Genomewide Identification And Characterization Of Functional Noncoding Rnas In Regulating Osteogenic Differentiation And Bone Tumor Development

Background/Aims:The disruption of osteogenic differentiation and proliferation balance may result in bone tumorigenesis.However the mechanisms have not been fully understood.With the completion of human genome project,the important regulatory functions of non-coding RNAs(nc RNAs)have been gradually recognized.However,the identification and functional annotation of nc RNAs remains a daunting challenge.We have recently developed a novel short RNA library,which expresses completely-randomized 19-mer(i.e.,n19 RNA library).The RNA library was used to identify potential regulatory non-coding RNAs that control osteogenic differentiation and possible bone tumor development.This knowledge should allow us to further understand the mechanism underlying osteogenic differentiation and bone tumor development,which will ultimately aid us to effectively treat primary bone tumors.Methods:(1)Retrovirus transfection system was used to introduce n19 RNA library into the i MEF cells,resulting i MEFn19.Based on BMP9-induced osteogenic differentiation model of MSCs,Ad BMP9 was used to induce osteogenic differentiation of the i MEFn19 cells in vitro.After several passages of in vitro selections,the survived i MEFn19 cells,which were under BMP9 stimulation,were subcutaneously injected to nude mice.After 3-4 weeks post implantation,the nude mice were sacrificed,and the subcutaneous masses that contained undifferentiated cells were recovered,which was named as i MEF osteogenic resistance(OR)1A cells(i.e.,i MEFOR1A).The i MEFOR1 A cells were further expanded in vitro and infected with Ad BMP9 for several passages.The survived i MEFOR1 A cells were subcutaneously injected to nude mice,and the masses containing undifferentiated cells were recovered,yielding i MEFOR1 B line.We conducted four rounds of such in vitro and in vivo selections,and obtained four cell lines,called i MEFOR1 A,i MEFOR1 B,i MEFOR1 C and i MEFOR1 D,while the unselected i MEFn19 cells were used as a control.(2)i MEFOR cell lines and i MEFn19 cells were cultured in vitro,Ad BMP9 was used to induce the cell lines for osteogenic differentiation,ALP staining and ALP reading were performed to detect the ALP activities while Ad GFP was used as control.Alizarin Red S staining was used to determinate calcium deposit upon Ad BMP9 or Ad GFP treatment.Osteogenic markers were detected by Tq PCR.Ectopic bone formation in nude mice was used to determinate the osteogenic differentiation abilities in vivo.(3)Genomic DNA was isolated from i MEFOR and i MEFn19 cell lines.The genomic DNA fragments containing the 19-mer sequences were amplified by PCR with distinct Illumina NGS indices.Next generation sequencing(NGS)was carried out to identify the 19 mer sequences.The NGS data were analysed and the highly enriched fragments were tabulated and named as Disruptors of Osteogenesis induced by BMP9(DON).Piggy Bac transposon system was used to express the single 19 mer DON sequences in i MEFs,resulting i MEFDON2,i MEFDON3,i MEFDON5,i MEFDON6,i MEFDON7 cell lines.ALP staining and ALP reading were carried out to detect the ALP activities in these cell lines stimulated with BMP9.Alizarin Red S staining was used to detect the calcium deposit in these cell lines upon BMP9 stimulation.Osteogenic markers were analyzed by Tq PCR.Ectopic bone formation in nude mice was used to assess the osteogenic differentiation abilities of these cell lines in vivo.(4)Total RNA was isolated from i MEFn19 and i MEFOR1 B cell lines and subjected to RNA-seq to identify the transcriptomic differences genomewide.Tq PCR was used to further verify the changes in expression levels in i MEFOR cell lines.RNA pull-down was used to determine if DON3 directly binds to the identified lnc RNAs.(5)According to the RNA-seq results,lnc RNA H19 was selected for further investigation.The expression levels of lnc RNA H19 and osteogenic differentiation markers upon BMP9 stimulation in i MEFn19 and i MEFOR1 B cell lines were detected by Tq PCR.The effect of overexpression of lnc RNA H19 on BMP9-induced osteogenic differentiation in i MEFOR1 B was assessed both in vivo and in vitro.The effect of exogenous activation of Notch signaling pathway on BMP9-induced osteogenic differentiation in i MEFOR1 B cells were also be analyzed.(6)Lnc RNA H19 and osteogenic differentiation markers upon BMP9 stimulation in i MEFs were measured by Tq PCR.The effects of overexpression or silencing lnc RNA H19 in BMP9 induced early and late osteogenic differentiation markers were also determined in vitro.The effects of overexpression or silencing lnc RNA H19 in BMP9-induced ectopic bone formation were analyzed.Stable overexpression and silencing lnc RNA H19 cell lines were established using retroviral system.Ad NICD1 was used to activate Notch signaling pathway.The effects of Notch signaling on BMP9-induced osteogenic differentiation in i MEF-Sim H19 and i MEF-H19 cells were analyzed both in vitro and in vivo.The activation of Notch signaling in i MEF-Sim H19 and i MEF-H19 cell lines was identified by Western blot and immunofluorescence staining.The expression levels of Notch ligands and receptors in i MEF-Sim H19 and i MEF-H19 cell lines were determined by Tq PCR.The expression of Notch signaling associated micro-RNAs in BMP9 stimulated in i MEFs,or i MEF-Sim H19 and i MEF-H19 cell lineswere assessed by Tq PCR.(7)The expression of Notch ligands and receptors in BMP9-stimulated MSCs were assessed by Tq PCR.Ad NICD1 was used to activate Notch signaling pathway,the effect of Notch signal pathway activation on BMP9 induced osteogenic differentiation and angiogenic signaling in MSCs was performed.Addn Notch1 was used to inhibit Notch signaling pathway,and the effect of downregulated Notch signal pathway on BMP9-induced osteogenic and angiogenic differentiation of MSCs were characterized in vitro.To further explore the effect of Notch signaling pathway on BMP9-induced osteogenic and angiogenic differentiation in vivo,ectopic bone formation with or without PPCNg was carried out for 4 and 6 weeks,respectively.Results:(1)We constructed an innovative n19 RNA library,which has more than 2.4 x 107 unique sequences as revealed by deep sequencing.The n19 RNA library can be delivered into mammalian cells stably and effectively through retroviral system.After four rounds of in vitro expansions and in vivo selections,bone tumor-like osteogenic resistance cells were obtained successfully.(2)Compared with the control group,BMP9-induced early osteogenic differentiation abilities,late osteogenic differentiation abilities and osteogenic differentiation markers expressions were significantly decreased in i MEFOR cell lines in vitro.In vivo assays revealed that the osteogenic differentiation capacities decreased gradually and accompanied many undifferentiated cells with the increasing rounds of selections.Furthermore,the i MEFOR lines proliferated automously without BMP9 stimulation.All these phenotypes are similar to that of bone tumors.(3)Bassed on the NGS(next generation sequencing)data analysis,we selected five relatively highly enriched 19 mers,namely DON2,DON3,DON5,DON6 and DON7.We established i MEFDON2,i MEFDON3,i MEFDON5,i MEFDON6,and i MEFDON7 cell lines successfully.In vitro studies demonstrated that the DON lines were not responsive to BMP9-induced osteogenic differentiation,and the extent of osteogenic resistance was related to the enrichment levels of the DONs.We further demonstrated that DON3 and DON6 exhibited dramatically high proliferative capability while strongly inhibited osteogenesis in vivo.(4)Genomewide RNA-seq results demonstrated that 174 genes were upregulated while 517 genes were downregulated in i MEFOR1 B cells compared with that in i MEFn19 cells.At the noncoding RNA level,12 noncoding RNAs were upregulated and 14 non-coding RNAs were downregulated in i MEFOR1 B cells,compared with that of i MEFn19 cells.We further analyzed the expression levels of noncoding RNAs in i MEFOR cell lines and confirmed that lnc RNA H19 as one of the most important candidate lnc RNAs that were differentially expressed in i MEFOR cells.RNA pull-down further confirmed the direct interactions of DON3 and several candidate lnc RNAs.(5)Upon the stimulation of BMP9,the expression level of lnc RNA H19 maintained in a relatively low level in i MEFOR cell lines,which was consistent with the osteogenic differentiation deficiency of i MEFOR1 B cells.However,overexpression of lnc RNA H19 in i MEFOR1 B cells did not rescue the early and late osteogenic differentiation deficiency in vitro.We further showed that overexpression of lnc RNA H19 alone was not sufficient to rescue the osteogenic differentiation deficiency of i MEFOR1 B.However,exogenously activating Notch signaling pathway partially rescued the osteogenic differentiation deficiency of i MEFOR1 B cells.(6)Compared with the control group,BMP9 induced biphasic expression of lnc RNA lnc RNA H19,which correlated with the expression of osteogenic regulators and late osteogenic markers in MSCs.Silencing lnc RNA H19 expression diminished BMP9-Induced osteogenic differentiation of MSCs in vitro.Exogenous expression of lnc RNA H19 blocked BMP9-induced osteogenic differentiation of MSCs in vitro.Both overexpression and silencing of lnc RNA H19 inhibited the terminal differentiation of BMP9-Induced ectopic bone formation from MSCs.Activation of Notch signaling restored the osteogenic differentiation impaired by the constitutive expression of lnc RNA H19 in MSCs.lnc RNA H19 regulated BMP9-induced osteogenic differentiation through modulating the expression of Notch ligands.We further identified both silencing lnc RNA H19 expression and overexpression lnc RNA H19 expression led to the increased expression of Notch signaling-associated micro-RNAs.(7)BMP9 up-regulated the expression of Notch receptors and ligands at an intermediate stage of BMP9-induced osteogenic differentiation of MSCs.Activation of Notch signaling by the Notch1 intracellular domain(NICD1)enhanced BMP9-induced osteogenic differentiation.A dominant-negative Notch1(dn Notch1)diminished BMP9-induced osteogenic differentiation in MSCs.Activation of Notch signaling by NICD1 significantly improved the maturity and quality of BMP9-induced ectopic bone formation by increasing angiogenic activity and neovascularization.Conclusions :(1)On the basis of BMP9 induced osteogenic differentiation of MSCs,we utilized the n19 RNA library and successfully obtained the bone tumor-like osteogenic resistance phenotype,which further validates the relationship between disruption of osteogenic differentiation and bone tumor development.(2)Using our novel n19 RNA library approach,the important functional lnc RNAs that control osteogenic differentiation and bone tumor development can be effectively identified.(3)The biphasic expression of lnc RNA H19 is essential in BMP9 induced osteogenic differentiation of MSCs.The regulatory function of lnc RNA H19 may play an important mediater role in BMP9-regulated Notch signaling pathway during osteogenic differentiation,and the lnc RNA H19 may accomplish its regulatory function on Notch signaling by modulating the expression of a panel of Notch receptors and ligands-associated mi RNAs.(4)Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis.It is conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering.