RNA-seq Analysis Of High And Low Yielding Deer Antler Cartilage Layer And Effects Of Arachidonic Acid On The Proliferation Of Deer Antler Chondrocytes

As a typical traditional animal medicine,deer antler has a long history of use in China.For a long time,increasing the output and quality of deer antler has been the focus of attention in deer breeding.The growth and development of deer antler is the main influencing factor of antler yield,and there are many factors that affect the growth and development of velvet antler,such as genetics,nutrition,environment and so on.In this study,the chondrocytes layer of sika deer antler were taken as the research object,and we used RNA-seq technique to screen differentially expressed genes in the cartilaginous layer tissues of high-and low-yielding sika deer antlers of the same age with significant yield differences under the same feeding conditions..The expression of fatty acid metabolic pathway genes such as arachidonic acid was significantly up-regulated in the high-yielding group of sika deer,indicating that fatty acids such as arachidonic acid play an important role in the growth of antlers.The effects of arachidonic acid on antler chondrocytes was further investigated in vitro by CCK8,Edu,proliferation staining,toluidine blue and alisin blue staining,q RT-PCR and WB.The main results of this study were as follows:(1)RNA-seq analysis of velvet cartilage layer of high and low-yield sika deer: three high yielding(G403,G972,G396)and three low yielding(D946,D962,D961)sika deer were selected for non-reference transcriptome analysis.The clean reads after sequencing quality were assembled using the Trinity software to obtain reference sequences,and a total of 194067 Transcript and 102023 Unigene were obtained,and the average comparison rate between clean reads and assembled reference sequences of samples was86.35%.(2)Compared with low-yield sika deer,there were 505 genes up-regulated and 278 genes down-regulated in high-yield group.The enrichment analysis of GO and KEGG showed that the gene expression of Rap1,Ras,PI3K-Akt,Hippo and other value-added related pathways and amino acid and fatty acid metabolic pathways were significantly increased in the high-yield group.Ten differentially expressed genes of proliferationrelated pathways and fatty acid metabolism pathways were selected for q RT-PCR validation,which was consistent with the results of sequencing analysis.(3)The chondrocytes of pilose antler were isolated,frozen and resuscitated.The CCK8 gradient test shows that after treating with exogenous addition of 10μM arachidonic acid for 24 h,chondrocytes viability were highly significantly increased in the treated group(P<0.01).The results of Edu cell proliferation showed that the number of proliferating chondrocytes increased significantly after treatment.The results of q RT-PCR showed that the expression of Cyclin D-1 gene increased significantly(P<0.01),CDK2 increased significantly(P<0.05)and p21 decreased significantly(P<0.05).The WestenBlot results showed that the expression of proliferation protein PCNA increased significantly(P<0.05).(4)The staining results of Alisin Blue and Toluidine Blue showed that after 24 hours of treatment with 10μM arachidonic acid,the content of glycosaminoglycan secreted by chondrocytes in the treatment group increased significantly,and the m RNA expression of chondrocyte-related genes COL2α and RUNX2 increased significantly(P<0.05),and COMP increased significantly(P<0.01).The protein expression of COL2α in the treatment group increased significantly(P<0.05).In conclusion,there were a large number of differentially expressed genes in the high-yielding group of sika deer compared to the low-yielding group and the expression of genes in fatty acid metabolism pathways such as Arachidonic Acid was significantly upregulated.Exogenous addition of 10μM arachidonic acid to chondrocytes improved their cell vitality,increased expression of cell cycle genes,and increased intracellular glycosaminoglycan content,which promoted the proliferation and growth of antler chondrocytes,and can be used to further explore the effect of adding unsaturated fatty acids such as arachidonic acid on deer antler yield and quality in feed.