The Different Expression Profiles Of LncRNAs During The Osteogenic And Chondrogenic Differentiations Of Human Bone Marrow Mesenchymal Stem Cells

Background Mesenchymal stem cells (MSCs) are capable of self-renewal and can be differentiated into the osteogenic, chondrogenic, and adipogenic lineages. They are a source of cells in bone and cartilage tissue engineering. Human bone marrow MSCs have wide use in numerous clinical applications, including tissue engineering, autoimmune disease and wound repair. The osteogenic and chondrogenic differentiation is the focus of stem cell researches. However, the precise molecular mechanisms of differentiation remain unclear.Long noncoding RNAs (lncRNAs) belong to a novel heterogeneous class of ncRNAs that includes thousands of different species. lncRNAs have crucial roles in gene expression control during both developmental and differentiation processes. They have been found to play crucial regulatory roles in a diverse range of biological pathways and cellular processes and human diseases. However, whether the lncRNAs also involve in the osteogenic and chondrogenic differentiation of MSCs is unkown and few studies referred to the problem.Objectives1. To conduct the osteogenic and chondrogenic differentiation of human bone marrow MSCs respectively and prepared the cell samples for microarray analysis.2. To respectively detect differentially expressed lncRNAs and mRNAs between the osteo-with chondro-differentiated and non-differentiated MSCs.3. To analyze and predict the potential functions of differentially expressed lncRNAs involved in differentiations with bioinformatics analysis.Methods1. MSCs at P3 were induce to osteogenic and chondrogenic differentiations with different methods. The differentiation inductions were validated with cell dying and testing of differentiation related genes using qRT-PCR at Day 14 after inductions.2.3 osteo-differentiated,3 chondro-differentiated and 3 non-differentiated cell samples at Day 14 were selected. Total RNA was extracted and tested with Agilent human lncRNA+mRNA Array V4.0. The data of differentially expressed lncRNAs and mRNAs were analyzed.3. Bioinformatic analyses (gene ontology, pathway and co-expression network analysis) were applied for further study of these differentially expressed lncRNAs. Target regulated by lncRNAs was predicted with bioinformatic prediction.4. The qRT-PCR was used to validate microarray data and observe the dynamic expression levels of related lncRNAs.Results1. MSCs at P3 were induce to osteogenic and chondrogenic differentiations successfully and validated, which provided the ideal cell samples for later array tests.2.1206 differentially expressed lncRNAs were identified during the process of osteogenic differentiation (fold change>2.0 or<-2.0; P<0.05). Among these lncRNAs,687 were up-regulated and 519 were down-regulated.2143 mRNAs were found to be differentially expressed, including 957 that were up-regulated and 1186 that were down-regulated. The validated results of qRT-PCR were consist with microarray.3. For the chondrogenic differentiation,3638 differentially expressed lncRNAs were identified (fold-change> 2.0 or<-2.0, P<0.05), consisting of 2166 up-regulated and 1472 down-regulated.5560 mRNAs were found to be differentially expressed, including 1980 that were up-regulated and 3580 that were down-regulated.4. The differentiated expressed lncRNAs were further analyzed with gene ontology, pathway and co-expression network analysis. The dynamic expresson trends of lncRNA-H19 and ucO22axw.1 during osteo-differentiation and lncRNA-ZBED3-AS1 with CTA-941F9.9 during chondro-differentiation were observed.Conclusions1. The osteogenic and chondrogenic differentiations could be inducted successfully with different methods.2. This is the first time to reveal lncRNAs and mRNAs expression profiles in osteo-and chondro-differentiations of MSCs using microarray. The potential functions of differentiated expressed lncRNAs were further analyzed using bioinformatics analysis.3. LncRNAs were differentially expressed in osteo-and chondro-differentiation and may play a role in the pathogenesis of differentiation processes. Further researches are needed to explore the detailed functions of lncRNAs.