Screening And Interaction Of Key LncRNA And MRNA During The Differentiation Of Horse BMSCs Into Chondrocytes

Bone marrow mesenchymal stem cells(BMSCs)are one type of pluripotent stem cells existing in bone marrow stroma.They have become ideal seed cells in the field of tissue engineering and regenerative medicine due to their easy separation and rich content.Since cartilage or bone damage often occurs in horses during athletic competition,using BMSCs for cartilage and bone repairing as well as cell therapy have been used in clinical practice for many years.However,it is still unclear how regulatory mechanism works when BMSCs differentiate into chondrocytes in both vivo and vitro.Long non-encoded RNA(lncRNA)is a non-protein encoded transcription of length>200nt,which plays a vital role in the process of regulating a variety of cellular biology.Researches have confirmed that IncRNA can affect the differentiation of chondrocytes and the function of cartilage by regulating its target gene,but the study of its detailed regulation mechanism is seldom reported.Therefore,in this study,the differential expression of IncRNA and the target gene(mRNA)were initially identified through the differentiation process of horse BMSCs to chondrocytes using transcription group sequencing technique.Further,the regulatory network between the involved IncRNA and its target gene was preliminarily established.Additionally,the molecular regulatory mechanism for regulating the differentiation of BMSCs into chondrocytes was revealed.The main research results are as follows:1.The BMSCs cell line was established in vitro,and the detection of BMSCs expressed the stem cell marker factor Sox2 and mesenchymal stem cell surface marker factor CD44,CD90,CD 105 and Integrin CD29.2.The isolated and purified BMSCs were induced and further successfully differentiated into chondrocytes after 14d and 21d induction culture.The differentiated cells showed the unique morphology of chondrocytes and expressed the specific marker genes of chondrocytes,aggrecan,collagen? and Sox9.3.High-throughput Transcription group sequencing was carried out on BMSCs and the chondrocytes from which they were differentiated,and the obtained data were analyzed by bioinformatics-related software.The results showed a total of 3,592 lncRNA were identified,and the differential expressions of IncRNA between BMSCs and differentiated chondrocytes were 356.Several potential candidate factors involved in regulating the development of horse chondrocytes were identified,such as lnc-cpm?lnc-traf3?lnc-22173?lnc-acvr2a?Inc-pde4d,and their interacted target genes were MDM2,TRAF3,USP25,ACVR2A,and PDE4d.The expression trends of these differences of IncRNA and mRNA in BMSCs and differentiated chondrocytes were consistent with the sequencing results by real-time quantitative PCR.4.Based on GO and KEGG analysis,the differential expression of IncRNA and mRNA interaction regulation network was constructed,and it was also found that these target mRNAs were mainly enriched in Wnt,MAPK of p53 and NF-kb signaling pathways.These genes controlled the proliferation,differentiation and apoptosis of chondrocytes,and prevented the formation of osteoclasts,which in turn maintained the development of chondrocytes.Among all,the gene of wnt5a,wntll,plcdl in wnt signaling pathway,MAPK20,MAP2K,MAPK7/8 in MAPK signaling pathway,and MDM2,Bcl-2,WAF1,SIRT1,NRF2 in P53 signaling pathway with its lncRNA were closely related to the differentiation of chondrocytes.The above results build a preliminary foundation for further revealing the interaction mechanism between IncRNAs and target genes in the process of chondrocyte differentiation and provide a scientific basis for the cell therapy using BMSCs of cartilage repair.