LncRNA MINCR attenuates osteoarthritis progression via sponging miR-146a-5p to promote BMPR2 expression

Abstract

The purposes of this study are to explore the function and regulatory mechanism of a novel lncRNA MYC-Induced Long non-coding RNA (MINCR) in osteoarthritis (OA). The expression of lncRNA MINCR, miR-146a-5p, and bone morphogenetic protein receptor 2 (BMPR2), Sry-type high-mobility-group box 9 (SOX9), collagen type II alpha 1 (COL2A1), Aggrecan, metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), Matrix metalloproteinase 3 (MMP3), MMP13, COL2A1, and Aggrecan were determined using quantitative real-time PCR (qRT-PCR), western blot, immunohistochemistry (IHC) and immunofluorescence (IF) in vitro and in vivo. And distribution and expression of MINCR were examined by fluorescence in situ hybridization (FISH). Cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) assay, 5-Ethynyl-2'-deoxyuridine (EdU) staining, Annexin V-FITC/Propidium Iodide (PI), and Terminal Deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) staining in vitro and in vivo. The anterior cruciate ligament transection (ACLT) rat model was constructed to analyze the MINCR/miR-146a-5p/BMPR2 axis in vivo. The cartilage degeneration was determined by pathological staining with Hematoxylin and Eosin (H&E) and Safranin O staining. The binding relationship between MINCR and miR-146a-5p, and between miR-146a-5p and BMPR2 were determined by a dual-luciferase reporter gene, RNA Immunoprecipitation (RIP) assay, and RNA-pull down assays. Here, MINCR and BMPR2 were downregulated whereas miR-146a-5p was upregulated in OA cartilage tissues compared with control as well as IL-1β-induced chondrocytes compared with normal chondrocytes. Function experiments indicated that MINCR upregulation promoted cell proliferation and inhibited apoptosis and extracellular matrix (ECM)-degeneration. We also proved the binding relationship between MINCR and miR-146a-5p, and the BMPR2 acted as a target of miR-146a-5p. Mechanism analysis using rescue experiments in vitro and in vivo, MINCR silencing reversed the effects of miR-146a-5p downregulation in OA. Overexpression of miR-146a-5p also reversed the function of BMPR2 overexpression in OA. These data indicated that MINCR prevented OA progression via targeting miR-146a-5p to promote BMPR2 expression.

Keywords: BMPR2; MINCR; Osteoarthritis; inflammation; miR-146a-5p.

Figure 1.

Downregulation of MINCR in OA tissues and IL-1β-induced chondrocytes. (a) FISH was utilized to examine the expression and subcellular distribution of MINCR in cartilage tissues from OA patients and normal. (b)-(c) QRT-PCR was used to detect the mRNA expression of MINCR in OA cartilage tissues compared with normal as well as IL-1β-induced chondrocytes compared with normal chondrocytes. **P < 0.01, ***P < 0.001.

Figure 2.

Upregulation of MINCR promotes cell proliferation whereas suppresses apoptosis and ECM-degeneration in IL-1β-induced chondrocytes. (a) QRT-PCR was used to detect the mRNA expression of MINCR after pcDNA3.1-mediated MICNR overexpression. (b) CCK-8 assay was used to examine cell viability after IL-1β stimulation, MINCR overexpression, and IL-1β stimulation + MINCR overexpression. (c)-(d) EdU/Hocheast33258 double staining was used to detect the living cells and apoptotic cells after IL-1β stimulation, MINCR overexpression, and IL-1β stimulation + MINCR overexpression. (e) Annexin V-FITC/PI was used to analyze apoptotic cells after IL-1β stimulation, MINCR overexpression, and IL-1β stimulation + MINCR overexpression. (f)-(h) QRT-PCR and western blot were used to examine mRNA and protein levels of SOX9, COL2A1, Aggrecan, ADAMTS-4, MMP3, and MMP13 after IL-1β stimulation, MINCR overexpression, and IL-1β stimulation + MINCR overexpression. (i)-(j) IF staining was used to detect the levels of ADAMTS-4, MMP13, COL2A1, and Aggrecan after IL-1β stimulation, MINCR overexpression, and IL-1β stimulation + MINCR overexpression. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

miR-146a-5p Serves as A Target for MINCR. (a) The binding site sequences of MINCR on miR-146a-5p. (b) Dual-luciferase reporter gene assay was used to detect the binding relationship between MINCR and miR-146a-5p after wild/mutant type of MINCR-recombination reporter gene co-transfection with miRNA mimics into cells. (c) The MINCR enrichment was detected by qRT-PCR after RNA-pull down performing. (d) The MINCR expression was analyzed by qRT-PCR after RNA samples were bound to Ago2 antibody by RIP assay. (e) The miR-146a-5p expression was detected by qRT-PCR after overexpression of MINCR. (f)-(g) The miR-146a-5p expression was examined by qRT-PCR in OA cartilage tissues compared with normal as well as IL-1β-induced chondrocytes compared to normal chondrocytes. **P < 0.01, ***P < 0.001.

Figure 4.

Downregulation of MINCR reverses effects of miR-146a-5p silencing on IL-1β-induced chondrocytes. (a) The efficiency of knockdown of MINCR using shRNA was detected by qRT-PCR. After IL-1β stimulation, IL-1β stimulation + miR-146a-5p inhibitor, and IL-1β stimulation + miR-146a-5p inhibitor + MINCR silencing, (b) CCK-8 assay was used to examine cell viability. (c)-(d) EdU/Hocheast33258 double staining was used to detect the living cells and apoptotic cells. (e) Annexin V-FITC/PI was used to analyze apoptotic cells. (f)-(g) QRT-PCR and western blot were used to examine mRNA and protein levels of SOX9, COL2A1, Aggrecan, ADAMTS-4, MMP3, and MMP13. (h) IF staining was used to detect the levels of ADAMTS-4, MMP13, COL2A1, and Aggrecan. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

BMPR2 Acts as A Potential Target of miR-146a-5p, And MINCR Promotes BMPR2 via Inhibiting miR-146a-5p. (a) The binding sites of BMPR2 on miR-146a-5p. (b) The luciferase activity was performed after recombination reporter genes which contained wild/mutant type of BMPR2 and miRNA mimics transfected into cells. (c)-(d) The mRNA and protein expression of BMPR2 were detected by qRT-PCR and western blot after being transfected with miR-146a-5p mimic/inhibitor. (e)-(g) The expression of BMPR2 in OA cartilage tissues compared with normal as well as IL-1β-induced chondrocytes compared with normal chondrocytes. (H)-(I) The mRNA and protein expression of BMPR2 were detected by qRT-PCR and western blot after being transfected with miR-146a-5p mimic and miR-146a-5p mimic + BMPR2. *P<0.05, **P<0.01, ***P<0.001.

Figure 6.

Upregulation of BMPR2 inverts the effects of miR-146a-5p on cell proliferation, apoptosis, and ECM-degeneration in IL-1β-induced chondrocytes. After IL-1β stimulation, IL-1β stimulation + BMPR2, and IL-1β stimulation + miR-146a-5p mimic + MINCR, (a) CCK-8 assay was used to examine cell viability. (b)-(c) EdU/Hocheast33258 double staining was used to detect the living cells and apoptotic cells. (d) Annexin V-FITC/PI was used to analyze apoptotic cells. (e)-(g) QRT-PCR and western blot were used to examine mRNA and protein levels of SOX9, COL2A1, Aggrecan, ADAMTS-4, MMP3, and MMP13. (h)-(i) IF staining was used to detect the levels of ADAMTS-4, MMP13, COL2A1, and Aggrecan. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

Upregulation of MINCR Prevents pathological alterations in ACLT rat model. (a)-(b) The expression of MINCR and miR-146a-5p was detected by qRT-PCR in normal rats (normal), OA rats (OA), pcDNA3.1-transfected OA rats (OA+OE-NC), MINCR-overexpressed OA rats (OA+OE-MINCR). (d) Column charts of the statistical results of BMPR2 positive cells in the normal group, OA group, OA+OE-NC group, and OA+OE-MINCR group. (e) Column charts of the statistical results of TUNEL positive cells s in the normal group, OA group, OA+OE-NC group, and OA+OE-MINCR group. (f) IHC, Safranin O, and H&E staining were respectively used to examine BMPR2 levels, collagen-containing, and cartilage-degeneration s in the normal group, OA group, OA+OE-NC group, and OA+OE-MINCR group. (g) TUNEL staining was used to examine apoptotic cells in cartilage tissues s in the normal group, OA group, OA+OE-NC group, and OA+OE-MINCR group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8.

Upregulation of MINCR Prevents ECM-degeneration in ACLT rat model. (a)-(b) The mRNA and protein expression of SOX9, COL2A1, Aggrecan, ADAMTS-4, MMP3, and MMP13 were analyzed by qRT-PCR and western blot in the normal group, OA group, OA+OE-NC group, and OA+OE-MINCR group. (c)-(e) IF staining was utilized to detect the levels of ADAMTS-4, MMP13, COL2A1, and Aggrecan in the normal group, OA group, OA+OE-NC group, and OA+OE-MINCR group. *P < 0.05, **P < 0.01, ***P < 0.001.