TMF inhibits miR-29a/Wnt/β-catenin signaling through upregulating Foxo3a activity in osteoarthritis chondrocytes

Abstract

Background: miR-29a, a downstream factor of Wnt/β-catenin signaling, promotes the activity of the Wnt/β-catenin signaling in a positive feedback loop. Our previous work showed that 5,7,3',4'-tetramethoxyflavone (TMF), a major constituent from Murraya exotica L., exhibited chondroprotective activity by inhibiting the activity of Wnt/β-catenin signaling. Purpose: To investigate whether TMF showed the inhibitory effects on miR-29a/β-catenin signaling by up regulation of Foxo3a expression. Methods: Rat knee OA models were duplicated by using Hulth's method. TMF (5 μg/mL and 20 μg/mL) was used for administration to cultured cells, which were isolated from the rat cartilages. Analysis of chondrocytes apoptosis, gene expression, and protein expression were conducted. In addition, miR-29a mimics and pcDNA3.1(+)-Foxo3a vector were used for transfection, luciferase reporter assay for detecting the activity of Wnt/β-catenin signaling, and co-immunoprecipitation for determining proteins interaction. Results: TMF down regulated miR-29a/β-catenin signaling activity and cleaved caspase-3 expression and up regulated Foxo3a expression in OA rat cartilages. In vitro, miR-29a mimics down regulated the expression of Foxo3a and up regulated the activity of Wnt/β-catenin signaling and cleaved caspase-3 expression. TMF ameliorated miR-29a/β-catenin-induced chondrocytes apoptosis by up regulation of Foxo3a expression. Conclusion: TMF exhibited chondroprotective activity by up regulating Foxo3a expression and subsequently inhibiting miR-29a/Wnt/β-catenin signaling activity.

Keywords: Foxo3a; TMF; Wnt/β-catenin; chondrocytes apoptosis; miR-29a; osteoarthritis.

Figure 1

The gross observation, hitomorphological examination, and immunofluorescence assays in rat joint cartilage. The groups were divided in NC (negative control group), Model (OA model group), Low (OA model +25 mg/kg TMF treatment), and High (OA model +100 mg/kg TMF treatment). After 8-week OA model establishment, rats were sacrificed. (A) Joint cartilage was collected for gross observation (the first array), hitomorphological examination by HE staining (the second array), and immunofluorescence assays of Foxo3a (the third array) and β-catenin (the fourth array). The fluorescence intensities of Foxo3a and β-catenin were indicated in the column figure (B), of which the data in the model and TMF-treated groups were compared with those in the NC group. Cartilage tissues were collected for extracting miR-29a, which was determined by qRT-PCR (C). Data were presented by mean ± SD of 6 replicates. P<0.05 was considered to be statistically significant (*P<0.05, **P<0.01, ***P<0.001). Abbreviations: OA, Osteoarthritis; NC, negative control.

Figure 2

Changes of β-catenin, Foxo3a, and cleaved-caspase-3 expression in miR-29a mimics-transfected chondrocytes. Notes: (A) miR-29a mimics and miRNA mimic negative control (miR-NC) were transfected with chondrocytes. The expression of miR-29a was quantified by qRT-PCR. (B) After transfection of miR-29a mimics, the mRNA expressions of β-catenin, Foxo3a, and cleaved-caspase-3 were determined by qRT-PCR. (C and D) After transfection of miR-29a mimics, the protein expressions of β-catenin, Foxo3a, and cleaved-caspase-3 were determined by Western blot. The intensity of protein bands was indicated in the column. 1 (miR-NC group) (miR-NC), 2 (miR-29a mimics-transfected group) (miR-29a), 3 (miR-29a mimics-transfected group +5 μg/mL TMF treatment) (miR-29a + low), and 4 (miR-29a mimics-transfected group +20 μg/mL TMF treatment) (miR-29a + high). (E) Chondrocytes were co-transfected with miR-29a mimics and Topflash or Fopflash luciferase reporters. Transfected cultures were treated with 5 μg/mL (low) or 20 μg/mL TMF (high) for 24 hrs. The values were indicated as Firefly/Renilla ratio in the column. Data were presented by mean ± SD of 3 replicates. P<0.05 was considered to be statistically significant (*P<0.05, **P<0.01).

Figure 3

Inhibitory effects of TMF on apoptosis in miR-29a mimics-transfected chondrocytes. Group (A) was normal untreated chondrocytes. Group (B) was miRNA mimic negative control. Group (C) was miR-29a mimics-transfected chondrocytes. Group (D) was miR-29a mimics-transfected chondrocytes +5 μg/mL TMF (low) treatment. Group (E) was miR-29a mimics-transfected chondrocytes +20 μg/mL TMF (high) treatment. Figure (F) was the summarized data indicating the rate of chondrocytes apoptosis detected by flow cytometry. Data were presented by mean ± SD of 3 replicates. P<0.05 was considered to be statistically significant (*P<0.05, **P<0.01).

Figure 4

miR-29a targeted to degrade the expression of Foxo3a in chondrocytes. Chondrocytes were co-transfected with miRNA mimic negative control (miR-NC), miR-29a mimics, pcDNA3.1(+)-empty vector (EV), and pcDNA3.1(+)-Foxo3a. The expressions of miR-29a (A) and Foxo3a (B) were detected by qRT-PCR. The protein level of Foxo3a was determined by Western blot (C). Summary of the protein bands intensity (D). Data were presented by mean ± SD of 3 replicates. P<0.05 was considered to be statistically significant (*P<0.05, ***P<0.001).

Figure 5

Foxo3a overexpression attenuated the activity of Wnt/β-catenin signaling by competing with TCF4 for binding to β-catenin. Chondrocytes were co-transfected with miR-29a mimics and pcDNA3.1(+)-Foxo3a. After treatment with 5 (low) or 20 μg/mL (high) TMF for 24 hrs, the mRNA (A) and protein (B) expression were detected by RT-PCR and Western blot, respectively. (C) was the summary of protein bands intensity. (B) The co-immuno-precipitation assay was conducted for detecting the interaction between β-catenin and Foxo3a or TCF4. 10% of chondrocytes protein extracts were used as the input, which was subjected to Western Blot. The remaining protein extracts were subjected to IP by using control goat IgG or β-catenin antibody, followed by IB with anti-Foxo3a, anti-TCF4, and anti-β-catenin. (D) Chondrocytes were co-transfected with miR-29a mimics, pcDNA3.1(+)-Foxo3a, and Topflash or Fopflash luciferase reporters. Transfected cultures were treated with 5 or 20 μg/mL TMF for 24 hrs. The values were indicated as Firefly/Renilla ratio in the column. Data were presented by mean ± SD of 3 replicates. P<0.05 was considered to be statistically significant (*P<0.05, **P<0.01).

Figure 6

TMF inhibited miR-29a/Wnt/β-catenin signaling activity by up regulating Foxo3a expression. Wnt/β-catenin signaling up regulated the expression of miR-29a, which in turn promoted the activity of Wnt/β-catenin signaling. miR-29a targeted to degrade Foxo3a. However, TMF up regulated the activity of Foxo3a, leading to attenuation of miR-29a/Wnt/β-catenin signaling.