Study Of Differentially Expression Profile Of Long Noncoding RNA In Osteogenic Differentiation Of Human Marrow-derived Mesenchymal Stem Cells

Trauma, tumor and congenital malformations of maxillofacial often cause bone defects. It not only can cause facial deformity, but also can cause language, chew, and respiratory dysfunction. Then the patient’s physical, psychological and so on will have negative influence. So how to repair bone defects both functional and beautiful at the same time, it is the goal of people continue to study improvement. Traditional methods of bone defect repair have artificial bone graft, bone autograft, allograft bone transplantation and so on, but these methods all have their own drawbacks. In order to overcome these disadvantages, people build tissue engineering bone graft. It provides a new method for bone defect repair. Bone tissue engineering requires three basic biological factors, these are seed cells, growth and differentiation factors, extracellular matrix materials. The most fundamental is the choice of seed cells, it is the main source of active ingredient of tissue engineering. Ideal sources of seed cells should be made easy, broad, little damage to the body and try to reduce the immune rejection, more importantly must have the ability of directional differentiation of osteoblast. Human marrow-derived mesenchymal stem cells (hMSCs) have many advantages, such as osteogenetic differentiation potential, conveniently, from the self, strong ability of self-renewal and no immune rejection problem when porting. So it has been widely used in the construction of tissue engineering bone graft. Therefore, further study of osteogenetic differentiation mechanism of hMSCs will contribute to its clinical application in bone tissue engineering.At present, the study of molecular regulation mechanism of the osteogenesis of mesenchymal stem cells have made significant progress. It is regulated by many factors, such as growth factors (Wnt family, BMP), transcription factor (β-catenin, Runx2) and miRNA.And recent studies have found a new regulatory mechanism outside of DNA sequence, the long non-coding RNA (lncRNA) also affect the osteogenesis of mesenchymal stem cells. The length of lncRNA is more than 200nt, they are not directly involved in the protein coding. At first they was thought to be "noise" transcription of the genome and a by-product of transcription, did not have biological functions. But in recent years, along with the wide application of gene chip technology and RNA sequencing technology, a growing number of lncRNA were identified, and many studies confirmed that it has the complex biological function and it regulated cell differentiation and individual development and other important life processes on multiple levels. As studies show it related to differentiation of neurons, muscle, skin and fat. Although in recent years, a small amount researches also reported that lncRNA regulated osteogenesis of mesenchymal stem cells. But so far, differentially expression profile of lncRNA in osteogenic differentiation of hMSCs has no literature reports.Therefore, this study will first using human lncRNA expression profile chip detection technology to screen differentially expressed lncRNA in osteogenic differentiation of hMSCs and further analysis. This will provide new lncRNA targets for the research on osteogenesis mechanism of hMSCs, thus to build a more ideal tissue engineering bone graft provides a new theoretical basis.This experiment will be divided into the following three parts.Part I cultivation and identification of hMSCs and cells of hMSCs osteogenesis ObjectivehMSCs are a kind of cells with multi-directional differentiation potential. They have strong ability of division and proliferation and can differentiate into osteoblasts, adipocyte, chondrocyte, muscle cells, neurocyte, myocardial cells under different induction conditions. So They are considered to be an important seed cells in bone tissue engineering. This experiment culture hMSCs in vitro, then use mesenchymal stem cell osteogenic differentiation medium(MODM) to make it to the direction of osteoblast differentiation and identification. These are in preparation for the subsequent lncRNA expression profile microarray testing experiment.Methods1. To purchase the original generation of hMSCs and culture to P2 generation, We put the P2 generation of hMSCs in MODM cultivate 7、14、21 days later and observe the cell morphology.2. Take the osteogenetic differentiated hMSCs at 7、14 and 21 days as the experimental group (respectively for D7、D14 and D21), take the hMSCs as the control group (DO).3. Extract total RNA of the samples of D0、D7、D14 and D21 group, then identify the quality of RNA.4. Using real-time fluorescent quantitative PCR (RT-PCR) detect expression of osteogenesis specific transcription factor (Osterix, ALP, OPN).Results1. The original generation of hMSCs subculture to P2 generation, cell growth condition is good and present the uniform long fusiform shape. Cell morphology change from long fusiform shape to polygon after osteogenesis induction training. Along with the extension of incubation time, increased cell proliferation, the induction of 7 days can see cell aggregation, the induction of 14 days and 21 days can see the brownish black calcium nodules.2. Take P2 generation of hMSCs to ALP staining and alizarin red staining and the results were negative. Take the osteogenetic differentiated hMSCs at 7、14 and 21 days to ALP staining and alizarin red staining, found cytoplasmic appear red and deposition of red calcium salt, these prompt the results were positive.3. A260/A280 of the RNA samples of DO、D7、D14 and D21 group are 1.8 to 2.0, these show that the total RNA have high purity. RNA integrity:electrophoresis of RNA on gel containing formaldehyde show that electrophoresis stripe are clear and stripe brightness of 28s:18s rRNA are greater than or close to 2:1. All of these suggest that the total RNA quality accord with requirements of further RT-PCR and subsequent expression profile microarray experiment.4. RT-PCR detect expression of Osterix、ALP、OPN during osteogenesis of hMSCs, the results show that compared with hMSCs, the expression of Osterix, ALP、OPN increased significantly after the hMSCs osteogenesis and results have significant difference (P<0.05).Part Ⅱ Screening and verification of differentially expressed lncRNA during osteogenesis of hMSCsObjectiveThis experiment using lncRNA expression profile microarray detection technology to screen differentially expressed lncRNA during osteogenesis of hMSCs and explore the role of lncRNA played in hMSCs osteogenesis. But there were some false positive rate, microarray detection must be verified by other way, so we randomly selected five differentially expressed lncRNA (2 up-regulated lncRNA: ENST00000523786.1 and ENST00000436715.1,3 down-regulated lncRNA: ENST00000532315.1, HIT000218960 and ENST00000502125.2) from the microarray results. We further validate the accuracy of the microarray results by RT-PCR.Methods1. After quality inspection of RNA samples of D0、D7、D14 and D21 group qualified, amplify and tag RNA samples, then make them cross with people lncRNA V3.0 microarray of agilent company overnights. Scanned the hybridization chip using agilent microarray scanner to get the hybridization images. Analyzed and extract data of hybridization images using the agilent feature extraction (vl0.7) software. Normalized and analyze these data using the agilent genespring software (set difference standard is fold change of no less than 2 times).2. Randomly selected five differentially expressed lncRNA validate the accuracy of the microarray results by RT-PCR.Results1. The microarray results show that compared with hMSCs, the osteogenetic differentiated hMSCs at 7,14 and 21 days respectively have 923 lncRNA,1393 lncRNA,1338 lncRNA that up-regulated more than 2 times and have 993 lncRNA、 3843 lncRNA、3688 lncRNA that down-regulated more than 2 times and have 1462 mRNA、4093 mRNA、3354 mRNA that up-regulated more than 2 times and have 953 mRNA、2236 mRNA、1923 mRNA that down-regulated more than 2 times.433 lncRNAs were continuously upregulated significantly more than 2 times and 232 lncRNAs were continuously downregulated significantly more than 2 times during osteogenesis of hMSCs.956 mRNAs were continuously upregulated significantly more than 2 times and 229 mRNAs were continuously downregulated significantly more than 2 times during osteogenesis of hMSCs.2. The RT-PCR results show that compared with DO group, the expression of ENST00000523786.1 and ENST00000436715.1 were up-regulated significantly in D7、D14、D21 group (P< 0.05), the expression of ENST00000532315.1, HIT000218960 and ENST00000502125.2 were down-regulated significantly in D7、 D14、D21 group (P< 0.05). RT-PCR results consistent with microarray results.Part Ⅲ Bioinformatics analysis of differentially expressed lncRNA and mRNA during osteogenesis of hMSCsObjectiveTo explore continuously differentially expressed mRNA may associated with the changes in which genes function and biological pathways. To analyze protein-coding genes 300kb area near lncRNA of larger different expression in microarray results and predict target genes of lncRNA. This will help to reveal a new osteogenesis differentiation mechanism that particular lncRNA regulate.MethodsGene Ontology analysis (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (KEGG) are used for continuously differentially expressed mRNA during osteogenesis of hMSCs. To select some lncRNA of larger different expression from microarray results, analyze protein-coding genes 300kb area near lncRNA according to its sequence information combined with differentially expressed mRNA sequence information.Results1.Gene Ontology (GO) analysis shows that with respect of biological process (BP), 680 differentially expressed mRNA during osteogenesis of hMSCs enriched in biological process, among which, the system development is the highest, contains 217 differentially expressed genes. In respect of cellular component(CC),702 differentially expressed mRNA during osteogenesis of hMSCs enriched in cellular component, among which, the extracellular matrix is the highest, contains 57 differentially expressed genes. In respect of molecular function (MF),631 differentially expressed mRNA during osteogenesis of hMSCs enriched in molecular function, among which, the carbohydrate derivative binding is the highest, contains 28 differentially expressed genes.2. KEGG analysis shows that 427 differentially expressed mRNA during osteogenesis of hMSCs enriched in 31 biological pathways, among which, the Complement and coagulation cascades is the highest, contains 21 differentially expressed genes.3.We select larger up-regulated lncRNA ENST00000585537.1 and larger down-regulated lncRNA eHIT000015952 from microarray results, combined with differentially expressed mRNA sequence information found that lncRNA ENST00000585537.1 located on chromosome 17, there are three protein-coding genes (MAP2K6, TEKT1, ABCA5) near its 300 kb area. LncRNA eHIT000015952 located on chromosome 3, there are seven protein-coding genes (such as PAK2, MFI2, DLG1) near its 300 kb area.Conclusions1. hMSCs have strong osteogenesis ability in vitro and can be differentiated into osteoblast under the appropriate condition.2. The use of lncRNA expression profile microarray detection technology firstly screened differentially expressed lncRNA during osteogenesis of hMSCs. The confirmation by RT-PCR of randomly selected lncRNA with different expression, improve reliability of the microarray results.3. GO analysis shows that among differentially expressed mRNA during osteogenesis of hMSCs,680 mRNA enriched in biological process,702 mRNA enriched in cellular component,631 mRNA enriched in molecular function. KEGG analysis shows that 427 differentially expressed mRNA during osteogenesis of hMSCs enriched in 31 biological pathways.4. For the adjacent protein-coding genes of some differentially expressed lncRNA, bioinformatics analysis indicate that target genes of differentially expressed lncRNA during osteogenesis of hMSCs.5. To provide new lncRNA targets for the research on osteogenesis mechanism of hMSCs, thus to build a more ideal tissue engineering bone graft provides a new theoretical basis.