| Objectives:Chondrogenic differentiation is a sequential complicated process which begins with the condensation of mesenchymal stem cells?MSCs?that are derived from the mesoderm germ,followed by lineage commitment into prechondrocyte and differentiation to yield the chondrocytes.It is well acknowledged that chondrogenic differentiation is critical for cartilage development and repair as well as tissue regeneration engineering which is determined by temporal and spatial expression of multiple growth factors and activation of transcription factor and signaling pathways.However the underlying precise cellular mechanisms in chondrogenic differentiation remain largely unknown.Long noncoding RNAs?lncRNAs?refer to a category of non-protein-coding RNAs longer than 200 nucleotides in length.Growing evidences demonstrate that IncRNAs can regulate various biological processes,including development,chromosome inactivation,genomic imprinting and epigenetics via their interaction with DNA,RNA or protein from transcriptional and post-trascriptional levels.However,the potential regulatory roles of IncRNAs in chondrogenic differentiation remain largely unclear.In this study,microarray was performed to detect the differential expression profiles of IncRNAs during chondrogenic differentiation of murine chondrogenic cell line ATDC5.Then,the putative functions of candidate IncRNAs were predicted by bioinformatics methods.Finally,functional experiment was also utilized to explore the regulatory roles of lncRNA AK136902 in chondrogenic differentiation which might provide experimental evidence on the regulatory mechanisms of lncRNAs in cartilage development and treatment of cartilage diseases.Methods:1.1%ITS?insulin-transferrin-sodium selenite?was utilized to induce the murine chondrogenic cell line ATDC5 to establish the chondrogenic differentiation model in vitro.The chondrogenic and hypertrophic markers,including Aggrecan,Col2al,Sox9,Runx2 and Coll0al,were investigated at different time points?0d,1d,7d,14d,21d,28d?by Alcian Blue Staining,qRT-PCR and Western Blot.2.After 14 days of chondrogenic induction,the chondrogenic markers of differentiatd and undifferentiated ATDC5 cells,including Aggrecan,Col2al and Sox9,were detected by Alcian Blue Staining,qRT-PCR,Western Blot and Immunohistochemistry.3.The differential lncRNA expression profiles of differentiatd?14d?and undifferentiated?0d?ATDC5 cells were identified with Arraystar Mouse lncRNA Microarray V3.0 and the most dysregulated eleven lncRNAs were verified by qRT-PCR analysis.4.Gene ontology?GO?,Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway analysis,Coding-noncoding co-expression?CNC?and competing endogenous RNA?ceRNA?networks were performed to explore the functions of the differentially expressed lnRNAs and mRNAs.5.The expressions of three lncRNAs,including AK136902,AK016344 and ENSMUST00000180767 at different time points?0d,3d,7d,10d,14d?after chondrogenic differentiation of ATDC5 cells were detected and correlation analysis was performed to confirm the correlation between these lncRNAs and chondrogenic markers,including Aggrecan,Col2al and Sox9.The expressions of lncRNA AK136902 from different cartilage of C57BL/6 mouse,such as knee joint cartilage,mandibular condylar cartilage,intervertebral cartilage and auricular cartilage were also investigated by qRT-PCR.6.Stable lncRNA AK136902 knockdown ATDC5 cell lines were established by lentiviral vectors.Cell viability of lncRNA AK136902-depleted ATDC5 cell after chondrogenic induction were detected by Sulphorhodamine B?SRB?assay.The chondrogenic markers,including Aggrecan,Col2al and Sox9 of lncRNA AK136902-depleted ATDC5 cell after chondrogenic induction of 0,7 and 14 days,were investigated by Alcian Blue Staining,qRT-PCR and Western Blot.Aggrecan was detected by qRT-PCR and Western Blot after overexpression of Sox9 in AK136902-depleted ATDC5 cell of chondrogenic induction of 0 and 14 days.7.Cellular sublocalization of IncRNA AK136902 were confirmed by nuclear and cytoplasmic fractionation.Protein stability assay were carried out to determine the stability of Sox9 protein expression.Results:1.The Alcian Blue staining intensity was significantly increased after 7d when treated with 1%ITS.The expressions of chondrogenic markers,including Aggrecan,Col2a1 and Sox9,were gradually increased after 1%ITS induction.The expressions peaked at 14d and then went down till 28d.The hypertrpphic markers,such as Runx2 and Coll0al,gradually went up and peaked at 28d.2.Compared with undifferentiated?0d?ATDC5 cells,the chondrogenic specific genes of differentiated?14d?ATDC5 cells,including Aggrecan,Col2al and Sox9,were significantly upregulated from both mRNA and protein levels.3.1009 lncRNAs and 1206 mRNAs were differentially regulated during chondrogenic differentiation.The qRT-PCR results of the most dysregulated eleven lncRNAs were consistent with the microarray analysis.4.GO and KEGG pathway analysis indicated the principal functions of the dysregulated mRNAs were associated with system development and extracellular matrix interaction,TGF-? signaling and PI3K-Akt signaling pathway.The CNC network showed that lncRNA AK136902?AK016344?ENSMUST00000180767?ENSMUST00000181776?NR015466 and AK035926 were correlated with chondrogenic related genes,including Col2al,Ibsp and Ptgfr.The ceRNA network covered 3 lncRNAs,121 miRNAs and 241 edges.The upregulated lncRNA AK136902,AK016344 and ENSMUST00000180767 might promote chondrogenic differentiation by acting as ceRNAs.5.The qRT-PCR results showed that the expessions of lncRNA AK136902,AK016344 and ENSMUST00000180767 were gradully increased during chondrogenic diffrentiation of ATDC5 cells and peaked at 14d which was also positively correlated with chondrogenic related genes,such as Aggrecan and Sox9.Candidate lncRNA AK136902 were selected for further analysis and exhibited a spatio-temporal expression pattern in knee joint cartilage,mandibular condylar cartilage and intervertebral cartilage of C57BL/6 mouse.6.Stable lncRNA AK136902 knockdown ATDC5 cell lines were successfully established.Knockdown of lncRNA AK136902 suppressed cell viability of ATDC5 cell after chondrogenic induction.Knockdown of lncRNA AK136902 inhibited the expression of Aggrecan from both mRNA and protein levels at day 7 and 14 during chondrogenic differentiation of ATDC5 cells.Knockdown of lncRNA AK136902 suppressed the protein expression of Sox9 but did not affect the mRNA expression at day 7 and 14 during chondrogenic differentiation of ATDC5 cells.Knockdown of lncRNA AK136902 inhibited the expression of Col2al from both mRNA and protein levels at day 7 but did not affect the mRNA and protein expression at day 14 during chondrogenic differentiation.Overexpression of Sox9 could rescue down-regulated expression of Aggrecan in AK136902-depleted ATDC5 cell during chondrogenic differentiation.7.LncRNA AK136902 was predominantly localized in the cytoplasm and could promote the protein stability of Sox9,thereby contributing to the chondrogenic differentiation of ATDC5 cells.Conclusions:1.The murine ATDC5 cells can go through a sequential process analogy to chondrogenic differentiation and chondrocyte hypertrophy when treated with 1%ITS which is a promising in vitro model to study chondrogenic differentiation.After 14 days of induction,the ATDC5 cells differentiate into chondrocytes.2.High throughput microarray combined with qRT-PCR is an efficient approach to investigate lncRNAs.Constructing the CNC and ceRNA networks offers an effective method to predict the potential functions of lncRNAs.3.LncRNA AK136902 displayed a spatio-temporal expression pattern in diverse cartilage of C57BL/6 mouse and was siginificantly upregulated during chondrogenic differentiation of ATDC5 cells which indicated that lncRNA AK136902 could be viwed as a novel chondrogenic maker.4 LncRNA AK136902 was a stable cytoplasmic lncRNA which could promote cell viability and protein stability of transcription factor Sox9,thereby regulating the chondrogenic differentiation of ATDC5 cells. |
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