Fig 2: Replenishment of FBXL16 alleviates CS-induced experimental COPD in mice. C57BL/6 mice were normally ventilated (Air) or exposed to chronic smoking (CS) for 16 weeks to induce experimental COPD. Mice were intranasally administered with control (Luc, AAV6-expressing luciferase reporter) or FBXL16 expressing AAV6 twice. (A) The relative mRNA level of FBXL16 in mice after AAV6-FBXL16 treatment. (B) The percentage of body weight gain of mice after CS challenge with or without AAV6-FBXL16 treatment. (C) Macrophages and neutrophils were enumerated in bronchoalveolar lavage fluid (BALF) (n = 8 mice per group). (D) Histological examination of lung tissues stained by hematoxylin-eosin staining. Scale bar, 100 μm. (E) The mean linear intercept of lung tissues calculated based on (D). The change of TNF-α (F) and CXCL1 (G) protein in whole-lung homogenates. (H-I) Pulmonary function, including FEV100/FVC and FRC were determined (n = 8 mice per group). Data are presented as means ± SD. ns, no significance. *p < 0.05; **p < 0.01; ***p < 0.001

Fig 3: HIF1ɑ mediates FBXL16 regulated fibroblast activation. (A) The mRNA expression of HIF1ɑ and FBXL16 in isolated primary human lung fibroblasts derived from COPD patients and normal counterparts (n = 6 per group). (B) MRC-5 were transfected with wild type and point mutated (S31D) HIF1ɑ and treated as indicated. The expression of HIF1ɑ was determined by western blotting. HA-tag was detected to confirm ectopic expression of HIF1ɑ. (C) The markers of fibroblast transdifferentiation from (B) were determined by real-time PCR (n = 3). The values were normalized by β-actin expression. (D) C57BL/6 mice were normally ventilated (Air) or exposed to chronic smoking (CS) for 16 weeks to induce experimental COPD. Mice were intranasally administered with control or FBXL16 expressing AAV6 twice. The protein abundance of HIF1ɑ and FBXL16 in the lungs of mice were measured. Left, the representative result was shown; right, the intensity of bands was calculated by ImageJ and normalized by β-actin expression (n = 3). (E) MRC-5 cells were transfected with HIF1ɑ and/or FBXL16 expressing plasmid and treated by TGF-β as indicated. The markers of lung fibroblast activation (ɑ-SMA and fibronetion) were determined. Left, the representative result was shown; right, the intensity of bands was calculated by ImageJ and normalized by β-actin expression (n = 3). (F) MRC-5 cells were transfected with control or miR-1307-5p agomir (miR) and treated by TGF-β as indicated. The protein abundance of HIF1ɑ was measured. Left, the representative result was shown; right, the intensity of bands was calculated by ImageJ and normalized by ɑ-tubulin expression (n = 3). (G) C57BL/6 mice were normally ventilated (Air) or exposed to chronic smoking (CS) for 16 weeks to induce experimental COPD. MiR-1307-5p agomir (miR) or scrambled (Scr) agomir were treated intranasally weekly. The protein abundance of HIF1ɑ and FBXL16 in the lungs of mice were measured by western blotting. Left, the representative result was shown; right, the intensity of bands was calculated by ImageJ and normalized by β-actin expression (n = 3). Data are presented as means ± SD. n.s., not significant. *p < 0.05; **p < 0.01; ***p < 0.001

Fig 4: miR-1307-5p directly targets FBXL16 mRNA. (A) The seed region of miR-1307-5p predicted to target the 3’ UTR of FBXL16 in human. (B) MRC-5 cells were transfected with control or miR-1307-5p agomir (miR). The mRNA level of FBXL16 was measured by real-time PCR 24 h after transfection (n = 3). (C) Depiction of luciferase reporter construct for wild-type (WT) and mutated (MT) 3’-UTR of the FBXL16 seed region. (D) 293T cells were transfected with 50 ng of either pMirTarget vector carrying WT or mutated 3’ UTR of FBXL16 with or without 50 nM miR-1307-5p agomir. (E) The lung fibroblasts derived from normal or COPD lung tissues were transfected with luciferase reporter construct carrying either WT or MT 3’ UTR of FBXL16 (n = 3). Data are presented as means ± SD. n.s., not significant. **p < 0.01

Fig 5: miR-1307-5p enhances lung fibroblast differentiation through inhibition of FBXL16. (A) MRC-5 were treated as indicated. The expression of FBXL16 was measured by real-time PCR (n = 3). (B) MRC-5 cells were transfected with siRNA targeting FBXL16 (siFBXL16) and/or miR-1307-5p antagomir (Ant-1307) for 72 h after TGF-β treatment. Representative images of immunofluorescence staining and quantification of α-SMA expression were shown. (C) MRC-5 cells were transfected with miR-1307-5p agomir (Agomir) and control (Luc) or FBXL16 expressing plasmids for 72 h after TGF-β treatment. The markers of lung fibroblast differentiation were determined by real-time PCR (n = 3). The values were normalized by β-actin expression. Data are presented as means ± SD. n.s., not significant. *p < 0.05; **p < 0.01