Role Of Notch Signaling During The Process Of BMP9-induced Osteogenetic Differention Of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are adult stem cells, with self-renewaland multiple differentiation potential, has become a research hotspotin bone disease therapy and tissue engineering. Bone morphogeneticprotein9is currently considered has the best capacity of Osteoinductionaamong BMPs family, but it is still unclear which way it plays.The Notchpathway is a conserved signaling pathways in organisms and plays animportant role during the organism embryonic and postnatal development,regulation and control of proliferation and differentiation of variouscell.Meanwhile，There are also a number of studies indicate that Notchsignaling pathway is involved in mesenchymal stem osteogenicdifferentiation and plays an indispensable role in the development ofskeletal system. This study aims to clarify the Notch1signal onBMP9-induced mesenchymal stem cells into osteoblasts.(Part1);Theinfluence of osteogenic differentiation when specifically block the entireclassic Notch signal by Construction adenovirus target Rbpj,the keytranscription factor of Notch downstream)(Part2);Preliminaryinvestigation of Notch-related micro RNA in BMP9-induced osteoblastdifferentiation of MSCs (Part3). PART1ROLE OF NOTCH1SIGNALING DURING THE PROCESS OFBMP9-INDUCED OSTEOGENETIC DIFFERENTION OFMESENCHYMAL STEM CELLSObjective To Research on the role of Notch1for BMP9-induced bonedifferentiation.Methods treatment of MEF with dominantnegative mutant adenovirus vector (AdRdnNotch1),analysis of earlyosteogenic index ALP (alkaline phosphatase, ALP) with cell chemicalstaining; late osteogenic index OPN and OCN with RT-PCR;p-Smad1/5/8protein level and Smad binding element(SBE) activity,marker for BMP classical pathway activation,with Western blot andluciferase detection; analysis of its effects on cell cycle and apoptosis withCrystal violet staining and flow cytometry; the expression of downstreamtarget genes of BMP9with RT-PCR. Results The3,5,7-day ALP activityof AdRdnNotch1and BMP9co-treated group were decreased(P <0.05),compared with control group and present a dose-dependent manner,so asthe OPN,OCN,the level of p-Smad1/5/8,the activity Luciferase activity ofSBE(P <0.05),but not the level of Smad1/5/8.Crystal violet and flowcytometric presented that AdRdnNotch1can inhibit cell proliferationinduced by BMP9, so as the targets blow BMP9,but notapoptosis.Conclusion The Osteogenic ability of BMP9decreasedsignificantly when AdRdnNotch1competitively inhibits Notch1signaling.The reason for this phenomenon might be AdRdnNotch1Impaired theability of BMP9on cell proliferation and the activation of BMPsignaling,which may futher suppressed osteoblast differentiation.In aword,Notch1may plays an important role in BMP9-induced osteogenicprocess of MSCs. PART2CONSTRUCTION AND FUNCTIONAL IDENTIFICATION OFSMALL INTERFERING RNAS AGAINST MOUSE NUCLEARTRANSCRIPTION FACTOR RBPJ BY RECOMBINANTADENOVIOUSObjective To construct recombinant adenovirus against mouse nucleartranscription factor Rbpj, downstream of Notch, with the purpose toprovide a powerful molecular tool for exploring the molecular mechanismof Notch signaling during osteogenesis.Methods Three pairs ofdouble-stranded DNA sequence targeted mouse Rbpj were designed andsubcloned to pSES-HUS vector to obtain pSES-HUS-siRbpj used AdEasysystem, then reorganized with pAdEasy-1plasmid in E.coli BJ5183.pAd-siRbpj was transfected into HEK293to package recombinantadenovirus AdsiRbpj and screened charge by RT-PCR、 Western bloting、Cytochemistry and RT-qPCR.Results: Screening out AdsiRbpj-3, but notvery satisfactory.Conclusion: AdsiRbpj construction is not ideal.PART3EXPLORE THE ROLE OF NOTCH-RELATED microRNA INBMP9-INDUCED OSTEOGENIC DIFFERENTION OF MSCsObjective To explore the role of Notch-related micro RNA inBMP9-induced osteogenic differentiation of MSCs.Methods Use softwareto predict Notch-related miRNA. Analysis of the expression of thoseNotch-related miRNA in C3H10T1/2cells with BMP9treated.ResultNotch-related micro RNA Involved mir133a、 mir133b and mir21.Meanwhile, BMP9can increase the expression of mir133a and mir133b, downregulate the expression of mir21.In the above-describedmicroRNA, mir133is likely regulation and control the Notch signaling pathway. Analysis of the targets of mir133, fortunately, MAML1,MAML3,the co-activator inhibitor of Notch signaling pathway, CTBP2,the co-inhibitor of Notch signaling pathway and RBPJ, the keytranscription factor of Notch all Shortlisted. conclusion mir133is likelyregulated the Notch signaling pathway, especially mir133a.