Mir 503 agomir

Male C57BL/6 mice (4–6 weeks of age, 18–20 g) were purchased from Shanghai Laboratory Animal Center (SLAC, Shanghai, China), and were fed in sterile-free conditions of the animal center for one week before experiment to adapt to the environment. All animal studies were approved by the Ethics of Committee of Nanjing Medical University (Nanjing, China). Silica-induced mouse pulmonary fibrosis model was performed by intratracheal instillation of 50 μl 50 g/l silica turbid liquids for the three experimental group, and with equal amount of saline for the control group (n = 10 for each group). The mice were sacrificed on day 7, 14 and 28 after the treatment. The lungs were collected, quick-frozen in liquid nitrogen and store at −80 °C for further study. The miR-503 overexpression mouse lung fibrosis model was conducted by intratracheal instillation of 200 nmol/kg miR-503 agomir (RiboBio Co, Ltd, Guangzhou, China) after silica instillation. And on day 7, 14, 21 after the molding, 120nmol/kg miR-503 agomir were given to the miR-503 over expression group via tail vein injection. The mice were sacrificed at day 28.

C57BL/6 male mice (4‐6 weeks of age) were purchased from SLAC Laboratory Animal Co., Ltd., (Shanghai, China). All experimental protocols were approved by the Animal Care and Use Committee at Nanjing Medical University. The mice were anaesthetized with an intraperitoneal injection of 1% pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan). A 0.05 mL sterile saline containing silica (50 mg/kg, Sigma‐Aldrich, St. Louis, MO, USA) was directly instilled intratracheally. The control group was instilled with 0.05 mL saline. The lung tissues were harvested on day 7, 14 or 28 after silica instillation. The miR‐503 up‐regulation mouse model was conducted by a co‐instillation of 200 nmol/kg miR‐503 agomir (RiboBio Co., Ltd., Guangzhou, China) with silica instillation. Subsequently, 120 nmol/kg miR‐503 agomir was injected via the tail vein weekly. The lung tissues were harvested at day 28.

Dulbecco's modified Eagle's medium (DMEM), opti-MEM, fatal bovine serum (FBS), 0.25% Trypsin-EDTA, and penicillin/streptomycin were ordered from GIBCO, Thermo Fisher Scientific (Waltham, MA, USA). The primers of miR-503 and U6 snRNA, the miR-503 agomir, antagomir, and their corresponding negative control (NC) were ordered from RiboBio (Guangzhou, China). The rabbit polyclonal anti-Bcl-2 antibody (ab196495) was purchased from Abcam (Cambridge, UK). The anti-PI3 Kinasep85 (4292S), anti-phospho-Akt (T450) (9267S), anti-phospho-Akt (T308) (4056S), anti-JAK2 (3230S), anti-phospho-JAK2 (Y1007/1008) (3771S), anti-phospho-Stat3(Y705) (9131S), anti-Stat3 (9139S), anti-Akt1 (2938S), anti-Akt2 (3063S), anti-Bax(2772S), anti-β-actin (4967S), and anti-GADPH (2118S) antibodies were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA). All other analytical grade chemical reagents were purchased from standard commercial companies.