| Vascular smooth muscle cells(VSMC)play important roles in the physiological functions of vessels.VSMCs and the excellular matrix synthesized and secreted by VSMCs are the main regulators of vascular contraction.They can maintain the peripheral vascular resistance,keep the blood pressure and control the distribution of blood flow pumped by heart.VSMCs are not only the regulators,but also the effectors.They are the sensors of vasoactive substances and mechanical forces,and then transduce them into various signals which can be recognized by the cells.Therefore,VSMCs are the focus of vascular studies due to their vital structures and functions.Hypertension,which has affected more than a quarter of the world's adult population in 2000,and the proportion is expected to rise to 29%by 2025,is a worldwide public-health problem.As a leading risk factor for mortality,hypertension can result in the development of various related complications,such as stroke and vascular diseases.The mechanisms that underlie hypertension are widely investigated but still remain unclear.VSMC contraction which leads to increased vascular resistance is one of the mechanisms of hypertension.Cellular senescence is traditionally defined as a permanent and irreversible proliferative arrest state of cells.Senescent cells exhibit large and flattened morphological characteristics,increased gene expression of p53,p21and p16,which are negative regulators of the cell cycle,as well as increased activity of senescence-associated?-galactosidase(SA-?-gal),a biomarker of cellular senescence.Senescence-associated secretory phenotype(SASP)is also included.Emerging data indicate that hypertension and atherosclerosis are significantly associated with senescence of VSMCs.Circular RNAs(circRNA)are a novel type of noncoding RNAs,characterized by covalently closed loop structures without free terminals.The circular structure,which is formed by the backsplicing,makes circRNAs more stable than linear m RNAs.circRNAs play key roles in many biological and pathological processes.Most of circRNAs located in the cytoplasm act as miRNA sponges,protein sponges,or protein scaffolds,a few circRNAs stay in the nucleus regulating splicing and transcription.Furthermore,circRNAs containing particular sequences can even be translated into proteins or peptides.Emerging studies demonstrate that circRNAs are implicated in cardiovascular diseases,such as hypertension.For instance,the circulating levels of c ZNF609 were down-regulated in patients with hypertension.circRNAs are also involved in cellular senescence.circ-Foxo3 promotes cardiac senescence by binding to anti-senescent proteins.circACTA2 is a circular RNA found in VSMCs by our laboratory,and the mechanism underlying circACTA2-regulated VSMC functions is still unknown.In this study,we focused on the roles of circACTA2 in VSMC contraction and senescence and investigated the underlying mechanisms of circACTA2 action.Part?FN1 mediates circACTA2-regulated VSMC contractionObjective:To explore the effect of Ang?stimulation on circACTA2expression and the roles of circACTA2 in VSMC contraction.Methods:1 VSMCs were treated with Ang?or PDGF-BB and the expression of circACTA2 was detected by qRT-PCR.2 The expression of circACTA2 in vessels of hypertension patients was detected by qRT-PCR and fluorescence in situ hybridization(FISH).3 VSMCs were transfected with circACTA2 and/or treated with Ang?.The actin stress fibers were observed by confocal microscopy after phalloidin staining.4 circACTA2-interacting proteins were captured and identified by combining RNA antisense purification(RAP)or U-biotin-labeled RNA pull-down with quantitative liquid chromatography-mass spectrometry(LC-MS).5 circACTA2 and FN1 interaction was examined by RNA pull-down and RNA binding protein immunoprecipitation(RIP).6 VSMCs were transfected with circACTA2,or treated with Ang?accompanied by si FN1 transfection.The expression of FN1 was examined by Western blot,and actin stress fibers were observed by confocal microscopy after phalloidin staining.7 VSMCs were transfected with circACTA2 accompanied by si FN1transfection.Cell contraction induced by acetyl choline(Ach)was examined.8 Cells were treated with Ang?,and vessels were obtained from hypertension patients.Immunofluorescence staining was performed to detect the co-localization and interaction between circACTA2 and FN1.Results:1 The expression of circACTA2 is upregulated by Ang?We first demonstrated that circACTA2 expression was up-regulated by Ang?treatment and down-regulated by PDGF-BB treatment.Both qRT-PCR and fluorescence in situ hybridization(FISH)indicated that circACTA2expression in the vessels of hypertension patients was significantly higher than that in normal controls.Then we explored whether circACTA2 influences cytoskeleton organization.VSMCs were treated with Ang?or transfected with lentivirus encoding circACTA2 alone,or treated with them together,then phalloidin staining was performed.We found a strong F-actin cable formation in Ang?-treated or circACTA2-overexpressed cells,and the effect was further strengthened in cells treated with them together.2 circACTA2 interacts with FN1 and ILF3To identify specific interactors with circACTA2,we quantitatively compared the proteins captured with antisense oligonucleotides or U-biotin-labeled circACTA2 with their corresponding control-captured proteins.We analyzed biological replicate purifications in the two methods,quantifying 181 proteins that were identified by more than two unique peptides in each method.We reproducibly identified the part of strongly enriched proteins that met our enrichment criteria(mean log2>2.0,P<0.05,moderated t-test),and confirmed that circACTA2 interaction with FN1,VIM and ILF3,but not?-actin,was highly specific.Their interaction was further validated by RNA-binding protein immunoprecipitation(RIP).3 circACTA2 regulates VSMC contraction by stabilizing FN1To explore whether circACTA2 affects the expression of the proteins interacting with it,VSMCs were transfected with circACTA2.The results showed that the expression of FN1 significantly increased while the expression of ILF3 and VIM did not.When cells were treated with different concentrations of Ang?(0,10-7,2×10-7,4×10-7mol/L),the expression of FN1 was increased in an Ang II dose-dependent manner.The results indicate that FN1 is stabilized by circACTA2.Then we designed a si FN1 to knock down FN1 expression.As shown by Western blot,the elevation of FN1expression caused by Ang?was abrogated by FN1 knockdown,and VSMCs transfected with circACTA2 exhibited the same results.We further examined the effect of FN1 on VSMC contraction.We found that increased F-actin cable formation in circACTA2-overexpressing cells was decreased by knockdown of FN1,as shown by phalloidin staining.And the Ach-induced cell contraction was alleviated by FN1 knockdown.Then we explore the mechanism whereby VSMC contraction is regulated by circACTA2.FISH experiments showed that circACTA2 and FN1 were co-localized in the cytoplasm,and Ang?treatment enhanced their interaction.Summary:Ang?induces circACTA2 formation and the latter regulates VSMC contraction by interacting with FN1.Part?circACTA2 promotes VSMC contraction via regulating FN1 and SPTBN1 interactionObjective:To explore the molecular mechanism whereby circACTA2upregulates FN1 expression and regulates the interaction of FN1 with SPTBN1.Methods:1 RNA pull-down and high throughput sequencing were used to detect miRNAs binding to circACTA2.2 miRNAs binding to circACTA2 were confirmed by RNA pull-down and qRT-PCR.3 The binding sites of miR-149 on the FN1 3'UTR were predicted by bioinformatic analysis.The expression of FN1 in cells transfected with miR-149 mimic or inhibitor was detected by Western blot analysis.4 Dual luciferase reporter gene assay was used to confirm the interaction between miR-149 and FN1 3'UTR.5 VSMCs were transfected with circACTA2 accompanied by knockdown of miR-149,cell contraction induced by Ach was examined,and the formation of actin stress fibers was observed by confocal microscopy after phalloidin staining.6 The binding sites between FN1 and circACTA2 were predicted by bioinformatic analysis.7 The expression of GFP-FN1(183-275)and FN1 in VSMCs transfected with GFP-FN1(183-275)was examined by Western blot analysis.8 VSMCs were transfected with GFP-FN1(183-275)accompanied by Ang?treatment,and the expression of FN1 was examined by Western blot analysis.9 VSMCs were transfected with GFP-FN1(183-275),and circACTA2binding to FN1(183-275)was detected by RIP.10 VSMCs were transfected with circACTA2 accompanied by GFP-FN1(183-275)or blocking oligonucleotide transfection,circACTA2 binding to FN1 was detected by RIP.11 VSMCs were transfected with circACTA2 accompanied by GFP-FN1(183-275)transfection,actin stress fibers were observed by confocal microscopy after phalloidin staining.12 Proteins binding to FN1 were detected by Co IP-MS.13 VSMCs were transfected with GFP-FN1(183-275)or treated with Ang?,the interaction of FN1 with SPTBN1 was detected by coimmunoprecipitation(Co IP).14 VSMCs were transfected with circACTA2 accompanied by FN1knockdown,the co-localization and interaction of FN1 with SPTBN1 were examined by immunofluorescence staining.Results:1 circACTA2 upregulates the expression of FN1 by sponging miR-149RNA pull-down and high throughput sequencing were used to identify miRNAs sponed by circACTA2.There were 24 miRNAs enriched by circACTA2 probe.Then qRT-PCR was used to examine the expression of miRNAs in the precipitates of oligo pull-down.The results showed that miR-27b-3p,miR-27b-5p,miR-149,and miR-197 were significantly elevated in the precipitates of circACTA2 probe.The interaction of circACTA2 and miR-149 was further verified by dual luciferase reporter assay.Furthermore,the expression of miR-149 was upregulated in Ang?-treated VSMCs and downregulated in PDGF-BB-treated cells.We found that there is a binding site of miR-149 on FN1 3'UTR.Then we designed miR-149 mimic and inhibitor and confirmed that miR-149 mimic inhibited the expression of FN1 while the inhibitor promoted its expression.The interaction of FN1 and miR-149 was further verified by dual luciferase reporter assay.These results suggested that circACTA2 upregulates FN1expression via sponing miR-149.Moreover,increased F-actin cable formation in circACTA2-overexpressing VSMCs was further increased by miR-149inhibitor,as shown by phalloidin staining.And the Ach-induced cell contraction in circACTA2-overexpressing VSMCs was enhanced by miR-149inhibitor.2 VSMC contraction induced by circACTA2 is inhibited by interfering with circACTA2 and FN1 interactionWe predicted that the oligo-peptide residing in 237-252aa of FN1 might be a binding site of circACTA2.To test this,a DNA sequence,which could be translated into this oligo-peptide,was recombined with GFP-C1,and named GFP-FN1(183-275).After transfected with GFP-FN1(183-275),cells could express the oligo-peptide containing 183-275aa of FN1,as confirmed by Western blot.Then VSMCs were transfected with GFP-C1 or GFP-FN1(183-275),and RIP-PCR was performed to examine circACTA2 enrichment in the precipitates pulled down by anti-GFP antibody.circACTA2 enrichment in GFP-FN1(183-275)–overexpressing cells was much higher than that in GFP-C1 control cells,indicating that GFP-FN1(183-275)binds to circACTA2.Importantly,transfection of GFP-FN1(183-275)inhibited the expression of FN1.And transfection of GFP-FN1(183-275)alleviated the upregulation of FN1 expression induced by Ang?.RIP experiment showed that transfection of GFP-FN1(183-275)reduced circACTA2 enrichment which was enhanced by circACTA2 overexpression.These results indicated that GFP-FN1(183-275)disturbed the interaction of circACTA2 with FN1.Oligonucleotide was used to block the binding site in another RIP experiment,and it did inhibit circACTA2 enrichment which was enhanced by circACTA2 overexpression.Then we examined the cytoskeleton organization after GFP-FN1(183-275)transfection.After phalloidin staining,we found that increased F-actin cable formation in circACTA2-overexpressing cells was abolished by GFP-FN1(183-275)transfection.3 The contraction of VSMCs is regulated by FN1 and SPTBN1interactionTo identify the proteins interacted with FN1,we treated VSMCs with or without Ang?.There were 14 proteins interacted with FN1 in Ang?treated-VSMCs,including SPTBN1 and CALU among them.As SPTBN1belongs to a cytoskeletal protein,it is of great interest to us.Co IP experiment confirmed that FN1 and SPTBN1 interacted with each other,and Ang?treatment enhanced their interaction.Immunofluorescent staining exhibited the colocalization of FN1 with SPTBN1 in the cytoplasm,and their interaction was enhanced by circACTA2 overexpression while inhibited by si FN1.Moreover,Co IP experiment also showed that the interaction of FN1 with SPTBN1 was reduced by GFP-FN1(183-275)transfection.Summary:circACTA2 promotes the expression of FN1 via sponging miR-149,and FN1 interacts with SPTBN1 to regulate VSMC contraction.Part? circACTA2 promotes Ang?-induced VSMC senescence via interacting with ILF3Objective:To explore the molecular mechanism whereby circACTA2medietes Ang?-induced VSMC senescence.Methods:1 VSMCs were treated with Ang?,and/or were transfected with circACTA2 or sh-circACTA2,VSMC senescence was examined by SA-?-gal staining.The senescent cells were observed by light microscope.The expressions of p21,p16 and CDK4 were detected by Western blot analysis.2 VSMCs were transfected with ILF3-expressing vector or sh-ILF3accompanied by Ang?treatment,VSMC senescence was examined by SA-?-gal staining.The senescent cells were observed by light microscope.The expressions of p21,p16 and CDK4 were detected by Western blot analysis.3 VSMCs were transfected with ILF3-expressing vector or sh-ILF3,and the expression of CDK4 m RNA was detected by qRT-PCR.4 VSMCs were transfected with ILF3-expressing vector or sh-ILF3accompanied by actinomycin D treatment,and the expression of CDK4m RNA was detected by qRT-PCR.5 The interaction of CDK4,cyclin D1,and cyclin E1 m RNA with ILF3was detected by GST pull-down.6 The interaction of CDK4,cyclin D1,and cyclin E1 m RNA with ILF3was detected by RNA pull-down.7 The interaction between CDK4 m RNA or circACTA2 and ILF3 was detected by GST pull-down and RNA pull-down.8 VSMCs were transfected with circACTA2 accompanied by ILF3overexpression,the expression of CDK4 m RNA was detected by qRT-PCR.9 VSMCs were transfected with circACTA2 accompanied by ILF3overexpression,and VSMC senescence was examined by SA-?-gal staining.The senescent cells were observed by light microscope.Results:1 circACTA2 mediates Ang?-induced VSMC senescenceVSMCs were treated with Ang?,then SA-?-gal staining was used to detect cellular senescence.The results showed that there were more senescent cells in Ang?-treated group than in control group.And the expression of p21and p16 was upregulated.The expression of CDK4 was downregulated.These results indicated that Ang?induces VSMC senescence.Then we explored the function of circACTA2 in cellular senescence.We found that the expression of p21 and p16 was also upregulated in circACTA2-overexpressing VSMCs.These results suggested that circACTA2 also induces VSMC senescence.Then circACTA2 expression in VSMCs was knocked down,and at the same time VSMCs were treated with Ang?.SA-?-gal staining showed that circACTA2 knockdown abrogated the cellular senescence induced by Ang?treatment.The upregulation of p21 expression was reduced,while the expression of CDK4 exhibited the opposite alteration.These results suggested that circACTA2 is involved in VSMC senescence induced by Ang?treatment.2.Ang?-induced VSMC senescence is mediated by circACTA2 and ILF3 interactionWe speculated that circACTA2 could exert its function in VSMC senescence through interaction with proteins.Because ILF3 was one of the proteins that interact with circACTA2,we examined its effect on cellular senescence.When VSMCs were transfected with sh ILF3 to knock down the expression of ILF3,the proportion of cells positive for SA-?gal activity was increased.Western blot analysis showed an increased expression of p21 and p16 and reduced level of CDK4.Brd U incorporation assay indicated that cell proliferation was inhibited by ILF3 knockdown.On the contrary,the proportion of cells positive for SA-?gal activity was decreased by ILF3overexpression.The expression of p21 and p16 in ILF3-overexpressing VSMCs was downregulated,and the expression of CDK4 was increased.These results indicated that ILF3 inhibits cellular senescence.Then ILF3-overexpressing VSMCs were treated with Ang?.We found that ILF3overexpression significantly inhibited cellular senescence promoted by Ang?.These results suggested that circACTA2 mediates Ang?-induced VSMC senescence via interacting with ILF3.3 ILF3 inhibits VSMC senescence via stabilizing CDK4 m RNAThen we explored the molecular mechanism whereby ILF3 inhibits cellular senescence.Because ILF3 increased the expression level of CDK4,we speculated that CDK4 could be involved in cellular senescence induced by Ang?.The level of CDK4 m RNA was upregulated by ILF3 overexpression while downregulted by ILF3 knockdown.As a RNA-binding protein,ILF3can bind various types of RNA molecules.It was reported that ILF3 was able to bind to the 3'UTR of cyclin E1,thus stabilizing cyclin E1 m RNA.We speculated that ILF3 was also able to bind to the 3'UTR of CDK4 m RNA.After actinomycin D was used to block transcription,ILF3 knockdown obviously decreased CDK4 m RNA level,while ILF3 overexpression increased CDK4 m RNA level.We further examined whether ILF3 bound to 3'UTR of CDK4 m RNA.Firstly,Co IP was performed to confirm that enough endogenous ILF3 was pulled down by anti-ILF3 antibody.Next,in vitro RIP experiments were performed to examine m RNA binding to ILF3.qRT-PCR showed that m RNA of cyclin E1 and CDK4 was enriched in the precipitates,but cyclin D1 m RNA was hardly detected in the precipitates.The results were further confirmed by RNA pull-down.These results indicated that 3'UTR of cyclin E1 and CDK4m RNA,but not cyclin D1 was able to bind to ILF3.4 circACTA2 promotes VSMC senescence by competing with CDK4m RNA for ILF3 bindingBecause both circACTA2 and CDK4 m RNA binds to ILF3,we speculated that circACTA2 could compete with CDK4 m RNA for ILF3binding.Thus,RIP experiments were performed after Ang?treatment.The results showed that increased enrichment of circACTA2 but reduced enrichment of CDK4 m RNA could be found in the precipitations of Ang?-treated VSMCs.The enrichment of cyclin D1 m RNA did not change.Then RNA pull-down was performed.The results demonstrated that the interaction of circACTA2 with ILF3 was enhanced while the interaction of CDK4 m RNA with ILF3 was reduced in Ang?-treated VSMCs.These indicated that circACTA2 competes with CDK4 m RNA for ILF3 binding.We then examined the stability of CDK4 m RNA affected by circACTA2 and ILF3and found that circACTA2 overexpression reduced CDK4 m RNA stability while ILF3 overexpression facilitated CDK4 m RNA stability.These findings indicated that circACTA2 decreases CDK4 m RNA stability via reducing interaction of ILF3 with CDK4 m RNA.Finally,we examined VSMC senescence affected by circACTA2 and ILF3.The results showed that ILF3overexpression significantly suppressed cellular senescence promoted by circACTA2.Summary:In summury,circACTA2 competes with CDK4 m RNA for ILF3 binding,then decreases the stability of CDK4 m RNA and thus promotes VSMC senescence.Conclusion:1 Ang?induces circACTA2 formation and the latter regulates VSMC contraction by interacting with FN1.2 circACTA2 promotes the expression of FN1 via sponging miR-149,and FN1 interacts with SPTBN1 to regulate VSMC contraction.3 circACTA2 competes with CDK4 m RNA for ILF3 binding,then decreases the stability of CDK4 m RNA and thus promotes VSMC senescence. |
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