CIAPIN1 promotes proliferation and migration of PDGF-BB-activated airway smooth muscle cells via the PI3K/AKT and JAK2/STAT3 signaling pathways

Abstract

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is an essential anti-apoptotic protein; however, its role and associated molecular pathways in asthma remain largely unexplored. This study aimed to investigate the potential effects of CIAPIN1 on the proliferation and migration of platelet-derived growth factor BB (PDGF-BB)-induced ASMCs and the underlying mechanisms involved. Considering these aspects, ASMCs are activated with PDGF-BB as a cellular model for asthma. CIAPIN1 is then downregulated using small interfering ribonucleic acid (siRNA). Western blot analysis was performed to assess protein expression. Elevated levels of CIAPIN1 were observed, demonstrating a positive correlation with cytokine levels. CIAPIN1 expression is significantly increased in PDGF-BB-induced human ASMCs. In addition, CIAPIN1 knockdown inhibited proliferation, inflammatory cytokine production, and migration ability, while elevating apoptosis in PDGF-BB-induced human ASMCs. Moreover, CIAPIN1 knockdown inhibited phosphorylated phosphoinositide 3-kinase (p-PI3K), phosphorylated protein kinase B (p-Akt), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein expression. In conclusion, the results indicate that CIAPIN1 regulates the proliferation and migration of human ASMC in response to PDGF-BB by inhibiting the PI3K/AKT and JAK2/STAT3 pathways.

Keywords: CIAPIN1; JAK2/STAT3; PI3K/AKT; asthma; migration; proliferation.

FIGURE 1

Serum CIAPIN1 is highly expressed in patients with asthma. (a) Serum CIAPIN1 level in healthy subjects and asthma patients was measured by ELISA (n = 30 in each group). Serum CIAPIN1 levels are positively correlated with (b) TGF‐β1 (r = 0.469, p = 0.000), (c) MMP‐2 (r = 0.424, p = 0.020), and (d) MMP‐9 (r = 0.648, p < 0.001). Pearson correlation test was performed.

FIGURE 2

CIAPIN1 knockdown inhibits human ASMC proliferation in the presence of PDGF‐BB. CIAPIN1 siRNA (siCIAPIN1) and control siRNA (siNC) were transfected to ASMCs for 48 h. (a) RT‐qPCR was performed to assess the mRNA expression of CIAPIN1. (b) A western blot was performed to assess the protein expression of CIAPIN1, and representative gel blots of CIAPIN1 were shown. The #2 sequence shows the best inhibitory effect and was chosen for the next experiments. ASMCs were transfected with siCIAPIN1 or siNC and incubated with 20 ng/mL PDGF‐BB for 24 h. (c) Cell viability was assessed by CCK‐8 assay. (d) 5‐ethynyl‐20‐deoxyuridine (EdU) assay was used to determine cell proliferation of ASMCs, and representative images are shown. (e) Quantification of positive cells relative to DAPI‐positive cells. Data are presented as mean ± SD in triplicates and analyzed using one‐way ANOVA, and the Bonferroni test was used for the post‐hoc test.

FIGURE 3

CIAPIN1 knockdown suppresses PDGF‐BB‐stimulated inflammatory cytokines production. RT‐qPCR was used to assess mRNA expression of pro‐inflammatory cytokines (a) TNF‐α, (b) IL‐1β, and (c) IL‐6 in ASMC supernatants stimulated by 20 ng/mL PDGF‐BB. ELISA was used to detect the levels of (d) TNF‐α, (e) IL‐1β, and (f) IL‐6 in ASMCs supernatants stimulated by 20 ng/mL PDGF‐BB. Data are presented as mean ± SD in triplicates and analyzed using one‐way ANOVA and Bonferroni test for the post‐hoc test.

FIGURE 4

CIAPIN1 knockdown inhibits human ASMC migration in the presence of PDGF‐BB. (a) Transwell assay was used to assess the migration ability of ASMCs with 0.1% crystal violet staining. Representative images of migrated cells are shown (magnification 100×). (b) Quantification of the number of migrated cells. RT‐qPCR was used to assess mRNA expression of genes related to migration (c) MMP‐2 and (d) MMP‐9. Data are presented as mean ± SD in triplicates and analyzed using one‐way ANOVA and Bonferroni test for the post‐hoc test.

FIGURE 5

CIAPIN1 knockdown modulates PI3K/Akt and JAK2/STAT3 pathway. (a) Representative gel blots depicting levels of phosphorylated PI3K (p‐PI3K, p85, Tyr458, normalized to total PI3K), phosphorylated Akt (p‐Akt, Ser473, normalized to total Akt), phosphorylated JAK2 (p‐JAK2, normalized to total JAK2), phosphorylated STAT3 (p‐STAT3, normalized to total STAT3). (b–e) Quantification analysis of p‐PI3K, p‐Akt, p‐JAK2, and p‐STAT3 protein bands. Data represent the average of three independent experiments (mean ± SD).

FIGURE 6

Schematic diagram of effect of CIAPIN1 on proliferation and migration of ASMCs.