MiR-29b-3p promotes chondrocyte apoptosis and facilitates the occurrence and development of osteoarthritis by targeting PGRN

Abstract

This study was aimed to explore the role of miR-29b-3p and PGRN in chondrocyte apoptosis and the initiation and progress of osteoarthritis (OA). Both miR-29b-3p and PGRN were up-regulated in cartilage tissue from patients with OA. Transfection of miR-29b-3p mimic into rat primary chondrocytes and SW1353 chondrosarcoma cells significantly suppressed PGRN expression and release, induced apoptosis, inhibited proliferation and scratch wound closure. By contrast, transfection of miR-29b-3p inhibitor exhibited the opposite effects. Moreover, the expression and secretion of cartilaginous degeneration-related molecules were also altered by miR-29b-3p. Luciferase reporter gene assay showed rat GRN mRNA is directly targeted and repressed by miR-29b-3p. The fact that recombinant PGRN or shPGRN-mediated PGRN interference abolished miR-29b-3p mimic-induced cell apoptosis and growth inhibition suggested miR-29b-3p affect the cellular functions of chondrocyte through regulating PGRN expression. In vivo, joint cavity injection of miR-29b-3p antagomir prior to surgical induction of OA significantly suppressed the upregulation of miR-29b-3p, whereas further promoted the increased expression of PGRN. Articular chondrocytes apoptosis and cartilage loss in the knee joint of surgically induced OA rats were also ameliorated by the injection of miR-29b-3p antagomir, demonstrated by TUNEL and safranin O-fast green staining. This work showed miR-29b-3p facilitates chondrocyte apoptosis and OA by targeting PGRN, and miR-29b-3p or PGRN may be the potential target for OA treatments.

Keywords: Chondrocyte; MiR-29b-3p; Osteoarthritis; Progranulin.

Figure 1

Expression levels of miR‐29b‐3p and PGRN in cartilage tissues from osteoarthritis and fracture patients. A. Relative miR‐29b‐3p levels in clinical specimens detected by qRT–PCR. B. Relative expression of GRN at transcriptional level detected by qRT–PCR. C‐D. Protein level of PGRN in clinical specimens measured by Western blotting and the semi‐quantitative densitometry results. *P < 0.05, **P < 0.01, Fracture versus Osteoarthritis.

Figure 2

miR‐29b‐3p negatively regulates PGRN in primary cultured rat chondrocytes and SW‐1353 human chondrosarcoma cells. A‐B. miR‐29b‐3p and GRN mRNA levels in the two types of cells after transfected with miR‐29b‐3p mimic or inhibitor were determined by qRT–PCR. C. After transfection with the mimic or the inhibitor, ELISA was performed to detect the secretion of PGRN. D‐E. Representative Western blots of intracellular PGRN of the primary chondrocytes and SW‐1353 cells and the corresponding semi‐quantitative densitometry results shown in histogram. *P < 0.05, **P < 0.01, compared with NC (negative control).

Figure 3

miR‐29b‐3p accelerated apoptosis of primary cultured rat chondrocytes and SW‐1353 cells. A. Photomicrographs (400 × ) of the nucleus the cells stained with DAPI. B. Cell apoptosis of the chondrocytes and chondrosarcoma cell line analysed using flow cytometry (FCM). The histogram showed the results based on three paralleled experiments. C‐D. The protein level of cleaved caspase‐3, Bax and Bcl2 measured with Western blotting. The histogram showed the relative level of cleaved caspase‐3 and the ratio of Bax to Bcl2. *P < 0.05, **P < 0.01, compared with NC; ^#P < 0.05, ^##P < 0.01, compared with TRAIL treatment group.

Figure 4

miR‐29b‐3p inhibited the progressions of primary rat chondrocytes and SW‐1353 human chondrosarcoma cells. A. miR‐29b‐3p mimic and inhibitor significantly altered the proliferation of the chondrocytes and SW‐1353 cells. B. miR‐29b‐3p mimic‐induced cell cycle arrest and miR‐29b‐3p inhibitor caused accelerated cell division. C. miR‐29b‐3p mimic hindered the closure of scratch wound, while the inhibitor promoted the healing of scratch wound. *P < 0.05, **P < 0.01, compared with NC.

Figure 5

miR‐29b‐3p regulated cartilaginous degeneration‐related molecules, including MMP‐1, MMP‐13, COL II and COL X, and direct targeted at 3′ UTR of rat GRN (rGRN) mRNA. A‐D. Concentrations of secreted MMP‐1, MMP‐13, COL II and COL X in the media of rat primary chondrocytes or SW‐1353 human chondrosarcoma cells after transfection with the miR‐29b‐3p mimic or the inhibitor measured by ELISA assay. E, F. mRNA expression of COL2A1 and COL10A1 of rat primary chondrocytes or SW‐1353 human chondrosarcoma cells after transfection with the miR‐29b‐3p mimic or the inhibitor measured by qRT–PCR assay. G. Schematic representation of miR‐29b‐3p's predicted binding site in the 3′UTR of rGRN mRNAs. H, I. The relative luciferase activity after the cotransfection of miR‐29b‐3p mimic and 3′ UTR of GRN mRNA into HEK293 and SW1353 cells respectively. *P < 0.05, **P < 0.01, compared with NC (negative control). NS: not significant.

Figure 6

Recombinant PGRN or shPGRN‐mediated PGRN interference antagonized miR‐29b‐3p mimic‐induced cell apoptosis and growth inhibition. A‐C/G‐I. The protein level of cleaved caspase‐3, Bax and Bcl2 measured with Western blotting. D/J. Percentage of apoptotic cells determined by FCM after treatment with miR‐29b‐3p mimic or combination of PGRN and the mimic. E/K. Cell proliferation measured with CCK‐8 kit. F/L. Percentage of wound closure measured on the basis of scratch wound assay. *P < 0.05, **P < 0.01, compared with NC (negative control); ^#P < 0.05, ^##P < 0.01, compared with Wild‐type mimic group.

Figure 7

Knock‐down of miR‐29b‐3p resulted in decreased cell apoptosis of articular chondrocytes of osteoarthritic rats. A. 2, 4 days, 3, 4 or 6 weeks after surgery, miR‐29b‐3p level in the tibial cartilage was detected by qRT–PCR. B‐C. Three, four or six weeks after surgery, PGRN in the tibial cartilage was measured with Western blotting, and the semi‐quantitative results were shown in the histogram. D‐E. Cleavage of caspase‐3 and the Bax/Bcl2 ratio in the cartilage were measured with Western blotting, and the histogram presented the semi‐quantitative results. F‐G. Apoptotic chondrocytes in the cartilage were identified by TUNEL analysis, and cell counting results of the positive stained were presented in the histogram. **P < 0.01, *P < 0.05, compared with OA/NC group; ^##P < 0.01, ^#P < 0.05, compared with Sham group.

Figure 8

Knock‐down of miR‐29b‐3p attenuated cartilage loss in knee joint of osteoarthritic rats. A‐B. Protein levels of catabolic biomarkers (ADAMTS‐5, ‐7, MMP‐1, ‐13 and COMP fragment), anabolic biomarkers (COL II and Aggrecan), the hypertrophic chondrocyte marker (COL X) and inflammatory cytokines (IL‐1β and TNF‐α). C. Representative safranin O‐fast green staining of the knee joint from each group. D. Cartilage thickness of tibia measured by histomorphometry. E. Analysis of degenerative changes by Mankin's scoring. F. **P < 0.01, *P < 0.05, compared with OA/NC group; ^##P < 0.01, ^#P < 0.05, compared with Sham group.