LncRNA HOTTIP Regulates Osteogenic Differentiation And Angiogenesis Of BMSCs By Promoting DLX2

Objective:Bone increment surgery has become the main treatment to jaw defects in today’s clinical oral and maxillofacial repair.However,there are still many disadvantages and shortcomings urgent to be optimized,such as complexity of the operation,the secondary injury to the sampling site,inflammation,rejection reaction et al.Bone marrow mesenchymal stem cells(BMSCs)are a sort of stem cells.BMSCs might promote bone defects and accelerate bone healing by differentiating into osteoblasts,and promoting angiogenesis,which may solve clinical difficulties including the failure of implanted tissue to promote bone regeneration after bone incremental surgery due to ischemia.Distal homeobox free 2 gene(Dlx2)is a member of Dlx homeobox gene family,which plays an important regulatory role in biological development.Once was stimulated by multiple signals,Dlx2 has been proved to Promote osteogenic differentiation and accelerate bone formation in vitro and in vivo.However,the specific upstream and downstream molecular mechanism of Dlx2 regulation on osteogenic process remains unclear and needs further research.After the success of human BMSCs isolation and identification,our project focused on DLX2 and its regulatory effects on osteogenic differentiation and angiogenesis of hBMSCs and the underlying molecular mechanism.Methods:1.Human bone marrow-derived mesenchymal stem cells(hBMSCs)were isolated,and the surface stem cell markers(CD29,CD34,CD44,CD45,CD90)were detected by flow cytometry.BMSCs were cultured and induced by s Pecific commercial culture medium of differentiation induction kits.qRT-PCR was used to detected osteogenic markers(OCN,BSP,RUNX2,ALP)expression,ALPenzyme activity assay were conducted,and alizarin red staining were used to identify calcium nodule formation,oil red O staining was used to observe li Pid dro Plet formation,alcian blue staining was used to observe cartilage glycogen formation,in order to identify multiple differentiation potential of BMSCs.At the same time,BMSCs conditioned medium was collected and co-cultured with vascular endothelial cells(HUVECs).The regulatory effect of BMSCs on angiogenesis was observed by tube forming experiment and CD31 immunofluorescence staining experiment.2.Firstly,BMSCs were cultured in osteogenic inducing medium,and the expression of DLX2 was detected by qRT-PCR and WB.Then BMSCs cells were transfected with DLX2 knockdown plasmid(shDLX2).The effects of DLX2 knockdown on osteogenic differentiation of BMSCs were detected by qRT-PCR,WB,ALPenzyme activity and alizarin red staining;qRT-PCR,CHIP and WB were further used to verify the effect of DLX2 on expression and activation of Wnt1 and its downstream Wnt1/GSK3β/β-catenin signaling pathway.Finally,DLX2 overexpression plasmid and Wnt / β-catenin pathway inhibitor FH535 were used to establish rescue experiments,followed by qRT-PCR,WB,ALPenzyme activity assay and alizarin red staining,to completely verify whether DLX2 regulates BMSCs osteogenic differentiation via Wnt/β-Catenin pathway.3.Sh DLX2 and Wnt/β-catenin pathway agonist HLY78 were used to treat BMSCs cells.On the one hand,treated cells were tested by qRT-PCR,WB,ELISA to observe the regulation of DLX2 and Wnt/β-catenin pathway on the expression and secretion of angiogenic factors(VEGFA,bFGF,TGFβ1,MMP2,MMP9)in BMSCs;On the other hand,the conditioned medium of BMSCs in each group was collected to treat HUVECs cells,and the tube formation experiment was carried out,and the expression of angiogenesis related markers(CD31,CDH5,vWF,VEGFR2)in HUVECs cells were detected by WB,to verify whether DLX2 regulates BMSCs angiogenesis via Wnt/β-catenin pathway.4.Osteogenic differentiation was induced again,and the expression of lncRNA HOTTIP was observed by qRT-PCR.Sh HOTTIP knockdown was used to reduce the expression level of lncRNA HOTTIP in BMSCs cells.qRT-PCR,WB,ALPenzyme activity detection,alizarin red staining,ELISA and tube formation experiment were used to observe the effect of lncRNA HOTTIP knockdown on DLX2 expression and DLX2-mediated osteogenic differentiation and angiogenesis of BMSCs.RIP,RNA pulldown and RNA degradation experiments were further used to observe the recruitment of lncRNA HOTTIP on TAF15,and the effects of their binding on DLX2.Finally,the rescue experiments were established by using DLX2 overexpression Plasmid and shHOTTIP.qRT-PCR,WB,alizarin red staining,ELISA and tube formation experiment were used to verify whether lncRNA HOTTIP could regulate osteogenic differentiation and angiogenesis stimulation of BMSCs via DLX2-Wnt/β-catenin pathway.Results:1.According to the latest definition of mesenchymal stem cells,we have completed the isolation and identification of BMSCs.By flow cytometry detection,isolated BMSCs highly ex Pressed CD29,CD44 and CD90,while low expressing CD34 and CD45.Alizarin red,oil red O and alcian blue staining experiments showed that the obtained BMSCs could differentiate into osteoblasts,adi[ocytes and chondrocytes in vitro.The expression of OCN,BSP,RUNX2 and ALPand the activity of ALPenzyme in BMSCs cells increased on day 0/ 1/ 3/ 7/ 14 after osteogenesis induction in a time manner.Finally,the results of tube formation experiment and CD31 fluorescence staining showed that BMSCs could promote the tube formation ability of HUVECs cells.2.qRT-PCR and WB results showed that the expression of DLX2 increased with the extension of osteogenesis induction time.The results of qRT-PCR,WB and ALPenzyme activity detection and alizarin red staining showed that the down-regulation of DLX2 could inhibit the expression of OCN,BSP,RUNX2 and ALP,ALPenzyme activity and calcium nodule formation in BMSCs cells.qRT-PCR results showed that DLX2 silencing could inhibit the expression of Wnt1,and CHIP experiments showed that DLX2 could combine with Wnt1.DLX2 could promote the expression of Wnt1 expression,phosphorylation levels of GSK3β,and the expression as well as nuclear translocation of β-catenin.Whereas,Wnt1 sh RNA can reverse these effects.The results of rescue experiments further confirmed that Wnt/β-catenin pathway inhibitor(FH535)could effectively deminish the promotion effects of DLX2 on Wnt/β-catenin signaling pathway activation and osteogenic differentiation of BMSCs.3.qRT-PCR and WB results indicated that the inhibitory effects of shDLX2 on DLX2 itself as well as β-catenin expression could be reversed by Wnt/β-catenin pathway agonist(HLY78).The results of WB,ELISA and tube formation experiments showed that shDLX2 could inhibit the expression and secretion of VEGFA,bFGF,TGFβ1,MMP2,MMP9 in BMSCs.The conditioned medium of treated BMSCs could further inhibit the expression of CD31,CDH5,vWF and VEGFR2 in HUVECs cells,and the tube formation ability of HUVECs cells.These effects could be courteracted by the addition of HLY78.4.The results of qRT-PCR and WB showed that the expression of lncRNA HOTTIP increased with the extension of osteogenesis induction time.LncRNA HOTTIP silencing could inhibit the increase of DLX2,OCN,BSP,RUNX2 and ALPexpression,ALPenzyme activity,and calcium nodule formation in BMSCs cells induced by osteogenesis induction;and the expression and secretion of VEGFA,bFGF,TGFβ1 MMP2 and MMP9 in BMSCs,and finally the expression of CD31,CDH5,vWF and VEGFR2 as well as tube formation ability in HUVECs cells RIP and RNA pulldown experiments confirmed the recruitment effect of lncRNA HOTTIP on TAF15 and their binding to DLX2.The results of RNA stability experiment showed that the combination of lncRNA HOTTIP / TAF15 with DLX2 could improve the RNA stability of DLX2.Finally,the rescue experiment established by shHOTTIP and DLX2 overexpression plasmid showed that DLX2 overexpression could reverse the inhibitory effect of shHOTTIP on the expression of DLX2,OCN,BSP,RUNX2 and ALP,as well as ALPenzyme activity and calcium nodule formation in BMSCs cells;Consistently,the expression and secretion of VEGFA,bFGF,TGFβ1,MMP2 and MMP9 in BMSCs inhibited by shHOTTIP also were rescued by DLX2 overexpression,followed by the recovery of CD31,CDH5,vWF and VEGFR2 expression and the tube formation ability in HUVECs cells.Conclusions:1.hBMSCs were successfully isolated,and their multidirectional differentiation potentials,especially the ability of osteogenic differentiation and angiogenesis promotion,were verified.2.In the process of osteogenic induction,DLX2 was highly expressed in BMSCs and promoted osteogenic differentiation by activating Wnt/β-catenin signaling pathway.3.The increased expression of DLX2 and Wnt/ β-catenin signaling pathway in BMSCs could also promote the activation of vascular endothelial cells and angiogenesis process by secreting angiogenic factor into the microenvironment.4.Upstream lncRNA HOTTIP could bind and improve the stability of DLX2 mRNA and activate its transcriptional expression by recruiting transcription factor TAF15.Finally,lncRNA HOTTIP could promote the osteogenic differentiation and angiogenesis stimulation of BMSCs,through stimulating DLX2 expression.