Differential expression of RNA-binding proteins in bronchial epithelium of stable COPD patients

Figures & data

Table 1 Study population providing bronchial and lung samples for immunohistochemical study

Participants	N Age, years	Sex	Smoking history	Pack- years	FEV1/FVC%
Smokers	12 68.6 (2.3)	10 M/2 F	6 ex, 6 current	48.2 (9.7)	79.3 (1.3)
COPD	12 71.9 (1.9)	11 M/1 F	6 ex, 6 current	52.9 (8.7)	65.7 (1.0)*

Notes: Predicted FEV₁% and FEV₁/FVC% are post-bronchodilator values. Data are expressed as mean (SD).

* P<0.01 compared to smokers. M, male; F, female.

Figure 1 Expression of RNA-binding proteins in human bronchial rings.

Notes: (A) Photomicrographs showing immunostaining for HuR, TTP, and AUF-1 in bronchial epithelium, mucous gland cells, and serous gland cells (left to right) from a CS with NLF and a patient with mild-to-moderate COPD (C) (representative of n=12 for each group, see the “Methods” section). Scale bar set at 20 µm. (B) Individual and median (line) cell counts of nuclear immunostaining for HuR, TTP, and AUF-1 in bronchial epithelial cells, mucous gland cells, and serous gland cells (left to right) of CS and COPD, normalized as indicated. Student’s t-test P-value for COPD vs CS is indicated in all panels. *P<0.05 vs CS. Scale bar set at 20 µm, magnification 1,000×.

Abbreviations: C, patients with COPD; HuR, human antigen R; TTP, tristetraprolin; AUF-1, AU-rich element-binding factor 1; NLF, normal lung function; CS, control smoker.

Figure 2 Expression of RNA-binding proteins in human peripheral lung.

Notes: (A) Photomicrographs showing immunostaining for HuR, TTP, and AUF-1 in alveolar macrophages and bronchiolar epithelium (left to right) from a control smoker and a patient with mild-to-moderate COPD (C) (representative of n=12 for each group, see the “Methods” section). Scale bar set at 20 µm, magnification 1,000×. (B) Individual and median (line) cell counts of nuclear immunostaining for HuR, TTP, and AUF-1 in alveolar macrophages and bronchiolar epithelium (left to right) of control smokers and COPD, normalized as indicated. Student’s t-test P-value for COPD vs CS comparison is indicated in all panels.
Abbreviations: C, patients with COPD; HuR, human antigen R; TTP, tristetraprolin; AUF-1, AU-rich element-binding factor 1; CS, control smoker.

Figure 3 Modulation of the expression of RNA-binding proteins in the human bronchial epithelial cell line BEAS-2B.

Notes: Western blot analysis of AUF-1, TTP, and HuR expression in nucleocytoplasmic lysates of BEAS-2B cells cultured with (A) cytomix (n=4, n=3 for HuR) and (B) 200 mM H₂O₂ (n=3, n=4 for AUF-1) for the indicated times. Upper panels show representative immunoblots for the RBPs and for lamin A/C and β-tubulin as nuclear and cytoplasmic loading controls, respectively; bar graphs show densitometric analysis of nuclear fraction (mean ± SEM of indicated n). *P<0.05 vs unstimulated cells; ^#P<0.05, ##P<0.01 between indicated time points.
Abbreviations: HuR, human antigen R; TTP, tristetraprolin; AUF-1, AU-rich element-binding factor 1; RBP, RNA-binding protein; SEM, standard error of mean.

Figure 4 Effect of CSE on RBP expression in BEAS-2B cells.

Notes: (A) Cell viability following 24-hour exposure to indicated CSE concentrations (n=3, *P<0.05 vs unstimulated [CT] cells). (B) Representative immunoblots of AUF-1, TTP, and HuR expression; lamin A/C and β-tubulin as nuclear and cytoplasmic loading controls, respectively. (C) Densitometric analysis of cytoplasmic fraction (mean ± SEM of n=3). *P<0.05 vs unstimulated cells; changes among concentrations not statistically significant.
Abbreviations: CSE, cigarette smoke extract; HuR, human antigen R; TTP, tristetraprolin; AUF-1, AU-rich element-binding factor 1; ND, not determined; RBP, RNA-binding protein; SEM, standard error of mean.

Figure 5 Expression of RNA-binding proteins in BEAS-2B cells following AUF-1 silencing.

Notes: (A) Representative Western blot analysis (of n=4) of AUF-1, TTP, and HuR expression in cells transfected with mock (Fugene only), AUF-1 siRNA, or scrambled siRNA, and treated for 48 hours with cytomix or medium control. (B) Densitometric analysis of cytomix-induced response (mean ± SEM of n=4 blots). *P<0.05; **P<0.01 compared to the corresponding medium control.
Abbreviations: HuR, human antigen R; TTP, tristetraprolin; AUF-1, AU-rich element-binding factor 1; SEM, standard error of mean.

Figure 6 Expression of AUF-1-targeted cytokines in BEAS-2B cells following AUF-1 silencing.

Notes: (A) CCL2 and IL-6 mRNA levels measured by real-time PCR (mean ± SEM of n=4) in cells transfected with mock (Fugene only), AUF-1 siRNA, or scrambled siRNA and then treated 48 hours with cytomix or medium control. To show the comparison of mRNA levels in unstimulated cells, results are expressed as fold over housekeeping mRNA levels (2^−ΔCt). (B) Levels of IL-6 and CCL2 protein detected by ELISA in cell supernatants of experiments shown in A (mean ± SEM of n=4). *P<0.05; **P<0.01 compared to the corresponding medium control. (C) BEAS-2B supernatants of a representative experiment from data set shown in A and B were screened using the Human Cytokine R&D Protein Arrays. Left: arrays for the indicated conditions; the grid identifies proteins as displayed on the array; in gray, proteins expressed in cytomix-treated cells; circled, proteins expressed also at baseline (all above arbitrary 5,000 densitometry unit cutoff). Right: bar graphs show densitometric analysis for the indicated conditions, expressed as percent of each blot’s positive control (mean of densitometry reading of the six spots).
Abbreviations: AUF-1, AU-rich element-binding factor 1; IL-6, interleukin 6.

Figure 7 Differential expression of RNA-binding proteins in transcriptomic analysis of primary human airway epithelial cells.

Notes: Expression of AUF-1, TTP, and HuR (corresponding gene names indicated in legend) was investigated in the GEO database GSE5058.³⁶ (A) Clinical characteristics of study population where epithelial cells were collected by bronchial brushing. Reprinted from, Cancer Research, 2018; 66(22):10729–10740, Carolan BJ et al, Up-regulation of Expression of the Ubiquitin Carboxyl-Terminal Hydrolase L1 Gene in Human Airway Epithelium of Cigarette Smokers, with permission from AACR.³⁶ (B) Scatterplot showing FC for RBP expression between smokers vs nonsmoker controls, COPD vs nonsmokers, and COPD vs smoker controls (left to right). FCs for RBPs were set at ≥2.0 with a false discovery rate of #0.05.

Abbreviations: HuR, human antigen R; TTP, tristetraprolin; AUF-1, AU-rich element-binding factor 1; FC, fold change; RBP, RNA-binding protein.

Figure 8 Expression of AUF-1-dependent genes in airway epithelial gene array database GSE5058.

Notes: (A) Heatmap showing FC of 152 annotated AUF-1-dependent genes investigated in the GEO database GSE5058, comparing expression from left to right: smokers vs nonsmoker controls; COPD vs nonsmokers; COPD vs smoker controls. Enlarged heatmaps show 30 upregulated and 22 downregulated genes displaying a FC >2.0 in COPD vs smoker controls. (B) Results of genome ontology analysis indicating the main pathways related to the expressed genes according to NCBI Panther tool.
Abbreviations: AUF-1, AU-rich element-binding factor 1; FC, fold change.

Figure S1 Expression of RBP mRNA in PBMC of COPD patients and control smokers.

Notes: Real-time PCR analysis for RBP mRNA in PBMC of control smokers (n=4) and COPD patients (n=5) (Table S1). Data are mean ± SEM of RBP mRNA normalized to GAPDH mRNA and expressed as fold over GAPDH (ctrl) (as 2^−ΔCt).

Abbreviations: RBP, RNA-binding protein; PBMC, peripheral blood mononuclear cell; SEM, standard error of mean.

Figure S2 Evaluation of treatment-induced apoptosis in BEAS-2B cells.

Notes: Western blot analysis of PARP and cleaved Caspase 3 expression in nuclear and cytoplasmic lysates obtained from BEAS-2B cells exposed to cytomix (left panel) and 200 µM (right panel) for the indicated times. Only the full-length bands of PARP and Caspase 3 were detectable in experimental samples, excluding treatment-induced apoptosis. Positive control (C+) is whole cell lysate of gefitinib-treated H1975 NSCLC cell line. Representative immunoblots of n=3 independent experiments are shown. Lamin A/C and tubulin are shown as nuclear and cytoplasmic loading controls, respectively.

Abbreviation: NSCLC, non-small-cell lung cancer.

Figure S3 Expression of RBP mRNA in BEAS-2B cells.

Notes: Real-time PCR analysis for RBP mRNA in nontransfected BEAS-2B cells stimulated with (A) cytomix and (B) hydrogen peroxide (200 M for the indicated times (mean ± SEM of n=3). (C) RBP mRNA in BEAS-2B cells stimulated 48 hours with cytomix following transfection with mock (F = Fugene), siRNA for AUF-1, scrambled siRNA (mean ± SEM of n=4). RBP mRNA was normalized to housekeeping mRNA levels and expressed as fold over corresponding unstimulated controls (2^−ΔCt). *P<0.05 versus controls.

Abbreviations: AUF-1, AU-rich element-binding factor 1; RBP, RNA-binding protein; SEM, standard error of mean.

Figure S4 Evaluation of treatment-induced apoptosis in BEAS-2B cells following AUF-1 silencing.

Notes: Western blot analysis of PARP expression in whole cell lysates obtained from BEAS-2B cells untreated and stimulated 48 hours with cytomix following transfection with AUF-1 siRNA, scrambled siRNA, and mock transfection (Fugene) (mean ± SEM of n=4). Positive control (Ctrl) showing cleaved PARP protein band is whole cell lysate of gefitinib-treated H1975 NSCLC cell line. Representative immunoblots of n=3 independent experiments are shown. β-Tubulin is shown as loading control.

Abbreviations: AUF-1, AU-rich element-binding factor 1; NLF, normal lung function; SEM, standard error of mean; NSCLC, non-small-cell lung cancer.

Table S1 Study population providing peripheral blood mononuclear cells (PBMC)

Participants	N	Age, years	Sex	Smoking history	Pack- years	FEV1 % pred	FEV1 / FVC%
Smokers	4	53.5 (9.3)	2 M/2 F	4 current	31 (11.2)	105.8 (11.4)	78.6 (1.3)
COPD	5	73.8 (7.5)*	4 M/1 F	1 current 4 ex	39.6 (10)	59.8 (21.0)	52.9 (10.5)*

Notes: Predicted FEV₁% and FEV₁/FVC% are post-bronchodilator values. M, male; F, female. Data expressed as mean (SD).

* P<0.01 compared to smokers.

CarolanBJHeguyAHarveyBGLeopoldPLFerrisBCrystalRGUp-regulation of expression of the ubiquitin carboxyl-terminal hydro-lase L1 gene in human airway epithelium of cigarette smokersCancer Res20066622107291074017108109

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