Effect And Mechanism Of SIRT1 On Apoptosis Of Human Dermal Fibroblasts Through MiR-27a-5p

IntroductionSirtuins are a class of histone deacetylase and have been associated with mammalian metabolism and longevity of lower organisms.SIRT1 can deacetylate histone and non-histone proteins in a nicotinamide adenine dinucleotide+dependent way to regulate expression of gene and activity of protein.SIRT1 can regulate numerous biological processes,such as metabolism,cellular cycle,DNA repair,cell survival and aging.A pile of evidence has demonstrated the close relationship between SIRT1 and Smad family members.In vitro and in vivo,SIRT1 has an interaction with resting and active Smad2,and SIRT1 interact Smad2 in specific location,and Smad2 acetylation can reinforce the interaction between SIRT1 and Smad2.In mice brown adipose tissues,SIRT1inhibits endoplasmic reticulum stress-induced apoptosis by reducing Smad3/ATF4.SIRT1plays an important role in preventing endothelial-mesenchymal transition through the TGF-?/Smad4 pathway to inhibit fibrosis in human endothelial cells.Smads are family proteins and critically important for cell proliferation,differentiation,and apoptosis.Smads are the primary signal sensors for TGF-?superfamily receptors.Smad2 and Smad3 are known as receptor-regulated Smads which are directly taken part in response to signals which from the TGF-?receptor.As the known common partner Smads in human,Smad4 has the partnering role with R-Smads.Phosphorylation of Smad2 and Smad3 causes itself exposing to a nuclear import sequence and promotes its interaction with Smad4.Then this Smad complex translocates into nucleus and binds their target genes with other associated proteins like TAZ.TAZ and yes-associated protein?YAP?act as transcriptional cofactors to regulate transcription factors activity in the nucleus.In human skin dermal fibroblasts,the decreased level of YAP/TAZ inhibits TGF-?/Smad3 signaling by inducing Smad7 through activation of activator protein-1.Besides,YAP and TAZ have been shown can be regulated by SIRT1.At the downstream of Hippo signaling pathway exists nuclear YAP acetylation/deacetylation cycle and YAP isregulated by SIRT1?mediated deacetylation in cancer cells.It had been reported that miR-27a function as a pivotal role in TGF-?signaling pathway in different cells.In cervical cancer,miR-27a is proved to directly target transforming TGF-?receptor I?TGF-?RI?by luciferase assays an downregulates the expression of TGF-?RI and the activity of TGF-?signaling as a tumor suppressor in vivo.In streptozotocin-induced diabetic rats,down expression of miR-27a targets PRKAA2 to activate the TGF-?1 and Smad3 thus reduces the fibrosis of detrusor.In human lymphatic endothelial cells,co-culturing with colon cancer cells induces miR-27a expressing increased,then enhances lymphatic vessels formation and migration through negatively regulating Smad4.In lung cancer,miR-27a inhibits cell cycle arrest induced by TGF-?through targeting Smad2 and Smad4 to promote cell proliferation and invasion.To sum up,SIRT1,Smads and miR-27a showed close connections directly or indirectly through TGF-?1 pathway or not.The present study was aimed to clarify whether SIRT1 regulates cell apoptosis and cell cycle through miR-27a via targeting Smads in human dermal fibroblast.It may provide a potential drug target for senescence prevention.Methods1.Cells culturePrimary fibroblasts were extracted from discarded foreskin tissues,which were obtained from department of urology surgery of the first hospital of China Medical University.The experiment was in conformity to the ethical standards of the Institutional Medical Ethics and Human Research Committee.These cells were cultured at 37°C 5%CO₂ humidified atmosphere with Dulbecco's Modified Eagle Medium?Hyclone,USA?containing 10%fetal bovine serum?Hyclone,USA?and 1%Penicillin-Streptomycin Solution?BI,Israel?,.2.Immunofluorescence stainingPrimary fibroblasts cells were fixed with 4%paraformaldehyde for 20 min,permeated with PBS containing 0.2%Triton X-100 and 1%BSA for 5 min,and blocked with 1%BSA in PBS for 1h.Then cells were incubated with anti-Vimentin?VIM?antibody?diluted1:100,BOSTER,China?or anti-alpha smooth muscle actin?a-SMA?antibody?diluted1:100,Abcam,UK?overnight at 4?,and then incubated with fluorescein-conjugated goat anti-rabbit IgG?H+L??diluted 1:50,ZSGB-BIO,China?or fluorescein-conjugated goat anti-mouse IgG?H+L??diluted 1:50,ZSGB-BIO,China?for 2 h.The nuclei were dyed with 4'-6-diamidino-2-phenylindole?DAPI?for 10 min in the dark.Then cells were observed under r a fluorescence microscopy.3.MTT assayCells were digested by trypsin at logarithmic phase,centrifuged and collected after termination,and cell suspension was made.Cell count,adjust cell suspension to desired concentration.200ul cell suspension was added to each well,and the number of cells per well was 1000-3000.The cells were adhered to the wall the next day and treated with different concentrations of drugs.5%CO2,37?for 24 h hours incubation,the effects of drugs were observed under inverted microscope.20ulMTT solution?5mg/ml,or0.5%MTT?was added into each well,and the culture continued for 4h.After 4h,the supernatant was removed,and 150ul dimethyl sulfoxide was added into each hole.The crystal was placed on a shaking table and oscillated at a low speed for 30min to make the crystal fully dissolved.The light absorption value of each hole was measured at OD490nm by enzyme-linked immunoassay.4.Transfections of SIRT1 small interfering RNAs,miRNA-27a/432 mimicSIRT1 small interfering RNAs?siRNAs?,miRNA-27a/432 mimic,miRNA-27a/432inhibitor,and normal control were all purchased from Thermo Fisher Scientific,USA.Twenty-four hours after plating,when cells were at a confluency of 70%,10 nM siRNA and 10nM mi RNA-27a/432 mimic were transfected using 5ul lipofectamine RNAiMAX?Invitrogen,USA?according to the instructions.Then qRT-PCR and western-blot were conducted to determine the efficiency of transfections.5.qRT-PCR assayTotal RNAs were extracted from fibroblasts with miRNeasy mini kit?Qiagen,Germany?.For mRNA,the cDNA synthesis was performed using GoScript?Reverse Transcription System?Promega,Germany?,then the cDNA subjected to real-time PCR with GoScript?qPCR Master Mix?Promega,Germany?following the protocol.For miRNA,the expression levels were detected using TaqMan?MicroRNA Reverse Transcription Kit?Thermo Scientific,USA?and corresponding primers?Thermo Scientific,USA?.The PCR amplification was carried out by 7900HT fast real-time PCR system?Applied Biosystems,USA?.Melt curve analysis was carried out to check the specificity of each primer-pair.2^-??^Ct was used to calculate relative gene expression.6.Western-blot analysisFibroblasts were harvested when cells were at a confluency of 80-90%,washed with PBS and lysed using RIPA?Beyotime,China?with a protease inhibitor PMSF?Beyotime,China?and a phosphatase inhibitor?Beyotime,China?.The protein content was measured with BCA protein assay kit?Beyotime,China?.The collected supernatant was mixed with5*SDS-PAGE Sample Loading Buffer?Beyotime,China?and heated at 100°C for 5 min.Denatured proteins?20ulg?were separated by 10%SDS–PAGE and transferred to 0.45ulm PVDF membrane,then blocked with TBST containing 5%skim milk or 5%BSA and incubated antibody at 4°C overnight.The membranes were washed using TBST for three times for 10 min each time and incubated with HRP-labeled goat anti-rabbit IgG?H+L??1:1000,Beyotime,China?or HRP-labeled goat anti-mouse IgG?H+L??1:1000,Beyotime,China?for 1.5h at room temperature.The blots were visualized with enhanced ECL?Beyotime,China?following exposure to X-ray films.7.Flow cytometry assayFor apoptosis analysis,cells were harvested and stained with 5ul FITC Annexin V?BD Bioscience,USA?and 10ul PI?BD Bioscience,USA?for 15 min keep in dark place according to the instructions.For cell cycle analysis,cells were harvested,washed with cold PBS three times,fixed with 1.5ml 75%ethanol at 4°C overnight.Stained with 3ul RNase A?Sigma,USA?for 30 min at 37°C and 10ul PI for 30min on ice,keeping in the dark.Flow cytometry assay were tested with BD LSRFortessa instrument?BD Bioscience,USA?,and cellular apoptosis or cellular cycle was analyzed by BD LSRFortessa software?BD Bioscience,USA?or ModFit software?Becton Dickinson,USA?respectively.8.MiRNA microarraysTotal RNAs were extracted from 3 si-SIRT1 cells and 3 normal cells for screening differentially expressed miRNA by TaqMan?Array Human MicroRNA A+B Cards ?Thermo Scientific,USA?,which enabling accurate quantitation of 754 human miRNAs.Three kinds of endogenous controls?U6,RNA44,RNA48?for data normalization and one negative control that not related to human were also included in the TaqMan?MicroRNA Array kit.Follow the manufacturer's instructions to conduct the experiment.9.Bioinformatics analysisPredicted target genes of differentially expressed miRNA were divided into three independent categories,i.e.biological process?BP?,cellular component?CC?,and molecular function?MF?by using Gene ontology?GO?enrichment analysis.Kyoto Encyclopedia of Genes and Genomes?KEGG?Pathway Database was used to assess the pathways of potential target genes.Besides,the association of potential target genes was mapped by regulatory network analysis system.GO enrichment analysis and KEGG Pathway were conducted with DAVID Bioinformatics Resources 6.8,and PPI network was conducted with STRING 10.5.Visualization results were achieved using R 3.5.0,Funrich 3.1 for window and Cytoscape 3.6.1.10.Luciferase reporter assaysThe 293T cells?Hanbio Biochemistry,Shanghai,China?were plated in 96-well plates24h prior to transfection.The pGV306 luciferase reporter plasmids containing pmiR-REPORT control vector,Luc-Smad2-wt vector or Luc-Smad2-mu vector and miR-27a-5p/miR-432-5p mimic or normal control were co-transfected into 293T cells,using0.8mg/ml transfection reagent?Hanbio Biochemistry,Shanghai,China?.The luciferase assay was carried out following the instructions of Promega Dual-Luciferase system.The assay was performed at least 3 times in independent experiments.11.Statistical analysisAll analyses were performed using Statistical Package for Social Sciences?SPSS?software?Version 20.0,Inc.,Chicago,IL,USA?.Paired groups were compared using paired t-tests.Pairwise multiple comparisons were performed by one-way ANOVA?two-sided?.A p-value<0.05 was considered to indicate statistical significance.Result1.Human skin fibroblasts were obtained and interfered with SIRT1 siRNAs.Fibroblasts were obtained from 3 subjects at the age of 17,24 and 25 years old,respectively.The immunofluorescence staining showed that the cultured cells were vimentin?+?DAPI?+??-SMA?-?,which demonstrated that the cells were human skin fibroblasts.Two different SIRT1 siRNAs?SIRT1 siRNA 1,SIRT1 siRNA 2?were respectively transfected into cells,and the transfected efficiency was tested by qPCR and western blot.According to the results,SIRT1 siRNA 2 and 72 h were the optimal SIRT1 siRNA and experimental time to be used in the following experiments.2.Knockdown of SIRT1 promotes apoptosis,leading to a decrease in G1 cells and an increase in G2 cellsThe apoptosis of fibroblasts was significantly increased in si-SIRT1 group compared to the control?p<0.05?,which indicated the knockdown of SIRT1 could increase the apoptosis of fibroblasts.The G1 phase fraction was reduced and the G2 phase fraction was increased in si-SIRT1 group?p<0.05?,but the S phase fraction remained unchanged.3.MiR-27a-5p and mi R-432-5p were screened by miRNA microarray and bioinformatics analysisBy TaqMan?Array Human MicroRNA A+B Cards,176 miRNAs were tested in allthe 3 paired-samples.In these miRNAs,15 miRNAs were shown to be differentially expressed between si-SIRT1 cells and normal cells?log2FC<-1 or>1 and p<0.05?.The heatmap and volcano plot displayed that the expressions of 14 miRNAs were decreased and the expression of 1 miRNA?miR-27a-5p?was increased.Seven miRNAs?miR-362-5p,miR-149-5p,miR-365a-3p,mi R-432-5p,miR-539-5p,miR-204-5p,miR-370-3p?which declined 3 times comparing to controls and mi R-27a-5p were chose to predict target genes and carry out bioinformatics analysis.Based on the results,mi R-27a-5p and miR-432-5p we screened for further experiments.The bioinformatics analysis results of predicted target genes of miR-27a-5p/miR-432-5p were showed,respectively.The GO enrichment analysis showed that the predicted target genes of miR-27a-5p/miR-432-5p mostly enriched in nucleus acted as molecular binding and regulated transcriptions.The KEGG pathway analysis revealed these genes mainly affected pathways in cancer.The protein-protein interaction network presents Smad2 to be the hub gene among these predict target genes.Among those predict target genes of mi R-27a-5p as well as miR-432-5p,Smad2 was found to be involved in many different biological processes with other genes indicating an important role.4.Silencing SIRT1 and overexpression of miR-27a-5p inhibited the expression of Smad2 and related proteins like Smad3,Smad4,TAZ and COL1After knockdown of the SIRT1,mi R-27a-5p increased,miR-432-5p decreased,and Smad2 decreased,verifying by qRT-PCR assay.The combined positions of mi R-27a-5p with Smad2 and miR-432-5p with Smad2 were predicted by TargetScan 7.2.The luciferase reporter assays showed that the Firefly-Luc activities of pGV306-Smad2 3'-UTR reporter was obviously inhibited by miR-27a-5p mimic?p<0.05?,but not by miR-432-5p mimic?p>0.05?.According to the results,we suspect that knockdown of SIRT1 could decrease Smad2through the increased mi R-27a-5p.So,we tested the protein levels of Smad2 in different conditions by western blot.Resveratrol?RSV??10uM,Sigma,USA?was used as the SIRT1 activator and SRT 1720?2uM,Selleck,China?was a selective activator of SIRT1.SIRT1 was significantly reduced by SIRT1 siRNA,in contrast,was raised by SRT 1720 or RSV.Smad2 was obviously decreased by SIRT1 siRNA and miR-27a-5p mimic?p<0.05?,and unchanged by SRT 1720 or RSV.Western-blot results showed that the expressions of Smad3,Smad4,TAZ and COL1proteins were declined in si-SIRT1 group or miR-27a-5p mimic group,compared with control group.No significant difference was seen for YAP or CTGF.5.Silencing SIRT1 and overexpression of miR-27a-5p promoted apoptosisThe western-blot showed that BAX was increased in si-SIRT1 group and mi R-27a-5p mimic group,and decreased in RSV group and SRT 1720 group?p<0.05?.In contrast,BCL2 was decreased in miR-27a-5p mimic group,and increased in RSV group and in SRT1720 group?p<0.05?.The flow-cytometry assay results present that cell apoptosis were significantly increased in si-SIRT1 group or miR-27a-5p mimic group?p<0.05?and obviously decreased in RSV group or in SRT 1720 group?p<0.05?,comparing to the control.The flow-cytometry assay for cell cycle showed no disciplinary results.6.Resveratrol can inhibit UVA-induced expressions of p38,JNK,MMP1 and MMP3At 6h,12h and 48h,mRNA expression of MMP1 and MMP3 increased in the UVA group.After resveratrol administration,the expression of MMP1 and MMP3 was inhibited by UVA.At 6h and 12h,the mRNA expression of c-fos was the same as that of MMP1and MMP3,that is,it was elevated in the UVA group,and the expression of UVA was inhibited after administration of resveratrol.At 12 h,the expression of JNK also changed the same.At the protein level,it was found that MMP1,MMP3,phosphorylated p38 and phosphorylated JNK were elevated in the UVA irradiation group,and the degree of elevation was inhibited after treatment with resveratrol,which was lower than that in the UVA group.No regular changes were found at other points in time.7.After SIRT1 was silenced,there was no significant change in UVA-induced expression of p38/JNK pathway componentsWestern results showed that resveratrol inhibited the elevation of MMP1 and MMP3induced by UVA at 24h.After SIRT1 silencing,the effect still existed,suggesting that the inhibition of resveratrol was not significantly related to SIRT1.8.SIRT1 can regulate UVA-induced changes in MMP1/COL1/BCL2 expression through miR-27a-5pWestern results showed that MMP1 increased and COL1/BCL2 decreased after UVA irradiation.In UVA+Sirtinol group and UVA+miR-27a-5p mimic group,the expression of MMP1 increased and the expression of COL1 and BCL2 decreased,and the changes were more obvious than that in UVA group.On the basis of these two groups,RSV were given,and it was found that the changes of MMP1,COL1 and BCL2 were inhibited.Conclusion1.SIRT1 inhibits the apoptosis of human dermal fibroblasts,reduces the G1 phase distribution,increases the G2 phase distribution,and regulates the expression of miR-27a-5p and miR-432-5p.2.SIRT1 inhibits the apoptosis of human dermal fibroblasts by regulating Smad2through miR-27a-5p.3.SIRT1 can regulate UVA-induced changes in MMP1/COL1/BCL2 expression through mir-27a-5p.4.Resveratrol can inhibit UVA-induced expressions of p38,JNK,MMP1 and MMP3,but not through SIRT1.