-mirna interaction plays a key role in the occurrence and development of DKD.The competing endogenousRNA(ceRNA)theory was introduced by Salmena et al.in2011.They believe that one miRNA can regulate multiple target genes and the same target gene can be regulated by different mirnas.Then theseRNA regulated by the same miRNA form a competitive relationship,which is called ceRNA.Such as coding protein mRNA,long chain noncodingRNA(long non codingRNA,lncRNA)and some false gene transcription etc.If you have the same micrornas response element(miRNA response element,MRE),they can exert competitive effects by binding the same kind of miRNA,that is,if the level of the above-mentionedRNA increase and its ability to attract miRNA increases.The inhibition of miRNA on otherRNAs will be weakened and the protein expression level of otherRNAs will be increased,thereby affecting the function of cells.ThisRNA with the same miRNA response element is called ceRNA.Currently,studies on ceRNA are mainly focused on tumor.In recent years,studies have confirmed that ceRNA is related to prostate cancer,colorectal cancer,lung cancer,breast cancer and other tumors,and regulates the occurrence and development of tumors.In eukaryotes,90% of the genome is transcribed,but only1%~2% of the genome encodes proteins,and the vast majority of the genome is transcribed into non-codingRNA.Therefore,non-codingRNA plays a certain role in regulating the life activities of organisms,especially the research on the relationship between miRNA and DKD,especially on the relationship between miRNA and LncRNA,is very popular,and it is expected to find new markers or therapeutic targets.microRNA/ miRNA in recent years,the emergence of short non-codingRNA allows DKD markers to choose a new direction.It can bind to the3’ untranslated region of its target messengerRNA(mRNA)by complementary command,resulting in posttranscriptional gene silencing.Many animal and cell experiments have recognized that abnormal miRNA expression is related to the pathological process of DKD occurrence and development.miRNA-21 acts on the kidney through PTEN-SMAD7,and the upregulation degree of miRNA-21 is positively correlated with the rate of renal fibrosis and renal function decline.In American Indian DKD patients,ACR was found to be positively correlated with miRNA-21 expression in glomeruli.In addition, Wang et al.also found that the expression of miRNA-21 in serum and kidney tissues increased with the progression of DKD,and the changes of miRNA-21 in serum and kidney tissues were consistent during the progression of DKD.In STZ-induced DKD mice,silencing of miRNA-21 expression can reveal decreased expression of podocyte injury,urinary albumin,fibrosis and inflammation genes.Our previous experiments showed that high glucose induced up-regulation of miRNA-21 expression in mesangial cells,thereby down-regulating the expression of its target gene PTEN,activating the Akt/ m TOR signaling pathway,promoting the proliferation of mesangial cells,inhibiting autophagy,and leading to the accumulation of extracellular matrix proteins.After transfection with miRNA-21 inhibitor,the expression of PTEN was upregulated,which inhibited the activation of Akt/ m TOR signaling pathway of highglucose-induced mesangial cells,improved hypertrophy and proliferation of highglucose-induced mesangial cells,enhanced autophagy,and reduced extracellular matrix protein accumulation.Besides,our team also found that diabetic nephropathy patients serum mi R-21 expression level and urine trace albumin levels,kidney function decline,lower serum albumin were positively correlated,and serum mi R-21 is the independent risk factors influencing the serum albumin,suggesting that mi R-21 overexpression in diabetic patients may promote the aggravation of pathological grading of diabetic nephropathy,leading to increased urine protein,decreased serum albumin,decreased kidney function.long non-codingRNA(LncRNA)LncRNA is a group ofRNA with more than200 nt nucleotides and cannot encode proteins,which plays an important regulatory role in many aspects.Recently,more and more reports have shown that the abnormal regulation of LncRNA is related to a series of diseases including DKD.Therefore,LncRNA can become a new gene regulator or prognostic marker involved in the pathogenesis of DKD.LncRNA MALAT1 is one of the first lncRNAs discovered.Liu et al.(2014)reported that LncRNA MALAT1 was up-regulated in retinal cells and high-glucose cultured endothelial cells of DM rats,indicating that LncRNA MALAT1 was involved in DM microvascular lesions.In the study of breast cancer patients,Zhang et al.found that mi R-21 in the tumor tissue was higher than that in the adjacent tissue,which could reduce the LncRNA GAS5,and the interference with GAS5 could increase the expression of mi R-21.At the same time,the paper also pointed out that mi R-21 not only had a targeted inhibitory effect on tumor proteincoding genes,but also had an inhibitory effect on LncRNA GAS5.Through theRNA22 program software(http: //cbcsrv.watson.ibm.Com/rna22.html)found that GAS5 and mi R-21 have complementary regions.Relevant studies showed that the expressions of mi R-21 and Gas5 in breast tumor specimens were negatively correlated,and LncRNA-GSA5,as a negative regulator of mi R-21,was involved in the regulation of chondrocyte survival in the occurrence of osteoarthritis.The previous experiments of our research group showed that the expression of lncRNA GAS5 in the diabetic group and diabetic nephropathy group was significantly down-regulated compared with the normal group.Fasting blood glucose and glycosylated hemoglobin are negatively correlated with serum lncRNA GAS5,and serum lncRNA GAS5 is an independent protective factor of fasting blood glucose,suggesting that the down-regulation of lncRNA GAS5 expression may promote the occurrence of hyperglycemia.GAS5 in the diabetic group and diabetic nephropathy group was significantly down-regulated compared with the normal group.Fasting blood glucose and glycosylated hemoglobin are negatively correlated with serum lncRNA GAS5,and serum lncRNA GAS5 is an independent protective factor of fasting blood glucose,suggesting that the downregulation of lncRNA GAS5 expression may promote the occurrence of hyperglycemia.In summary,according to previous experiments,the expression of mi R-21 in the serum of diabetic nephropathy patients was increased,while the expression of GSA5 was down-regulated.This study will take LncRNA-GSA5 / mi R-21 ceRNA as the entry point to explore the possible mechanism of ceRNA in the occurrence and development of diabetic nephropathy in animal and cell experiments.Methods: the 8-week-old db/db mice were used as the research object.The kidney tissue was taken after the high-sugar diet membrane formation,and the contents of LncRNAGSA5,mi R-21 and PTEN in the kidney tissue were determined by RT-PCR.western blot analysis of CCND1,CDK4,FN and IV collagen in renal tissue.Rat mesangial cells were divided into normal glucose group(5.5mmol/L glucose),hypertonic control group(5.5mmol/L glucose +19.5mmol/L mannitol)and hyperglycemia group(25mmol/L glucose)at different time points of 24,48,72 and 96 hours.CCK-8 method and MTT method were used to determine the value-added rate of mesangial cells induced by hyperglycemia.The contents of LncRNA-GSA5 and mi R-21 in mesangial cells induced by high glucose were determined by RT-PCR.western blot analysis of CCND1,CDK4,FN and IV collagen expression in mesangial cells induced by high glucose.The changes of mesangial cell cycle induced by high glucose were detected by flow cytometry.The transfection rate was determined by immunofluorescence.The contents of LncRNAGSA5 and LncRNA-GSA5 overexpressed plasmids were determined by RT-PCR after transfection of mi R-21 inhibited plasmids and LncRNA-GSA5 overexpressed plasmids in mesangial cells under high glucose induction.Western blot analysis of the expression of CCND1,CDK4,FN and IV collagen after transfection of mi R-21 inhibitory plasmid and GSA5 overexpressed plasmid in mesangial cells induced by high glucose.The changes of mesangial cell cycle induced by high glucose were detected by flow cytometry.Results: 1.Effects of high glucose on the renal tissue of db/db mice: the volume and weight of renal tissue increased;Compared with the db/m group,the difference was statistically significant(P<0.05).2.PRT-PCR was used to detect the changes inRNA expression in the db/db group and the db/m group: mi R-21 level was increased compared with the db/m group(P< 0.01),LncRNA-GSA5 level was decreased compared with the db/m group(P< 0.05),and PTEN level was decreased compared with the db/m group(P< 0.05).3.The results of western blot analysis of fibrosis degree in db/db group and db/m group showed that the level of FN and IV collagen were increased compared with the db/m group(P < 0.05).4.Western blot analysis of cyclin proteins changes in db/db group and db/m group showed that CCND1 and CDK4 level were increased compared with the db/m group(P < 0.05).5.The proliferation rate of mesangial cells induced by hyperglycemia was determined by MTT method.The results showed that after 24 hours of hyperglycemia culture,the proliferation of cells in the hyperglycemia group showed no significant change compared with the normal glucose group.However,the cell proliferation rate increased significantly from 48,72 and 96 hours,with the most significant increase at 48 hours(P < 0.01).6.RT-PCR was used to detect the changes ofRNA level in mesangial cells cultured with high glucose for 48 hours: mi R-21 level was increased compared with the normal glucose group(P< 0.01),LncRNA-GSA5 level was decreased compared with the normal glucose group(P< 0.05),PTEN level was decreased compared with the normal glucose group(P< 0.01).7.Western blot analysis of cell fibrosis in mesangial cells cultured with high glucose for 48 hours showed that the level of FN and IV collagen were increased compared with the normal glucose group(P< 0.05).8.Western blot analysis of cyclin changes in mesangial cells cultured with high glucose for 48 hours showed that CCND1 and CDK4 level were significantly increased compared with the normal glucose group(P < 0.05).9.Cell cycle measurement by flow cytometry showed that mesangial cells were stagnant at G0/G1 phase.10.48 h after transfection of mi R-21 inhibitory plasmid,cell proliferation rate decreased significantly(P< 0.05),LncRNA-GSA5 level increased compared with the normal glucose group(P< 0.05),PTEN expression increased significantly compared with the normal glucose group(P< 0.05),FN,IV collagen level decreased compared with the normal glucose group(P< 0.05),CCND1,CDK4 level decreased compared with the normal glucose group(P< 0.05),Cell arrest G0/G1 phase,G1 /S block;11.48 h after transfection of LncRNA-GSA5 overexpressed plasmid,cell proliferation rate decreased significantly(P< 0.05),mi R-21 level was decreased compared with the normal glucose group(P< 0.05),PTEN level was increased compared with the normal glucose group(P< 0.05),FN and IV collagen level was significantly decreased compared with the normal glucose group(P< 0.05),and CCND1 and CDK4 expression was decreased compared with the normal glucose group(P< 0.05).Cell stagnation G0/G1 phase,G1 /S block.Conclusion:(1)under the condition of diabetes,the overexpression of mi R-21 in glomerular mesangial cells and the low expression of LncRNA-GSA5 can inhibit PTEN,leading to the hypertrophy of mesangial mesangial cells,shortened cell cycle and proliferation,and the accumulation of extracellular matrix proteins.(2)expression of PTEN and LncRNA-GSA5 in mesangial cells of high-glucose group transfected with mi R-21 inhibitor was significantly up-regulated,and mesangial cell cycle arrest and proliferation were decreased,thus alleviating diabetic renal damage.(3)in cultured mesangial cells transfected with LncRNA-GSA5 overexpressed plasmid,the expression of mi R-21 was down-regulated,the cell cycle of mesangial cells was stagnated,the proliferation ability was decreased,the accumulation of extracellular matrix protein was reduced,and the diabetic kidney damage was improved. |
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