The Mechanisms Of C/EBPβ-Lin28a And DEPP1-let-7b Mediating Pathogenic And Protective Effects,respectively,in The Restenosis After Percutaneous Transluminal Angioplasty In Diabetic Lower Extremity Arterial Disease

Part Ⅰ:The mechanism of C/EBPβ-Lin28a positive feedback loop in the restenosis after percutaneous transluminal angioplasty in diabetic lower extremity arterial diseaseBackgroundThe prevalence of type 2 diabetes mellitus(T2DM)is increasing at an alarming rate.Diabetic lower extremity arterial disease(DLEAD)is one of the most prevalent chronic vascular complications of T2DM.Its incidence exceeds 50%in patients with T2DM.Percutaneous transluminal angioplasty(PTA)is widely used as a primary therapy for DLEAD.However,restenosis,mainly caused by excessive proliferation and migration of vascular smooth muscle cells(VSMCs),after PTA limits treatment efficacy.Lin28a,an RNA binding protein,is initially discovered in C.elegans and plays a vital role in growth and development.Overexpression of Lin28a leads to an increase in both body size and organ size of C.elegans.Furthermore,Lin28a is highly expressed in embryonic stem cells(ESCs)and participates in reprogramming.Our previous studies indicate that Lin28 a contribute to restenosis by promoting the proliferation and migration of VSMCs.However,the precise mechanisms regulating Lin28a expression remains unknown.The binding of transcription factors(TFs)to promoters is pivotal to transcriptional activation of genes.CCAAT/enhancer-binding proteins(C/EBPs)are a family of TFs that consist of six members,including C/EBPα,C/EBPβ,C/EBPδ,C/EBPε,C/EBPγ,and C/EBPζ.These TFs share a leucine-rich dimerization domain(leucine zipper;LZ)and a basic amino acid-rich DNA binding domain(basic region;BR)but differs in the N-terminal transactivation domains(TADs).In particular,C/EBPβ is considered a master regulator of multiple tissue types and has an emerging role in vascular function.It has been documented that C/EBPβ participates in vascular remodeling.Transactivation of C/EBPβ is responsible for advanced glycation end product(AGE)-induced migration of human aortic smooth muscle cells(HASMCs).However,whether and how C/EBPβ is involved in Lin28 mediated restenosis remains to be elucidated.DNA methylation,one of the most studied epigenetic events in eukaryotes,is a mechanism through which a methyl residue is covalently added to cytosine,bringing about the conversion of cytosine to 5-methylcytosine(5-mC).This process is catalyzed by DNA methyltransferases(DNMTs).In mammals,it occurs predominantly at cytosine-phosphate-guanine(CpG)dinucleotides.DNA methylation silences gene expression and has been confirmed to be a reversible process.Ten-eleven translocation 1(Tetl),an enzyme that can convert 5-mC to 5-hydroxymethylcytosine(5-hmC),has been implicated in DNA demethylation.In addition,decitabine(5-aza-2’-deoxycytidine,DAC),an inhibitor of DNA methyltransferase,mediates demethylation.Furthermore,it has been shown that global methylation level changes after VSMCs injury.In the context of restenosis,previous research illustrates that DNA methylation of Src homology 2 domain-containing PTP(SHP)-1 affect the proliferation of VSMCs involved in intimal hyperplasia and restenosis.In recent years,it has been demonstrated that mechanical stretch could promote phenotype transition from contractile to synthetic,leading to the excessive proliferation and migration of VSMCs in hypertension.However,the impact of mechanical stretch on DNA methylation and whether it plays a positive role in restenosis remains to be elucidated.Objectives1.Elucidate the expression and DNA methylation levels of C/EBPβ in restenosis by constructing rat models of type 2 diabetic atherosclerosis and restenosis.2.To clarify the role of mechanical stretch in the regulation of C/EBPβ DNA methylation and expression.3.Clarify the direct regulation of Lin28a expression by C/EBPβ and further effects on the proliferation and migration of VSMCs.4.Explore the feedback regulation mechanism between Lin28a and C/EBPβ and reveal the role of Tet1-mediated DNA demethylation in it.Methods1.Construction of animal models4-week-old male SD rats were fed with high-fat diet for 4 weeks,and streptozotocin(STZ)was injected intraperitoneally to construct T2DM rat models.Subsequently,the T2DM rats were randomly divided into atherosclerosis and restenosis groups,and the rats in both groups continued to be fed with high-fat diet for 4 weeks after balloon strain surgery.After establishing the atherosclerosis model by ultrasound detection,the atherosclerosis group underwent sham operation,and the restenosis group was then subjected to balloon dilation surgery.Then they were fed with high-fat diet for 4 weeks after surgery.The aerial tissues of restenosis group were harvested after establishing the restenosis model by ultrasound detection.2.Hematoxylin-eosin(HE)stainingParaffin sections were stained with hematoxylin and eosin and then sealed.The vascular morphology was observed with the microscope.3.Immunofluorescence double stainingThe expression and co-localization of C/EBPβ and α-SMC,Lin28a and α-SMC,C/EBPβand Lin28a in restenotic and atherosclerotic vessels were detected,respectively.4.Immunohistochemical stainingDetect the expression of C/EBPβ in restenotic and atherosclerotic vessels.5.Quantitative real-time polymerase chain reaction(qRT-PCR)RNA was extracted,and reverse transcribed into cDNA,and expression was calculated after amplification to detect C/EBPβ and Lin28a mRNA expression.6.Virus transfectionThe optimal multiplicity of infection(MOI)was determined,followed by transfection with C/EBPβ and Lin28a overexpression and interference lentivirus according to the determined optimal MOI.After cell confluence reached 70-80%,complete medium containing puromycin was added to screen the stably transfected cells.qRT-PCR and Western blot were used to detect transfection efficiency.7.Western BlotExtract total protein,measure protein concentration using BCA method and denature the protein by heating at 95℃ for 5 min.SDS-PAGE gels were prepared,followed by electrophoresis,membrane transfer and incubation with antibodies.C/EBPβ,Lin28a and Tet1 protein expression were detected.8.EdU proliferation assayApollo staining reaction solution and Hoechst 33342 reaction solution were used to stain proliferating nuclei and all nuclei respectively.The proliferation rates of VSMCs with different treatments were calculated after photographed with microscopy.9.Transwell migration assayThe cells were seeded into chambers after resuspension with serum-free medium,complete medium was added to the lower chambers.After incubated in incubator for 24 h,the chambers were fixed with 4%paraformaldehyde and stained with crystal violet,and finally photographed and counted to detect the migration number of VSMCs with different treatments.10.Chromatin Immunoprecipitation(ChIP)Immunoprecipitation was performed after ultrasound breaking of DNA,elute protein-DNA complexes,reverse cross-linking of protein-DNA complexes with free DNA,purify DNA,and finally PCR to detect whether C/EBPβ binds to Lin28a promoter.11.Transfection of plasmid and siRNAThe plasmid and siRNA of C/EBPβ,Lin28a and Tet1 were transfected into cells with LipofectamineTM 3000,incubated in incubator,and changed to complete medium after 6 h.qRT-PCR and Western Blot were used to detect the transfection efficiency.12.DNA methylation detectionDNA extraction followed by heavy sulfite treatment,multiplex PCR reactions on target fragments,sample addition of specific tag sequences,sample mixing and gel cutting for recovery,and finally library quantification and up-sequencing.DNA methylation levels of VSMCs in restenotic and atherosclerotic vessels and in different treatment groups were detected.13.Cell stretch assayThe cells were seeded in the plates dedicated to stretch and then placed on the r cell stretcher for cell stretching.14.Co-Immunoprecipitation(Co-IP)Detect the binding of Lin28a and Tet1 protein.15.Statistical analysisStatistical analysis was performed with GraphPad Prism 8.0.Student’s t test was used to assess difference in data within two groups.Statistical comparisons within multiple groups were analysed using one-way ANOVA.Data were presented as the mean±standard error of the mean(SEM),and the level of statistical significance was determined to be P<0.05.Results1.The expression of Lin28a in restenotic and atherosclerotic vesselsThe results of immunofluorescence double-staining confirmed that Lin2 8a expression was increased in restenotic vessels compared with the atherosclerotic vessels,and Lin28a mainly expressed in smooth muscle cells.2.Transcription factors prediction and screening of Lin28aThe PROMO database was used to predict the transcription factors of Lin28a.Combined with the results the whole transcriptome sequencing data,C/EBPβ,c-Jun,Pax-5,Ap2a1 and IRF-1 were differentially expressed between restenosis and atherosclerosis groups.Further validation by qRT-PCR was performed and C/EBPβ was selected for subsequent studies.3.Expression of C/EBPβ in restenotic and atherosclerotic vesselsThe results of immunohistochemical and immunofluorescence double-staining showed that C/EBPβ expression was increased in restenotic vessels compared with the atherosclerotic vessels,and C/EBPβ was mainly expressed in smooth muscle cells.4.C/EBPβ promoted proliferation and migration of VSMCsAfter the successful construction of C/EBPβ lentiviral stable cell line,the proliferation and migration of VSMCs were examined using EdU and Tranwell assays,respectively.The results showed that the proliferation and migration of VSMCs enhanced after C/EBPβoverexpression and decreased after C/EBPβ interference.5.C/EBPβ acted as a transcription factor to promote Lin28a expression and further promoted proliferation and migration of VSMCs via regulating Lin28aThe qRT-PCR and Western blot results showed that Lin28a expression was upregulated following C/EBPβ overexpression while downregulated following C/EBPβ knockdown.The ChIP assay confirmed that C/EBPβ could bind to the promoter of Lin28a directly.The results of EdU and Transwell experiments showed that C/EBPβ-overexpression-mediated enhanced VSMCs proliferation and migration were significantly reversed by co-transfection with si-Lin28a.Likewise,cell proliferation and migration inhibited by C/EBPβ depletion were reversed by co-transfection with the Lin28a overexpression plasmid6.DNA methylation levels of C/EBPβ in restenotic and atherosclerotic vesselsMethylTarget region methylation sequencing showed that the DNA methylation level of C/EBPβ was lower in restenotic vessels compared to atherosclerotic vessels.7.DAC promoted the expression of C/EBPβ and Lin28a,and enhanced proliferation and migration of VSMCsWestern blot results showed that C/EBPβ and Lin28a expression increased after 1 μM DAC treatment.Further EdU and Transwell assays showed that DAC promoted cell proliferation and migration.9.Mechanical stretch led to C/EBPβ hypomethylation and facilitated C/EBPβ and Lin28a expressionWestern blot results showed that C/EBPβ and Lin28a expression enhanced after mechanical stretch.The DNA methylation level of C/EBPβ was significantly reduced.10.Lin28a promoted proliferation and migration of VSMCs by regulating C/EBPβLin28a lentiviral stable cell line were successfully constructed.The results of Western blot showed that Lin28a promoted C/EBPβ expression.The results of EdU and Tranwell assays showed that interference with C/EBPβ reduced the increase in cell proliferation migration caused by overexpression of Lin28a,while C/EBPβ upregulation reversed the decrease of proliferation migration induced by Lin28a downregulation.11.Lin28a affects C/EBPβ expression by regulating Tet1Overexpression of Lin28a decreased C/EBPβ methylation levels,whereas inhibition of Lin28a expression increased C/EBPβ methylation levels.The results of Co-IP experiments showed that Lin28a could bind to Tet1.Co-transfection of si-Tetl and Lin28a-overexpressing lentivirus reduced C/EBPβ expression compared with overexpression of Lin28a alone.The above results suggest that Lin28a enhanced C/EBPβ expression by recruiting Tet1 to the promoter region of C/EBPβ and promoting C/EBPβ demethylation.Conclusions1.C/EBPβ was hypermethylated and highly expressed in restenotic vessels compared to atherosclerotic vessels.2.Mechanical stretch induced C/EBPβ demethylation and promoted its expression.3.As a transcription factor,C/EBPβ promoted Lin28a expression by binding to the Lin28a promoter,which further promoted proliferation and migration of VSMCs.4.Lin28a recruited Tet1 to the C/EBPβ promoter,leading to C/EBPβ demethylation,which in turn promoted C/EBPβ expression as well as proliferation and migration of VSMCs.Part Ⅱ:The protective effect and mechanism of DEPP1-let-7b in the restenosis after percutaneous transluminal angioplasty in diabetic lower extremity arterial diseaseBackgroundLower extremity arterial disease(LEAD)is one of the most common macrovascular complications of type 2 diabetes mellitus(T2DM),caused by partial or complete obstruction of lower extremity arteries.Inflammation,oxidative stress and advanced glycation end-products(AGEs)accelerates the process of LEAD in patients with T2DM.Percutaneous transluminal angioplasty(PTA)has become an effective intervention for LEAD.However,restenosis restricts the efficacy of PTA,the incidence of which is up to 40%-50%.Therefore,searching for novel treatments for restenosis is urgently needed.The main reason of restenosis is excessive proliferation of vascular smooth muscle cells(VSMCs)and migration from media to intima.Our previous research found that C/EBPβ-Lin28a contributed to the progression of restenosis by enhancing the proliferation and migration of VSMCs.However,although VSMCs show excessive proliferation and migration during restenosis,they do not exhibit aggressive proliferation and highly invasive properties.So it is of clinical significance to reveal the molecular mechanism of massive migration and proliferation of VSMCs in restenosis without "malignant transformation" to prevent and treat restenosis after PTA.Act as an RNA binding protein,Lin28a negatively regulates let-7 expression.Let-7 family belongs to microRNAs(miRNAs),which are lowly expressed in undifferentiated cells and the expression of which gradually decreases during cell differentiation.Let-7 is involved in many biological processes,such as angiogenesis,bone remodeling and inflammatory responses.Let-7 can also inhibit Lin28a expression through binding to 3’-UTR of Lin28a mRNA,forming a bidirectional feedback loop.However,let-7 family has many members,not all isoforms are involved in this loop.Our previous findings suggested that the Lin28a-let-7c/g feedback loop was involved in restenosis,whereas let-7a,let-7b,let-7e and let-7f were not regulated by Lin28a and highly expressed in restenotic vessels.However,whether they inhibit Lin28a expression in restenosis and thus play a protective role in restenosis has not been elucidated.Decidual Protein induced by Progesterone(DEPP1)is a protein that is induced by progesterone in endometrial stromal cells during decidualization,which is widely expressed in a variety of tissues,including ovary,heart,lung and arterial endothelial cells.A significant increase in DEPP1 expression has been reported after exposure to ultraviolet,ionizing radiation or energy deprivation,which indicated that DEPP1 may be involved in stress reaction.Whereas balloon dilation is a stimulus to the vessel,it is worth exploring whether DEPP1 plays a role in restenosis.In the present study,we elucidated the regulation of Lin28a by let-7a,let-7b,let-7e and let-7f,that are not regulated by Lin28a,and the effect if them on the proliferation and migration in VSMCs.Furthermore,the mechanisms underlying the specific upregulation of let-7 were explored via target gene prediction combined with sequencing analysis,and these findings shed new light on the treatment of restenosis.Objectives1.To clarify the roles of let-7a,let-7b,let-7e and let-7f,which are not regulated by Lin28a,on the proliferation and migration of VSMCs.2.To elucidate the roles of let-7a,let-7b,let-7e and let-7f on Lin28a,screen for let-7 isoforms that regulate Lin28a,and further clarify their effects on the proliferative and migration of VSMCs.3.To explore the molecular mechanism causing let-7 upregulation and clarify its role in restenosis.Methods1.Transfection of plasmid and siRNAThe plasmid and siRNA of DEPP1 as well as let-7a,let-7b,let-7e,let-7f mimics and inhibitors were transfected into cells with LipofectamineTM 3000,incubated in incubator,and changed to complete medium after 6 h.qRT-PCR and Western Blot were used to detect the transfection efficiency.2.Quantitative real-time polymerase chain reaction(qRT-PCR)RNA was extracted,and reverse transcribed into cDNA,and expression was calculated after amplification to detect let-7a,let-7b,let-7e,let-7f and DEPP1expression.3.EdU proliferation assayApollo staining reaction solution and Hoechst 33342 reaction solution were used to stain proliferating nuclei and all nuclei respectively.The proliferation rates of VSMCs with different treatments were calculated after photographed with microscopy.4.Transwell migration assayThe cells were seeded into chambers after resuspension with serum-free medium,complete medium was added to the lower chambers.After incubated in incubator for 24 h,the chambers were fixed with 4%paraformaldehyde and stained with crystal violet,and finally photographed and counted to detect the migration number of VSMCs with different treatments.5.Western BlotExtract total protein,measure protein concentration using BCA method and denature the protein by heating at 95℃ for 5 min.SDS-PAGE gels were prepared,followed by electrophoresis,membrane transfer and incubation with antibodies.Lin28a protein expression were detected.6.Construction of animal models4-week-old male SD rats were fed with high-fat diet for 4 weeks,and streptozotocin(STZ)was injected intraperitoneally to construct T2DM rat models.Subsequently,the T2DM rats were randomly divided into atherosclerosis and restenosis groups,and the rats in both groups continued to be fed with high-fat diet for 4 weeks after balloon strain surgery.After establishing the atherosclerosis model by ultrasound detection,the atherosclerosis group underwent sham operation,and the restenosis group was then subjected to balloon dilation surgery.Then they were fed with high-fat diet for 4 weeks after surgery.The aerial tissues of restenosis group were harvested after establishing the restenosis model by ultrasound detection.7.Statistical analysisStatistical analysis was performed with GraphPad Prism 8.0.Student’s t test was used to assess difference in data within two groups.Statistical comparisons within multiple groups were analysed using one-way ANOVA.Data were presented as the mean±standard error of the mean(SEM),and the level of statistical significance was determined to be P<0.05.Results1.The effects of let-7a,let-7b,let-7e and let-7f on the proliferation and migration of VSMCs.EdU assays demonstrated that let-7b,let-7e or let-7f overexpression suppressed the proliferation of VSMCs and vice versa.Transwell assays indicated that the number of migrating cells reduced when transfected with let-7b,let-7e let-7f mimics.Opposite effects were observed in let-7b and let-7f knockdown cells.While no significant difference was found in the migration and proliferation after regulating let-7a.2.Let-7b inhibited Lin28a-mediated proliferation and migration of VSMCs.Western blot results showed that let-7b inhibited Lin28a expression.No significant differences in Lin28a expression were observed when let-7a,let-7e or let-7f overexpression or interference.The proliferation and migration of VSMCs increased when upregulating both let-7b and Lin28a compared to let-7b overexpression alone.Conversely,Lin28a knockdown reversed let-7b inhibitor-induced enhancement of proliferation and migration3.Selection of DEPP1To further clarify the mechanism underlying let-7b upregulation,the lncRNA NONRATT020829.2 was screened by predicting the lncRNAs that might bind to let-7b combined with whole-transcriptome sequencing,and DEPP1 was further selected as the subsequent study molecule by sequence alignment and Sanger sequencing.4.Low DEPP1 expression in restenosis reduced let-7b inhibition and promotes proliferation and migration of VSMCs.The expression of let-7b was reduced after DEPP1 upregulation and increased after DEPP1 downregulation.The proliferation and migration of VSMCs enhanced after DEPP1 overexpression and decreased after DEPP1 knochdown.In addition,qRT-PCR results showed that DEPP1 was lowly expressed in restenosis compared with atherosclerosis.5.DEPP1 promoted Lin28a expression by inhibiting let-7bDEPP1 overexpression enhanced Lin28a expression,and the knockdown of DEPP1 had the converse effect.Furthermore,co-transfection with pcDNA3.1-DEPP 1 and let-7 mimics reversed the DEPP 1-induced increase in the expression of Lin28a.In contrast,reduced Lin28a expression due to DEPP1 silencing was restored following additional co-transfection with let-7 inhibitorsConclusions1.Let-7b,let-7e and let-7f could inhibit VSMCs proliferation and migration respectively,while let-7a exerted no regulation on proliferation and migration in VSMCs.2.Let-7b inhibited VSMCs proliferation and migration by downregulating Lin28a,while let-7a,let-7e and let-7f had no regulatory effect on Lin28a expression.3.The specific downregulation of DEPP1 in restenosis diminished the inhibitory effect on let-7b,resulting in increased expression of let-7b,which in turn inhibited Lin28a expression and reduced proliferation and migration of VSMCs,which exert the protective role in restenosis.