The Mechanism Of The Effects Of Oncogene C-myc On Proliferation And Differentiation Of Wapitis’ Anlter

Deer antlers are the only mammalian appendages that are subject to an annual cycle of epimorphic regeneration. It is not clear why an antler can grow rapidly after being cut like a tumor but not become cancerous, and does not cause canceration of surrounding tissues. Proto-oncogene c-myc plays a crucial role in cell proliferation and differentiation, especially in cancerous cells. C-myc has functions of regulating cell growth, proliferation, differentiation and apoptosis while maintaining normal cellular functions. Wapitis antler is good materials to explore the regulatory mechanism of c-myc on antler’s rapid growth but without carcinogenesis, and it is important to reveal the proliferation and differentiation characteristics of antler stem cells and construct the development model in cartilage, providing a model for the research on repair and regeneration of the bone.In this study, using techniques of RACE, real-time quantitative PCR, immunohistochemistry, adenovirus transfection and cell culture in vitro, the c-myc gene structure of Tarim wapitis antler was revealed and its gene expression characteristics in different growth periods of antlers was described. And effect of the c-myc gene on chondrogenesis of antler MSCs (mesenchymal stem cell) in vitro was studied through inhibiting Wnt by DKK1 overexpressing.The molecular cloning of the c-myc gene of Tarim wapitis antler showed that its full cDNA sequence was 1840bp long. The DNA binding domain of c-myc consisted of 80 amino acid residues in the bHLHZIP domain. An alignment of the amino acid sequences of bHLHZIP with other domestic animals showed that they differed at sites of the 16th and 46th amino acid residue. The results of quantitative real-time PCR (qPCR) showed that, on average in different zones (epidermis/dermis, mesenchyme, precartilages, and cartilage cell layers) of antler, the expression of c-myc was the highest at the growth stage of 30d after being cut, and decreased constantly along with different growth stages (30,40,60 and 70 days). However, it was lower than that in ear tissue. In particular,, the expressions of c-myc were significantly higher (p<0.05) in cartilage, epidermis, and mesenchyme than in other zones at stages of 30d,40d, and 60d, respectively. It was generally low at all stages in precartilage. By immunofluorescent histochemical analysis, it was founded that the positive c-myc protein primitively was located in inner root sheath of hair follicle, hair matrix of dermal layer, and vascular smooth muscle of arteries of the antler epidermis/dermis at stage of 30d and 40d. The fluorescent material also was seen within the cartilage cells at stage of 30d. Antler MSCs of adult Tarim wipptis were successfully induced to chondrogenesis by stimulating using growth factor betal (TGFβ1,10 ng/mL) in vitro, which simulated the antler’s natural growth process of endochondral ossification process. In the cartilage differentiation process, the expression of the c-myc gene in cells of the induced group was significantly lower than that of control group from the 7th day to 28th day (P<0.05), while there was no significant difference on the 35th day (P> 0.05). The results of blocking the upstream Wnt signaling of the c-myc gene using DKK1 overexpression showed that the c-myc gene could regulate the chondrogenic differentiation of antler MSCs by Wnt/beta catenin canonical pathway. The overexpression of DKK1 could inhibiting the initial expression level of c-myc, which could induce antler MSCs into differentiation status, but not inhibit the overall expressing level of MSCs c-myc, thus does not affect the proliferation activities of antler MSCs. The proliferation activity of antler MSCs at any level of c-myc was an important reason for the rapid growth of antler.In conclusion, the expression level of c-myc gene in antler was always lower than that in the surrounding tissues. C-myc affected chondrogenic differentiation of MSCs through the Wnt signaling pathway. Downregulation of c-myc was as trigger for cartilage differentiation, but had no effect on the proliferation of MSCs; and upregulation of c-myc expression promoted the proliferation of cartilage cells and mature, but also avoided differentiation of antler MSCs endlessly. Proliferation activity in any level of c-myc is an important reason for rapid growth of antler.