| Objective: Diabetes is an increasingly serious global health problem.It is a group of metabolic diseases characterized by high blood glucose caused by multiple causes.Longterm hyperglycemia damages large blood vessels and capillaries and endangers the eyes,heart,brain,kidneys,peripheral nerves,and feet.One of the most common microvascular complications is diabetic kidney disease(DKD).About 30-40% of diabetic patients develop DKD,and one third of them progress to end-stage renal disease(ESRD).Since2011,the proportion of patients with diabetes-related chronic kidney disease in tertiary hospitals in our country has surpassed that of chronic kidney disease related to glomerulonephritis,and the gap between the two has been increasing.DKD is characterized by decreased renal function,including proteinuria,glomerulosclerosis,and tubulointerstitial fibrosis.At present,the molecular mechanism leading to DKD is not fully understood.There is evidence that epigenetic mechanisms play an important role in the pathogenesis of DKD.Epigenetics includes cytosine methylation of DNA,posttranslational modifications of histones,and non-coding RNA.Non-coding RNA(ncRNA)is genomic RNA that does not encode genetic information into protein.ncRNAs are roughly divided into two categories: one is short ncRNAs,mainly micro RNAs(mi RNAs),and the other is long non-coding RNAs(lncRNAs).LncRNA is a type of non-coding RNA with a length greater than 200 nt,which base-pairs with DNA or RNA in a sequence-specific manner,thereby regulating gene expression.According to the position relative to nearby protein-coding genes,usually lncRNAs can be divided into sense lncRNA(sense lncRNA),antisense lncRNA(antisense lncRNA),bidirectional lncRNA(bidirectional lncRNA),intragenic lncRNA(intronic lncRNA)and intergenic lncRNA(intergenic lncRNA).Among them,antisense lncRNA is a type of lncRNA molecule which gene sequence is complementary to host gene m RNA and transcription direction is opposite.Antisense lncRNA can widely participate in the regulation of cell growth,differentiation,development and death and many other physiological and pathological processes through transcription,post-transcriptional modification and epigenetics.Our group previously used the Arraystar Human LncRNA/m RNA expression profiling chip(v3.0)to detect the serum lncRNA expression profile of patients with diabetic nephropathy,diabetic patients with normal albuminuria and healthy controls,and further expanded the clinical research cohort,using RT-q PCR to validate the microarray results.RT-q PCR results showed that the expression of natural antisense lncRNA-ARAP1-AS2 in the serum of patients with diabetes and diabetic nephropathy gradually increased,and the expression of its sense strand target gene ARAP1(Arf GAP with Rho GAP domain,ankyrin repeat and PH domain1)m RNA gradually increased.However,at present,the regulation mechanism of natural antisense lncRNA-ARAP1-AS2 to its sense strand target gene ARAP1 and mediating diabetic renal injury is still unclear.The natural antisense lncRNA-ARAP1-AS2 and its sense strand target gene ARAP1 are located on chromosome 11.ARAP1,also known as CENTD2,genome-wide association study(GWAS)determined that rs1552224 and rs11603334 in the 5'untranslated region of ARAP1 gene are risk susceptibility sites for type 2 diabetes.ARAP1 contains Arf GAP,Rho GAP,SAM,RAS,PH domains and ankyrin repeats.It also has Rho GAP and phosphatidylinositol(3,4,5)triphosphate(PIP3)dependent Arf GAP activity,and therefore ARAP1 may be the connection point of phosphoinositide,Arf and Rho-like signal,which could regulate cell activity.ARAP1 is an important mediator of epidermal growth factor receptor(EGFR)signaling pathway.EGFR is a member of the Erb B family and has tyrosine kinase activity.It is an important transmembrane receptor.Its continuous transactivation leads to the subsequent activation of the TGF-?/Smad3 signaling pathway and participates in the occurrence and development of DKD.However,the exact mechanism of persistent transactivation of EGFR in DKD is still unclear.Ubiquitin ligase(E3)Cbl and adaptor protein CIN85(also known as SH3KBP1)are important regulators of EGFR endocytosis and degradation.It has been reported that the adaptor protein CIN85 can regulate the endocytosis and ubiquitination of EGFR through the formation of a ubiquitin ligase Cbl–CIN85–endorphin complex.And ARAP1 can regulate the endocytosis and ubiquitination of EGFR in He La cells by competing with Cbl for the binding of CIN85,but the current regulation mechanism of ARAP1 and its natural antisense lncRNA-ARAP1-AS2 on the ubiquitination of EGFR in DKD is still unclear.The purpose of this study is to explore the regulatory mechanism of natural antisense lncRNA-ARAP1-AS2 on its sense strand target gene ARAP1,and the regulation mechanism of natural antisense lncRNA-ARAP1-AS2 and its sense strand target gene ARAP1 on EGFR ubiquitination in DKD,whether to maintain the continuous transactivation of EGFR by regulating the ubiquitination of EGFR to participate in diabetic renal injury.To explore whether the targeted inhibition of natural antisense lncRNAARAP1-AS2 and its sense strand target gene ARAP1 can inhibit the continuous transactivation of EGFR by regulating the ubiquitination of EGFR,and reduce the diabetic renal injury.Methods: The relative expression of natural antisense lncRNA-ARAP1-AS2 and its sense strand target gene ARAP1 in human proximal tubular epithelial cells(HK-2 cells)induced by high glucose(25mmol/L D-glucose)was determined by q RT-PCR.The full-length sequence of natural antisense lncRNA-ARAP1-AS2 in HK-2 cells was obtained by 5'3'RACE experiment.The subcellular localization of natural antisense lncRNA-ARAP1-AS2 was observed by fluorescence in situ hybridization(FISH)experiment.Bioinformatics methods verify the protein coding ability of natural antisense lncRNA-ARAP1-AS2 and obtain the protein regulatory network of its sense chain target genes ARAP1,CIN85 and EGFR.Transfection of natural antisense lncRNA-ARAP1-AS2 overexpression plasmid or small interfering RNA(si RNA)and its sense strand target gene ARAP1 knockdown plasmid in HK-2 cells cultured with normal glucose(5.5 mmol/L D-glucose+19.5 mmol/L mannitol)and high glucose,add EGFR tyrosine kinase inhibitor AG1478 to HK-2 cells cultured with high glucose,and co-transfect the natural antisense lncRNA-ARAP1-AS2 overexpression plasmid and ARAP1 knockdown plasmid or co-transfect the natural antisense lncRNA-ARAP1-AS2 overexpression plasmid and AG1478,the relationship between the natural antisense lncRNA-ARAP1-AS2 and its sense strand target gene ARAP1 and EGFR/TGF-?/Smad3 signaling pathway were verified by western blot and q RT-PCR.We measured the expression of extracellular matrix protein type I collagen(collagen I,Col I),type IV collagen(collagen IV,Col IV)and fibronectin(fibronectin,FN)and the proliferation level of HK-2 cells by western blot and CCK8.RNA pulldown and pulldown-seq analysis were used to verify the binding relationship between natural antisense lncRNA-ARAP1-AS2 and ARAP1 and CIN85 in HK-2 cells,and to obtain the m RNAs binding to natural antisense lncRNA-ARAP1-AS2.Use GO database to annotate RNA from RNA pulldown.Co-immunoprecipitation and double immunofluorescence were used to verify the regulatory mechanism between ARAP1 and CIN85 in HK-2 cells induced by high glucose,kidney tissues of DKD patients and 20-week-old db/db mice.Ubiquitination analysis was used to verify the effect of ARAP1 on EGFR ubiquitination in HK-2 cells induced by high glucose.Results: The expression of natural antisense lncRNA-ARAP1-AS2 and its sense strand target gene ARAP1 was significantly up-regulated in HK-2 cells cultured with high glucose(P<0.05).The 5'3'RACE experiment successfully obtained the full-length sequence of the natural antisense lncRNA-ARAP1-AS2 in HK-2 cells.FISH experiments showed that the subcellular localization of natural antisense lncRNA-ARAP1-AS2 was mainly distributed in the nucleus.Bioinformatics methods verify that the natural antisense lncRNA-ARAP1-AS2 has no protein coding ability,and obtain the protein regulatory network of ARAP1,CIN85 and EGFR.In HK-2 cells cultured with normal glucose and high glucose,after transfection with the natural antisense lncRNA-ARAP1-AS2 overexpression plasmid,the expression of its sense strand target gene ARAP1 was significantly up-regulated,and it significantly promoted the expression of extracellular matrix proteins and HK-2 cell proliferation by maintaining the continuous activation of the EGFR/TGF-?/Smad3 signaling pathway(P<0.05).On the contrary,in HK-2 cells cultured with normal glucose and high glucose,after transfection with lncRNA-ARAP1-AS2 si RNA,the expression of ARAP1 was significantly down-regulated,and it significantly inhibited the expression of extracellular matrix proteins and HK-2 cell proliferation by inhibiting the continuous activation of the EGFR/TGF-?/Smad3 signaling pathway(P<0.05).In normal and high glucose cultured HK-2 cells,after transfection of ARAP1 knockdown plasmid,the expression of ARAP1 was significantly down-regulated,and it significantly inhibited the expression of extracellular matrix proteins and HK-2 cell proliferation by inhibiting the continuous activation of the EGFR/TGF-?/Smad3 signaling pathway(P<0.05).After adding EGFR tyrosine kinase inhibitor AG1478 to HK-2 cells cultured with high glucose,the expression level of the EGFR/TGF-?/Smad3 signaling pathway decreased,and the expression of extracellular matrix proteins and HK-2 cells proliferation were significantly inhibited(P<0.05).After co-transfecting the overexpression plasmid of natural antisense lncRNA-ARAP1-AS2 and the knockdown plasmid of its sense strand target gene ARAP1 in HK-2 cells cultured with normal glucose,it significantly inhibited the up-regulated expression of ARAP1,EGFR/TGF-?/Smad3 signaling pathway,extracellular matrix protein expression and HK-2 cell proliferation caused by the overexpression plasmid of natural antisense lncRNA-ARAP1-AS2(P<0.05).After co-transfecting the natural antisense lncRNA-ARAP1-AS2 overexpression plasmid and AG1478 in HK-2 cells cultured with normal glucose,it significantly inhibited the up-regulated expression of the EGFR/TGF-?/Smad3 signaling pathway,extracellular matrix protein expression and HK-2 cell proliferation caused by the overexpression plasmid of natural antisense lncRNAARAP1-AS2(P<0.05),but had no effect on the up-regulation of ARAP1 expression.RNA pulldown and pulldown-seq analysis showed that ARAP1 was enriched in the complex pulled down by lncRNA-ARAP1-AS2(Fold change=1.515187062),but not enriched CIN85(Fold change=-1.415581462).The results of co-immunoprecipitation and double immunofluorescence showed that ARAP1 can bind with CIN85 in HK-2 cells cultured with high glucose,and the expression and co-localization of ARAP1 and CIN85 were both significantly increased(P<0.05).Compared with healthy controls,the results of double immunofluorescence showed that the expression and co-localization of ARAP1 and CIN85 in the kidney tissue of DKD patients were both significantly increased(P<0.05).Compared with the kidney tissue of normal control db/m mice,the double immunofluorescence results showed that the expression and co-localization of ARAP1 and CIN85 in the kidney tissue of db/db mice were both significantly increased(P<0.05).Ubiquitination analysis showed that in HK-2 cells induced by high glucose,after ARAP1 was knocked down,the ubiquitination level of EGFR was significantly increased,and the expression of EGFR was significantly decreased(P<0.05).Conclusion:(1)The full-length sequence of natural antisense lncRNA-ARAP1-AS2 in HK-2 cells was obtained for the first time.In HK-2 cells cultured with high glucose,the expression of lncRNA-ARAP1-AS2 was significantly up-regulated,and it was mainly distributed in the nucleus of HK-2 cells.It may be involved in the renal tubular interstitial injury process of DKD by promoting the accumulation of extracellular matrix proteins and cell proliferation.(2)Under the diabetic state,natural antisense lncRNA-ARAP1-AS2 may increase the expression of ARAP1 by binding to the m RNA of its sense strand target gene ARAP1,and then promote the increase of the binding between ARAP1 and CIN85.It may reduce the binding of CIN85 and Cbl to reduce the ubiquity of EGFR and increases the protein expression level of EGFR,thus providing more total EGFR protein that can be stimulated by high glucose,thereby maintaining the activation of the EGFR/TGF-?/Smad3 pathway and promoting the accumulation of extracellular matrix proteins and cell proliferation under the diabetic state.(3)Targeted inhibition of natural antisense lncRNA-ARAP1-AS2 and its sense strand target gene ARAP1 can inhibit the continuous transactivation of EGFR by increasing the ubiquitination of EGFR,and reduce the injury of proximal renal tubular cells and the accumulation of extracellular matrix proteins under the diabetic state. |
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