Whole-Transcriptome Sequencing of Antler Tissue Reveals That circRNA2829 Regulates Chondrocyte Proliferation and Differentiation via the miR-4286-R+1/FOXO4 Axis

Abstract

The antler is the unique mammalian organ found to be able to regenerate completely and periodically after loss, and the continuous proliferation and differentiation of mesenchymal cells and chondrocytes together complete the regeneration of the antler. Circular non-coding RNAs (circRNAs) are considered to be important non-coding RNAs that regulate body development and growth. However, there are no reports on circRNAs regulating the antler regeneration process. In this study, full-transcriptome high-throughput sequencing was performed on sika deer antler interstitial and cartilage tissues, and the sequencing results were verified and analyzed. The competing endogenous RNA (ceRNA) network related to antler growth and regeneration was further constructed, and the differentially expressed circRNA2829 was screened out from the network to study its effect on chondrocyte proliferation and differentiation. The results indicated that circRNA2829 promoted cell proliferation and increased the level of intracellular ALP. The analysis of RT-qPCR and Western blot demonstrated that the mRNA and protein expression levels of genes involved in differentiation rose. These data revealed that circRNAs play a crucial regulatory role in deer antler regeneration and development. CircRNA2829 might regulate the antler regeneration process through miR-4286-R+1/FOXO4.

Keywords: FOXO4; RNA sequencing; antler; ceRNAs; circRNA2829; miR-4286-R+1; sika deer.

Figure 1

Analysis of the differentially expressed circRNA, miRNA, and mRNA in tip tissue of antlers. (A) circRNA distribution on chromosomes. (B) The length distribution of circRNA. (C) Volcano diagrams of differential circRNA, miRNA, and mRNA. DEGs were selected with an absolute value of the thresholds of fold change ≥1 and p < 0.05, the red dots represent upregulated differential genes, the blue dots represent downregulated differential genes, and the black dots represent genes with no significant difference. (D) Clustering heatmap of the top 50 differentially expressed circRNA, miRNA, and mRNA; the pink represents downregulated and the green represents upregulated.

Figure 2

Validation of high-throughput sequencing results. (A,C,E) Analysis of the expression levels of circRNA, miRNAs, and mRNAs in high-throughput sequencing results. (B,D,F) RT-qPCR validation results of circRNA, miRNAs, and mRNAs. (G,H) RNase R digestion followed by RT-qPCR of circRNA and their host genes. ** p < 0.01.

Figure 3

GO and KEGG analysis of circRNA, miRNA, and mRNA. (A,B) Top 10 GO terms for differentially expressed circRNA and mRNAs in biological processes, cellular components, and molecular functions. (C,D) KEGG analysis of differentially expressed circRNA and mRNAs. Only the top 10 enriched KEGG pathways are shown in the figure.

Figure 4

Construction and Analysis of circRNA-miRNA-mRNA ceRNA Network. (A) Regulatory networks of circRNA-miRNA-mRNA interactions in mesenchymal and cartilage tissue of antlers. (B) The top 10 GO terms of the ceRNA network in biological process, cellular component, and molecular function. (C) KEGG analysis of the pathways of the ceRNA network. The figure shows only the top 10 enrichment KEGG pathways.

Figure 5

circRNA2829 was identified as a circular RNA related to antler regeneration and differentiation. (A) The circRNA-miRNA-mRNA interaction network is related to the development and regeneration of antlers. (B) The top five circRNA in the ceRNA network with relative expression levels were detected by RT-qPCR. (C) The agarose gel electrophoresis was performed using divergent primers and convergent primers to verify the existence and circular structure of circRNA2829. (D) Sanger sequencing was used to validate that the back splice site existed. (E,F) Relative expression levels of GAPDH and circRNA2829 after RNase R treatment or after Act-D treatment were examined by RT-qPCR. (G,H) Relative expression levels of circRNA2829 were detected by RT-qPCR after treatment of chondrocytes with OE-circRNA2829 or si-circRNA2829. ** p < 0.01, * p < 0.05.

Figure 6

circRNA2829 promotes chondrocyte proliferation and osteogenic differentiation. After transfection of OE-circRNA2829 or si-circRNA2829 in chondrocytes, CCK-8 (A,B) and EdU (C) were used to determine cell proliferation ability; BCIP/NBT staining (D) and ALP viability assay (E,F) to assess the osteogenic differentiation ability of chondrocytes; RT-qPCR (G,H) and Western blot (I,J) to detect changes in the transcriptional and translational levels of osteogenic marker-related genes. ** p < 0.01, * p < 0.05.

Figure 7

MiR-4286-R+1 inhibits chondrocyte proliferation and osteogenic differentiation. (A,B) Detection of miR-4286-R+1 expression level after transfection with mimic or inhibitor by RT-qPCR. (C) RT-qPCR detection of si-FOXO4 interference efficiency. After transfection of miR-4286-R+1 mimic or inhibitor in chondrocytes, CCK-8 (D,E) and EdU (F) were used to determine cell proliferation ability; BCIP/NBT staining (G) and ALP viability assay (H,I) to assess the osteogenic differentiation ability of chondrocytes; RT-qPCR (J,K) and Western blot (L,M) to detect changes in the transcriptional and translational levels of osteogenic marker-related genes. ** p < 0.01, * p < 0.05.

Figure 8

circRNA2829 promotes chondrocyte proliferation and differentiation via the miR4286-R+1/FOXO4 axis. (A,B) RT-qPCR to detect changes in FOXO4 expression levels in chondrocytes after transfection with inhibitor or si-FOXO4. After cotransfection of si-circRNA and inhibitor or cotransfection of OE-circRNA and si-FOXO4 in chondrocytes, cell proliferation ability was measured by CCK-8 (C,D) and EdU (E,F); osteogenic differentiation ability of chondrocytes was assessed by BCIP/NBT staining (G,H) and ALP viability assay (I,J); RT-qPCR (K,L) and Western blotting (M,N) were used to detect changes in the mRNA and protein of osteogenic marker-related genes. (O,P) Dual luciferase reporter gene assay to demonstrate the binding site of miR-4286-R+1 to circRNA2829 and FOXO4; (Q) RT-qPCR to detect the expression levels of circRNA2829 and miR-4286-R+1 in RIP antibody complexes. ** p < 0.01, * p < 0.05.