Deer antler reserve mesenchyme cells modified with miR-145 promote chondrogenesis in cartilage regeneration

Abstract

Deer antler-derived reserve mesenchyme cells (RMCs) are a promising source of cells for cartilage regeneration therapy due to their chondrogenic differentiation potential. However, the regulatory mechanism has not yet been elucidated. In this study, we analyzed the role of microRNAs (miRNAs) in regulating the differentiation of RMCs and in the post-transcriptional regulation of chondrogenesis and hypertrophic differentiation at the molecular and histological levels. The results showed that RMCs showed typical MSC differentiation potentials. During chondrogenic differentiation, we obtained the expression profile of miRNAs, among which miR- 145 was the most prominent candidate as a key microRNA involved in the balance of chondral and endochondral differentiation. Knockdown of miR-145 promoted chondrogenesis and inhibited hypertrophy differentiation in RMCs. Mechanically, by prediction through online databases combined with dual-luciferase reporter assay, SOX9 was suggested as a target of miR-145. Further validation experiments confirmed that knockdown of miR-145 contributed to the balance between endochondral versus chondral differentiation of RMCs by targeting SOX9. Additionally, RMCs transfected with the miR-145-knockdown-mediated lentiviral vector successfully promoted cartilage regeneration in vivo. In summary, our study suggested that the reciprocal negative feedback between SOX9 and miR-145 was essential for balancing between endochondral versus chondral differentiation of RMCs. Our study suggested that modification of RMCs using miRNAs transduction might be an effective treatment for cartilage defects.

Keywords: SOX9; cartilage regeneration; chondrogenesis; miR-145; reserve mesenchyme cells.

Figure 1

Identification of characteristics of RMCs and chondrocytes. (A) Morphology of RMCs. (B) Morphology of chondrocytes. (C) Alisin blue staining of chondrocytes. (D) IF analysis of RMCs surface markers. (E) Multilineage differentiation capacity. (F) Analysis of adipogenic, chondrogenic, and osteogenic gene expression after multilineage differentiation of RMCs by qRT-PCR. (G) Analysis of chondrogenic protein expression by WB.

Figure 2

miR-145 was assumed as a putative miRNA that might participate in the chondrogenic differentiation of RMCs. (A) Volcano map of differentially expressed miRNAs in the pairwise comparisons. (B) Venn diagram of differentially expressed miRNAs in the pairwise comparisons. (C) Clustering heatmap of 74 differentially co-expressed miRNAs. (D) Clustering heatmap of 12 candidate miRNAs related to antler growth. (E) Analysis of miR-145 expression after chondrogenic differentiation of RMCs by qRT-PCR. CP_0: RMCs induced chondrogenic differentiation for 0 days; CP_7: RMCs induced chondrogenic differentiation for 7 days; CP_14: RMCs induced chondrogenic differentiation for 14 days; CC: chondrocytes.

Figure 3

miR-145 inhibited chondrocyte differentiation of RMCs. (A) The relative expression of miR-145 in RMCs with different treatments was evaluated by qRT-PCR. (B) Alisin blue staining of RMCs with different treatments. (C) Quantitative analysis of Alcian blue staining. (D) The expression of chondrogenic genes of RMCs with different treatments was analyzed using qRT-PCR. (E) IF of COL II of the RMCs with different treatments. (F) Quantitative analysis of IF of COL II. (G) IF of COMP of the RMCs with different treatments. (H) Quantitative analysis of IF of COMP. (I) IF of COL X of the RMCs with different treatments. (J) Quantitative analysis of IF of COL X. (K) The expression of chondrogenic proteins of RMCs with different treatments was analyzed using WB; (L) Quantitative analysis of WB.

Figure 4

miR-145 targeted and suppressed SOX9 expression. (A) The target genes of miR-145 were predicted by online websites. (B) Venn diagram of differentially expressed target genes of miR-145 in the pairwise comparisons. (C) Clustering heatmap of 128 differentially co-expressed target genes of miR-145. (D) qRT-PCR analysis of SOX9 expression during RMC chondrogenic differentiation. (E) The expression trend of miR-145 and SOX9 was negatively correlated. (F) Normalized luciferase activity after co-transfection of miR-NC mimics or miR-145 mimics together with SOX9 3’UTR-WT or SOX9 3’UTR-MUT. (G) The expression of SOX9 of RMCs with different treatments was analyzed using IF. (H) Quantitative analysis of IF. (I) The expression of SOX9 of RMCs with different treatments was analyzed using qRT-PCR. (J) The expression of SOX9 of RMCs with different treatments was analyzed using WB. (K) Quantitative analysis of WB.

Figure 5

MiR-145-targeted SOX9 to inhibit chondrogenic differentiation of RMCs. (A) Alisin blue staining of RMCs with different treatments. (B) Quantitative analysis of Alcian blue staining. (C) The expression of chondrogenic genes of RMCs with different treatments was analyzed using qRT-PCR. (D) The expression of chondrogenic proteins of RMCs with different treatments were analyzed using WB. (E) Quantitative analysis of WB.

Figure 6

The expression of chondrogenic and hypertrophic markers in RMCs transfected with different lentiviruses were identified using IF. (A) IF of COL II of the RMCs with different treatments. (B) Quantitative analysis of IF of COL II. (C) IF of COMP of the RMCs with different treatments. (D) Quantitative analysis of IF of COMP. (E) IF of SOX9 of the RMCs with different treatments. (F) Quantitative analysis of IF of SOX9. (G) IF of COL X of the RMCs with different treatments. (H) Quantitative analysis of IF of COL X.

Figure 7

Low expression of miR-145 enhanced the repair efficacy of the RMCs and reduced their hypertrophic differentiation in the rat cartilage injury model. (A) Femoral was evaluated by Gross appearance. (B) Macroscopic ICRS scores of the femur. (C) Femoral section was evaluated using HE staining. (D) Pineda scoring of the HE staining. (E) Femoral section was evaluated using Safranin-O-Fast green staining. (F) Wakitani scoring of the Safranin-O-Fast green staining.

Figure 8

Immunohistochemical staining of SOX9, COL II, COL X, and COL I in femoral sections to evaluate repair efficacy.

Figure 9

Schematic to illustrate that deer antler reserve mesenchyme cells modified with miR-145 promotes chondrogenesis in cartilage regeneration by targeting SOX9.