MicroRNA Profiling Of MiRNAas In Proliferative Cell Layer And Hypertrophic Cell Layer Of Mandibularcondylar Cartilage

Mandibular condylar cartilage is an important growth area of temporomandibular joint,which is closely related to the morphological development of craniofacial bones and the formation of TMJ functional area.The hypertrophied process of mandibular condylar chondrocytes is the key process of condylar development,it is also the foundation of condylar endochondral ossification and is controlled by a series of genes and signaling molecules.If the cytokines or genes that regulate the hypertrophy process are mutated,it will lead to the imbalance of cell proliferation and hypertrophy,leading to abnormal condylar development and dysfunction.The condylar cartilage,as a non-vascular and non-nerve tissue,is very limited in its ability to repair and regenerate.So looking into genetic level of the hypertrophied process of cartilage cells wil help us to further understand the growth mechanism,pathology and repair process of condylar cartilage.It can also provide certain reference for basic,clinical research and new ideas for condylar cartilage injury repair.MiRNAs(microRNAs)are newly discovered small molecules RNA and play an important role in gene regulation.At present,many studies have shown that microRNAs play an vital role in the growth and differentiation of growth plates and the process of chondrocytes’ hypertrophy.The osteogenesis way of condylar cartilage is as same as growth plate,all belong to endochondral ossification,but they are different in many other parts,such as the organizational structure,growth mode and response to environmental factors.So for the differ in so many ways of condylar cartilage and growth plate cartilage,relevant miRNAs research obviously cannot be copied.The purpose of this paper is to explore the miRNAs that play a key role inthe process of condylar hypertrophy differentiation,and to reveal its regulatory mechanism in the hypertrophy process through advanced analysis such as target gene prediction.Methods:This study used fresh condylars of healthy adult pigs as the research object,which are 3 months old average.Then we divided the samples into the control group(proliferation cell layer)and the experimental group(hypertrophy cell layer)using the frozen section machine after stratification.The sample was tested by Illumina’s Solexa high-throughput sequencing technology after extracted RNA and quality inspection of two groups.Next,we screen differentially expressed miRNAs in two groups and then qRT-PCR verification was performed.Finally the Corresponding miRNA was used for target gene prediction,GO analysis and KEGG analysis To reveal the miRNAs and related mechanisms that play an important role in the hypertrophy of the condylar chondrocytes.Results:1.Screening of different miRNAs in the two groups of tissues.By comparing the miRBase 21.0 database,303 and 313 known miRNAs were identified in PCL and HCL libraries,among which 299 were identified in the two libraries together.In the two groups,41 significantly different expressing miRNAs were identified,15 of which were significantly increased,and 26 were significantly reduced.2.qRT-PCR verification of differential mi RNA.The results show that there is a good consistency between high-throughput sequencing and qRT-PCR verification results,which reflects the high reliability of high-throughput sequencing data obtained by this research.3.Prediction of target genes and bioinformatics analysis.By using the miRanda database to predict the target gene of 41 miRNAs with significant difference,the target gene was predicted to be 39247,and the subsequent GO and KEGG functional analysis were carried out for these target genes.GO analysis shows that these target genes are mainly involved in biological processes such as cell proliferation and metabolism,cell movement and protein binding,carbohydrate and lipid metabolism.KEGG showed that the signaling pathway closely related to the hypertrophy process included Notch signaling pathway(ssc04330),Wnt signaling pathway(ssc04310),and TGF-β signal pathway(ssc04350).Conclusions:1.MiRNA difference expression profiles between the experimental group and the control group included 14 up-regulated miRNAs and 27 down-regulated miRNAs.2.qRT-PCR confirms that high throughput sequencing results are reliable.3.GO functional analysis shows that these target genes are mainly involved in biological processes such as cell proliferation,metabolism and cell movement and protein,carbohydrate and lipid metabolism.KEGG pathway analysis indicates that Notch signaling pathways(ssc04330)may play an vital role in cartilage hypertroph.