Inhibition of AHNAK nucleoprotein 2 alleviates pulmonary fibrosis by downregulating the TGF‐β1/Smad3 signaling pathway

Abstract

Background Idiopathic pulmonary fibrosis (IPF) is a chronic and advanced interstitial lung disease with poor prognosis. AHNAK nucleoprotein 2 (AHNAK2) is a macromolecular protein that is important for cell migration and muscle membrane repair. The protein acts via epithelial–mesenchymal transition (EMT), which is a key mechanism in the pathogenesis of IPF. However, very few studies have elucidated the effect of AHNAK2 in the development of IPF. Therefore, we aimed to determine the role of AHNAK2 in IPF development. Methods C57BL/6 mice were induced with bleomycin, while A549 and Beas‐2b pulmonary epithelial cell lines were treated with TGF‐β1 to induce IPF model. The expression of AHNAK2 was detected using immunohistochemistry staining in vivo, and real‐time quantitative polymerase chain reaction (RT‐qPCR) and western blotting (WB) in vitro. C57BL/6 mice were injected with adeno‐associated virus (AAV)‐sh NC or AAV‐sh AHNAK2 and the pulmonary function and EMT marker expression were measured. The migratory abilities of the two transforming growth factor beta 1 (TGF‐β1)‐induced cell lines were examined using wound‐healing and Transwell assays after transfection with si‐NC, si‐AHNAK2‐1 and ‐2. EMT marker expression was detected using RT‐qPCR and WB. Smad3 and phosphorylated‐Smad3 of the two cells were examined using WB. Following Smad3 inhibition by Smad3 phosphorylation inhibitor (SIS3), TGF‐β1‐induced cell migration and EMT marker expression were evaluated again after different transfections. Results AHNAK2 expression was higher in the IPF model than in the normal model in vivo and in vitro. Partial inhibition of AHNAK2 suppressed the EMT process and improved pulmonary ventilation and compliance in the mouse model of IPF. Similarly, knockdown of AHNAK2 suppressed the migration of pulmonary epithelial cells and reversed EMT. Furthermore, Smad3 of the two TGF‐β1‐induced cell lines was not activated when AHNAK2 was inhibited. When SIS3 inhibited the activation of Smad3, the suppression of AHNAK2 had no effect on A549 and Beas‐2b, regardless of TGF‐β1 induction. Conclusions Inhibition of AHNAK2 alleviates pulmonary fibrosis and partially reverses EMT by inhibiting the TGF‐β1/Smad3 signaling pathway. Therefore, AHNAK2 is a potential therapeutic target for IPF.

Glycoprotein M6A Suppresses Lung Adenocarcinoma Progression via Inhibition of the PI3K/AKT Pathway

“…Human lung adenocarcinoma cell lines (A549, H1299) and normal pulmonary epithelial cells (BEAS-2B) were acquired from the Chinese Academy of Science (Shanghai, China) cell bank. Te cells were maintained in Dulbecco's modifed Eagle's medium (DMEM; Hyclone, Camarillo, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C in a 5% CO 2 incubator [17]. Te DMEM was replaced two to three times per week.…”

“…Te 2 -ΔΔCT method was used to analyse gene expression. Te primer sequences were obtained from Sangon (Shanghai, China) and listed in Table 1 [17].…”

“…Te two cell lines were seeded in 6-well plates at 70% confuence and transfected with siRNA using Lipofectamine 3000 ™ (Termo, Waltham, MA, USA). Te transfection efciency of siRNAs was detected after 48 h of transfection [17]. Te overexpression plasmid packaged into lentivirus (HBLV-h-GPM6A-3xfag-ZsGreen-PURO) (Lv-GPM6A), and also, the control plasmids (HBLV-ZsGreen-PURO) (vector) were synthesized by Hanheng Biotechnology (Shanghai) Co., Ltd. Te lentivirus was added to the cells after they reached 50% confuence in the 6well plates.…”

“…Te cells were imaged at 0 and 24 h after the wound gap was formed. Te relative migration area was measured using ImageJ software [17].…”

“…After the protein concentrations were measured, the collected supernatants were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difuoride membrane. After incubation with primary antibodies at 4 °C overnight, the membrane was incubated with secondary antibodies (1 : 10000; goat anti-rabbit IgG; ab6721; Abcam) and exposed using an ECL assay kit [17].…”