Effects Of Hsa-let-7b/MMP1 Axis On The Proliferation And Differentiation Of Human Stem Cells From Apical Papilla

MicroRNAs(miRNAs)are involved in the modulation of almost all cellular processes.Members of the let-7 family have been reported to play a crucial role in the proliferation and differentiation of mesenchymal stem cells(MSCs).Matrix metalloproteinases(MMPs)are involved in many developmental events by cleaving most of the extracellular matrix(ECM)components.However,the effects of hsa-let-7b/MMP1 axis on the proliferation and differentiation of stem cells from apical papilla(SCAPs)and the potential mechanism remain unclear.Part I.Effects of hsa-let-7b on the proliferation of SCAPsObjective: To isolate and incubate SCAPs and explore the effects of hsa-let-7b on the proliferation capacity of SCAPs.Methods: Non-carious third molars with open apex were collected in Oral Surgery Department of Jiangsu Provincial Stomatological Hospital after the informed consent was obtained.SCAPs were obtained by enzyme-digestion method.Cell morphology was observed under the inverted microscope.The groups of low expression(hsa-let-7b-inhibition and Con-inhibition)and overexpression(hsa-let-7b-over and Con-over)of hsa-let-7b were transfected into SCAPs,respectively.The gene expression level of hsa-let-7b was detected by real time quantitative reverse transcriptase polymerase chain reaction(real-time RT-PCR).The proliferation ability of transfected SCAPs was measured by cell counting kit-8(CCK8)and flow cytometry(FCM).Results: The isolated SCAPs presented the typical fibroblast-or spindle-like morphology.When SCAPs were transfected by low/over expression of hsa-let-7b(MOI=5)for 72 h,they grew well and had a high transfection efficiency by inversion fluorescence microscope.CCK8 and FCM showed hsa-let-7b had no effect on the proliferation of SCAPs(P>0.05).Conclusions: The model of hsa-let-7b low/over-expression SCAPs were successfully established.Hsa-let-7b had no effect on the proliferation of SCAPs.Part II.Effects of hsa-let-7b on the commited differentiation of SCAPsObjective: To explore the effects of hsa-let-7b on the odonto/osteogenic differentiation ability of SCAPs.Methods: To detect the expression change of hsa-let-7b when SCAPs were under osteogenic induction by using real-time RT-PCR.The groups of low expression(hsa-let-7b-inhibition and Con-inhibition)and overexpression(hsa-let-7b-over and Con-over)of hsa-let-7b were transfected into SCAPs,respectively.Alkaline phosphatase(ALP)activity,alizarin red staining,and quantitative calcium measurement were investigated.Furthermore,the expression of odonto/osteogenic markers(DSPP,ALP/ALP,RUNX2/RUNX2,OSX/OSX,OPN/OPN,OCN/OCN)was detected by real time RT-PCR and Western blot.Results: The hsa-let-7b expression was up-regulated during the osteogenic differentiation process of SCAPs(P<0.05).Repression of hsa-let-7b significantly increased the ALP activity and calcified nodules formation of SCAPs(P<0.05).Meanwhile,low expression of hsa-let-7b enhanced the expression of odonto/osteogenic markers(DSPP,ALP/ALP,RUNX2/RUNX2,OSX/OSX OPN/OPN,OCN/OCN)at both mRNA and protein levels(P<0.01 or P<0.05).However,the results in the overexpression of hsa-let-7b group were antipodal(P<0.01 or P<0.05).Conclusions: Overexpression of hsa-let-7b significantly reduced the osteo/odontogenic differentiation capacity of SCAPs,while repression of hsa-let-7b increased the osteo/odontogenic differentiation capacity of SCAPs,suggesting that hsa-let-7b could regulate the committed differentiation of SCAPs.Part III.Hsa-let-7b regulated the commited differentiation of SCAPs by targeting MMP1Objective: To investigate the interaction between hsa-let-7b and MMP1 as well as the mechanism of hsa-let-7b/MMP1 axis on the odonto/osteogenic differentiation of SCAPs.Methods: To investigate the hsa-let-7b target region in MMP1 mRNA,a dual luciferase reporter assay was conducted.Real time RT-PCR and Western blot were used to detect the expression of MMP1 after transfection of hsa-let-7b lentiviruses into SCAPs.After co-transfection of hsa-let-7b and MMP1,ALP activity,alizarin red staining,and quantitative calcium measurement were investigated.Furthermore,the expression of odonto/osteogenic proteins(DSPP,RUNX2,OCN)was detected by Western blot.Results: Luciferase activity was observed to be significantly down-regulated in MMP1-WT+ hsa-let-7b mimic group as compared with that in MMP1-MT+ hsa-let-7b mimic group(P<0.01).Real time RT-PCR and Western blot indicated that the repression of hsa-let-7b significantly increased the expression of MMP1 at day 3 and 7,while the overexpression of hsa-let-7b dramatically suppressed the expression level of MMP1 at day 3 and 7(P<0.01).ALP activity,mineralized nodules,and odonto/osteogenic proteins(DSPP,RUNX2,OCN)were up-regulated in hsa-let-7b-inhibition group as compared with those in Con-inhibition group but down-regulated in hsa-let-7b-inhibition+si MMP1 group as compared with those in hsa-let-7b inhibition+si MMP1-NC group(P<0.01 or P<0.05)..Conclusions: MMP1 is believed to be a direct target of hsa-let-7b.MMP1 partially reversed the effects of hsa-let-7b on SCAPs.