| Mongolian horse is an excellent local livestock breed in China.Many years of genetic improvement,a large number of excellent Mongolian horses participate in various competitions every year.The clinical treatment of sports injuries caused by training and competitions has become a practical problem that needs to be solved urgently.Orthopedic diseases have always been regarded as intractable diseases in the medical field,especially cartilage diseases.Because of fewer blood vessels around the cartilage,it is difficult for repair factors and nutrients to be transported to the injured site.Therefore,it is possible to develop the directional differentiation of stem cells in genetic engineering for the treatment of orthopedic diseases,among which bone mesenchymal stem cells(BMSCs)are the ideal source of seed cells.As one of the important non-coding family members,circRNA(circular RNA,circRNA)participates in many biological processes of the body and also plays an important role in the differentiation process of chondrocytes.In this study,the whole transcriptome data was obtained by high-throughput sequencing technology at three different time points(0d,14 d,21d)in the differentiation process of Mongolian horse BMSCs into chondrocytes.After bioinformatics analysis and molecular biology verification,screening the differential circRNA and m RNA in the differentiation of BMSCs into chondrocytes,combined with the results of micro RNA in the previous research,will further explore the regulatory role of circRNA in the differentiation of BMSCs into chondrocytes.The research results will provide the possibility of cell and molecular therapy for horse racing injuries.The results were as follows:1.Sequencing analysis results showed that in the 14d-0d group,21d-0d group and21d-14 d group,the differentially expressed circRNA were 15,23,and 15,respectively,and the differentially expressed m RNA was 4011,3592,and 1049,respectively.2.The GO classification showed that the differentially expressed circRNAs in the three groups were significantly enriched in biological processes such as transcription regulation,plasma membrane adhesion molecule binding,cell proliferation regulation,and cell recognition.KEGG enrichment showed that they were mostly enriched in different signaling pathway,such as the signaling pathway of Toll-like receptor,FcεRI,cell adhesion molecule,TNF,platelet activation,Erb Bc,osteoclast differentiation,Hippo,glycosaminoglycan biology synthesis and wnt,etc.However,circRNA transcription profiles and enrichment pathways were different among different groups.3.circRNA/mRNA target microRNA analysis and binding site prediction showed that3 circRNAs(circRNA299,circRNA688,circRNA440),3 micro RNAs(micro-PC-5p-87201＿5,micro-PC-3p-183030＿3,micro-PC-5p-113587＿4)and 4m RNAs(SOS1,PIK3R1,CREB1,BRAF)had mutual binding sites.4.The differentially expressed circRNA688,PIK3R1,CREB1,and FOXO1 were identified and analyzed by q RT-PCR,which were consistent with the sequencing results,and they were all up-regulated in the 21d-0d group.Based on the above analysis,four possible pathways that affect the differentiation of BMSCs into chondrocytes were screened out: circRNA688—micro-PC-3P-183030＿3—PIK3R1,circRNA688—micro-PC-3P-183030＿3—CREB1,circRNA299—micro-PC-5P-87201＿5—SOS1 and circRNA440—micro-PC-5P-113587＿4—BRAF,at the same time,an association analysis network diagram was drawn. |
