Background: The deer antler, a remarkable mammalian appendage, has a growth rate surpassing that of any other known osseous organ. Emerging evidence indicates that circRNA and MAPK1 play critical roles in chondrocytes. Thus, exploration of their functions in antler chondrocytes will help us to understand the mechanism regulating the rapid antler growth. Methods: qRT-PCR, western blot, and immunohistochemistry were used to assess the expression of mRNAs and proteins. CCK-8, EdU, Cell migration, ALP activity detection, and ALP staining examined the effects of MAPK1 in antler chondrocytes. FISH, RIP, and luciferase assays were performed to evaluate the interactions among circRNA3634/MAPK1 and miR-124486-5. RIP and RAP assays proved the binding interaction between circRNA3634 and RBPs. Me-RIP was used to determine the m6A methylation modification of circRNA3634. Results: This study revealed high MAPK1 expression in antler cartilage tissue. Overexpression of MAPK1 promoted the proliferation, migration, and differentiation of antler chondrocytes and increased the expression of MAPK3, RAF1, MEK1, RUNX2, and SOX9. The silencing of MAPK1 had the opposite effect. CircRNA3634 was found to act as a molecular sponge for miR-124486-5, leading to increased MAPK1 expression and enhanced proliferation and migration of antler chondrocytes through competitive miR-124486-5 binding. We discovered that METTL3 mediates m6A modification near the splicing site of circRNA3634 and is involved in the proliferation and differentiation of antler chondrocytes. The m6A reader YTHDC1 facilitated the nuclear export of circRNA3634 in an m6A-dependent manner. Our results indicate that m6A-modified circRNA3634 promotes the proliferation of antler chondrocytes by targeting MAPK1 and show that the nuclear export of circRNA3634 is related to the expression of YTHDC1, suggesting that circRNA3634 could represent a critical regeneration marker for the antler. Conclusions: Our results revealed a novel m6A-modified circRNA3634 promoted the proliferation and differentiation of antler chondrocytes by regulating MAPK1. The nuclear export of circRNA3634 was related to the expression of YTHDC1.

Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor β (TGF-β). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-β1 during the rapid growth of sika deer antler. Three CRISPR-Cas9 knockout vectors targeting the TGF-β1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-β1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-β pathway-related genes were determined using a PCR array. Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-β1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-β signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-β pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role. TGF-β1 is a newly identified regulatory factor of rapid growth in sika deer antler.

Long non-coding RNA (lncRNA) is a transcription product of the mammalian genome that regulates the development and growth in the body. The present study aimed to analyze the expression dynamics of lncRNA in sika antler mesenchymal and cartilage tissues by high-throughput sequencing. Bioinformatics was applied to predict differentially expressed lncRNAs and target genes and screen lncRNAs and mRNAs related to osteogenic differentiation, cell proliferation, and migration. Finally, the expression of the lncRNAs and target genes were analyzed by qRT-PCR. The results showed that compared to the cartilage tissue, the transcription levels of lncRNA and mRNA, 1212 lncRNAs and 518 mRNAs, in mesenchymal tissue were altered significantly. Thus, a complex interaction network was constructed, and the lncRNA-mRNA interaction network correlation related to osteogenic differentiation, cell proliferation, and migration was analyzed. Among these, the 26 lncRNAs and potential target genes were verified by qRT-PCR, and the results of qRT-PCR were consistent with high-throughput sequencing results. These data indicated that lncRNA promotes the differentiation of deer antler mesenchymal tissue into cartilage tissue by regulating the related osteogenic factors, cell proliferation, and migration-related genes and accelerating the process of deer antler regeneration and development.

Long non-coding RNA play an importantr role in the differentiation of chondrocytes. This study aims to explore the role of long non-coding RNA in the transcriptional regulation of Notch4. In previous studies, it has been found that Notch signal can be used as the downstream of TGF-β signal to affect the proliferation and differentiation of deer antler chondrocytes, but the specific mechanism remains unclear. Here we found that lncRNA27785.1 was involved in the regulation of TGF-β/ Smad3 signal and Notch4 gene. The overexpression lncRNA27785.1 can negatively regulate the expression of Notch4 to inhibit cell proliferation and differentiation, while interference with lncRNA27785.1 can promote the expression of Notch4 gene to promote the proliferation and differentiation of chondrocytes. Subsequently, through luciferase experiment and CHIP experiment, we found that lncRNA27785.1 is regulated by Smad3 transcription, and Smad3 inhibited the expression of lncRNA27785.1. In addition, activated TGF-β signaling can reduce the inhibitory effect of lncRNA27785.1 on Notch4 signaling. In summary, we found that lncRNA27785.1 and TGF-β/Smad3 play an important role in Notch4 signaling. Our findings provided evidence to explain how TGF-β signaling regulate the Notch signaling pathway to influence chondrocyte proliferation and differentiation by a specific lncRNA27785.1.