MiR-206 Influences Infantile Hemangioma Stem Cells Through Targeting To TGF?/Smad Signaling Pathway

BackgroundInfantile hemangioma is the most common tumor in infants that is characterised by postnatal appearance.It enlarges fast in the first year after birth,following slow involution.The course of IH can last 10-12 years without therapy.Although IH can spontaneously involute,the residual lesion often impairs appearance,functions and the health of psychology of patients,thus surgery is required for excising the focal lesions in a part of patients.There are three phases in the development of IH: proliferation phase,involuting phase and involuted phase.Proliferation phase is restricted in the first year after born,in which the pathological structural features includes a mass of endothelial cells without vascular structure.The involuting phase start at the second year,vascular structure appears during this period.In involuted phase,vascular structure is replaced by adipose tissue accompanied by little capillary.Notwithstanding the spontaneous involution trend,the timing and factors involve in involution is not clarified.There are three signaling pathways plays critical roles in angiogenesis,they are Notch VEGF and TGF? signaling pathway.We found the expression of Smad4 that belong to TGF? signaling pathway is variant in different phases of IH.The expression of Smad4 is higher in involuting phase compared to proliferation phase.We also found the miR-206 is higher in proliferation phase compared to involuting phase.The expression trends of Smad4 and miR-206 in IH is opposite.Thus,this study is try to demonstrate the functions of miR-206 and TGF?/Smads signaling pathway in IH through extracting hemangioma-derived stem cell(HemSCs)and establishing HemSCs cell lines with different expression levels of miR-206 to explore the roles of miR-206 and TGF?/Smads signaling pathway and the interact mechanism between miR-206 and TGF?/Smads signaling pathway in HemSCs,therefore to understand the molecular mechanism of the switch from proliferation phase into involuting phase,finally to provide new ideas for the therapy of IH.ObjectsPart 1: Hemangioma-derived stem cells(HemSCs)are extracted to induce multiple differentiation.The biomarker of HemSCs is identified by immunofluorescence and Flow cytometry.Part 2: MiR-206 is overexpressed or interfered via lentivirus in HemSCs.Taqman probe is used to detect the levels of miR-206 in different groups of HemSCs to make assurance the cell lines are successfully established.Part 3: The influence of miR-206 to HemSCs cell biology which include proliferation,migration,invasion,cell cycle and cell apoptosis is invested.The effects of TGF?1 to cell biology of HemSCs with different levels of miR-206 are also explored.Part 4: Molecular mechanisms of miR-206 and TGF?1 impact HemSCs is researched.MethodPart 1:(1)Adherent culture of IH tissue is used to amply primary cell,then magnetic-activated cell sorting(MACS)is applied to screen HemSCs for monoclonal culture.(2)Cell morphology is studied(3)Cell growth curve is detected by Cell count kit.(4)Cell Tubule Formation Assay,osteogenesis,adipogenesis and chondrogenesis essay are adopt to definite the multiple differentiation potential.(5)HemSCs markers GLUT-1,CD31 and CD133 are detected by immunofluorescence and flow cytometry to identify the cell phonetype.Part2:(1)Pre-miR-206 and TudRNA(hsa-mir-206)sequences are add to plasmids by double enzyme digestion,and the plasmids' sequences are verified by base sequencing.(2)The plasmids are packed into lentivirus.(3)The lentiviruses carrying variant plasmids are transfected into HemSCs and puromycin is used to filtrate cells which are successfully transfected.(4)Green fluorescent protein(GFP)expressions in different groups of HemSCs is observed by fluorescence microscope.(5)RNA is abstracted by Trizol for inverse transcription by stem-loop method,Taqman probe is applied to detect the expression of miR-206 in groups of HemSCs.Part 3: Groups of HemSCs are treated by phosphate buffered solution(PBS)or TGF?1,then the cell biology is monitored by methods as follows:(1)Annexin V and flow cytometry is adopted together to detect the cell apoptosis(2)Cell count kit-8 is used to test the cells proliferation.(3)The transwell assay is applied to detect migration and invasion ability of HemSCs.(4)Cell cycle is evaluated by PI staining and flow cytometry assay.Part 4: 1)The mRNA expression levels of TGF?/Smads signaling pathway,VEGF/VEGFR2 and endothlial-mesenchymal transition(EMT)related proteins in groups of HemSCs treated by PBS/TGF?1 is detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)(2)The protein expression levels of TGF?/Smads signaling pathway,VEGF/VEGFR2 and EMT related proteins in groups of HemSCs treated by PBS/TGF?1 is detected by Western-Blot.(3)The binding of miR-206 with Smad4 mRNA 3' Untranslated region(3'UTR)is detected by Dual Luciferase Reporter.(4)The impact of miR-206 to course of IH is confirmed by animal model.ResultsPart 1: Cells distracted from tissues by tissue adherent culture,MACS and monoclonal culture possess the capability of differentiated to vessel endotheliums,osseous cells and adipocyte.The cells proliferate rapidly.GLUT1 and CD133 are expressed positively,but CD31 is negatively expressed.It's confirmed that the cells are HemSCs.Part 2 The plasmid established with pre-miR-206 and is confirmed by base sequencing.HemSCs transfected with lentiviruses express GFP.The HemSCs overexpress miR-206 contain 31560±734.4 fold miR-206 compared to the normal control group by Taqman probe detection,and the HemSCs interfered miR-206 express 0.05±0.01 fold miR-206 compared to the normal control group.Part 3 To HemSCs,miR-206 promotes proliferation,inhibitors apoptosis and migration,it also reduces the proportion of G1 stage cells and enhances S stage cells.Whereas TGF?1 inhibitors proliferation,migration and promotes late stage apoptosis of HemSCs.Part 4 MiR-206 targets to Smad2/3/4 protein in TGF? signaling pathway,Smad4 is the mostly influenced protein,TGF? signaling pathway and miR-206 suppress each other in HemSCs,activation of TGF? signaling pathway downregulates the expression of miR-206,miR-206 inhibitors expression of the crucial proteins Smad2/3/4 in TGF? signaling pathway in turn.Activation of TGF? signaling pathway promotes endothlial-mesenchymal transition(EMT),miR-206 reversly restraint EMT,that manifested by upregulating E-Cadherin protein and downregulating,?-catenin.Dual luciferase reporter proves miR-206 binds to SMAD4 mRNA 3'UTR and inhibitors mRNA translation.Animal model confirmed that miR-206 facilitates HemSCs differentiation to adipose tissue while prevents angiopoiesis.