Increase in blood GDF15 level in patients with silicosis

Bioinformatics analysis indicated an increase in GDF15 expression in A549 cells treated with crystalline silica(Supplementary Data S1 and Figure S1).To study the role of GDF15 in silicosis, we measured the GDF15 level in the peripheral venous blood samples of patients with silicosis using ELISA. The GDF15 level in 46 patients with silicosis was 396.9 ± 150.6 pg/mL, whereas that in 10 control patients was 212.1 ± 13.66 pg/mL, which represented a statistically significant difference (P = 0.0003) (Fig. 1a).

Fig. 1

Increased blood GDF15 expression in patients with silicosis. A, Serum GDF15 expression in patients with silicosis was higher than that in controls (P = 0.0003, compared to control). B, Increased serum GDF15 expression did not relate to the FEV1% of patients with silicosis; P = 0.2002. C, Increased serum GDF15 expression did not relate to the clinical stages of silicosis, P = 0.5979

The FEV1% is an important indicator of lung function. According to the standard used by Pertzov and Ozkaya [19, 20], patients were divided into four groups: severe (FEV1 < 40%, n = 6), moderate (FEV1 between 40 and 60, n = 17), mild (FEV1 between 60 and 80, n = 18), and normal (FEV1 > 80%, n = 5). The GDF15 levels in the severe, moderate, mild and normal groups were 359.7 ± 151.9 pg/mL, 417.2 ± 137.2 pg/mL, 424.7 ± 170.4 pg/mL, and 273.6.2 ± 33.5 pg/mL, respectively. There was no statistically significant difference between the GDF15 levels in patients with different lung functions (R2 = 0.1034, P = 0.2002) (Fig. 1b).

Based on the results of imaging examinations, the 46 patients with silicosis were divided into stages 1–3 in clinical practice; this included 15 patients in stage 1, 11 patients in stage 2, and 20 patients in stage 3. The GDF15 level were 371.7 ± 139.4 pg/mL, 433.3 ± 165.7 pg/mL, and 395.9 ± 153.9 pg/mL in stage1,2 and 3 patients, respectively. However, no statistically significant difference was observed among the GDF15 levels in patients with stage-1, -2, or -3 disease (R2 = 0.0236,P = 0.5979). The data indicated that the increased serum levels of GDF15 were not associated with the clinical stages (Fig. 1c).

GDF15 activates human embryonic lung fibroblast MRC5 cells

To investigate the effect of GDF15 on human embryonic lung fibroblast MRC5 cells, MRC5 cells were treated with GDF15 with dosage and time course. The expression of α-SMA and col1a was observed using western blotting. In different dosage experiments, the expression levels of α-SMA and col1a were highest at 100 ng/mL. When MRC5 cells were treated with 100 ng/mL of GDF15 for 24 h, the expression of col1a and α-SMA increased most significantly (Fig. 2a and b).The CCK-8 assay revealed that GDF15 could induce MRC5 cell proliferation (Fig. 2c). The wound-healing scratch assay showed that GDF15 increased MRC5 cell migration (Fig. 2d). Thus, our results indicated that GDF15 induced MRC5 cell activation.

Fig. 2

GDF15 activates human embryonic lung fibroblast MRC5 cells. The effect of GDF15 on col1a and α-SMA protein expression in MRC5 cells was detected using western blotting. A, dosage course. B, time course. C, GDF15 increased the proliferation of MRC cells, as detected in the CCK-8 assay. D, GDF15 increased the migration of MRC cells, which was detected in the wound healing assay. Data represent the mean ± SD from three independent experiments.*,**,♯, P < 0.05

Effects of GDF15 on mRNA and miRNA expression in MRC5 cells

To study the regulation of GDF15 gene expression in human embryonic lung fibroblast MRC5 cells, the miRNA and mRNA expression profiles of MRC5 cells in the presence or absence of GDF15 were assessed using RNA sequencing.

The differentially expressed miRNAs were identified, which included nine upregulated genes and 18 downregulated genes (compared with those in the control group, genes showing a change in expression ratio greater than twofold or lesser than 0.5-fold change were identified; Fig. 3a-c). The upregulated miRNAs included has-miR-6850, has-miR-219a, has-novel-229, and has-novel-230. The top five miRNAs with successively decreasing expression included has-miR-338, has-miR-1, has-miR-133a, has-miR-5002, and has-miR-196a.

Fig. 3

Effects of GDF15 on the miRNA and mRNA expression in MRC5 cells. Effect of GDF15 on the miRNA profile of MRC5 cells. A, heatmap; B, Volcano plots; C, KEGG analysis. Effect of GDF15 on the mRNA profile of MRC5 cells. D, heatmap; E, Volcano plots; F, KEGG analysis

The differentially expressed mRNAs identified consisted of 230 upregulated genes and 238 downregulated genes (compared with those in the control group, genes showing a change in expression ratio greater than twofold or a lesser than 0.5-fold were identified, Fig. 3d-f). The upregulated genes included PLD3, CCNA2, MYD88, MARK2, NLRP1, STAT1, and VMP1. The downregulated genes included ZNF219, OGDH, ADD1, SMARCD2, LIPA, FHL1, and ATP5F1.

GDF15 reduced the expression of miR-338 in MRC5 cells

To explore the role of miR-338 in fibroblast activation induced by GDF15, we first detected the expression of miR-338 in MRC5 cells with or without GDF15 treatment using qRT-PCR. Our data showed that GDF15 significantly reduced miR-338 expression in MRC5 cells (P < 0.05, Fig. 4a). Following this, miR-338 mimics or inhibitors was transfected into MRC5 cells. Our data showed that miR-338 mimics reduced the expression of col1a and α-SMA in MRC5 cells both with and without GFD15 treatment. However, the miR-338 inhibitor promoted col1a and α-SMA expression in MRC5 cells with and without GFD15 treatment (Fig. 4b). CCK-8 assay data showed that miR-338 mimics inhibited MRC5 cell proliferation with and without GDF15 treatment. Meanwhile, the miR-338 inhibitor stimulated MRC5 cell proliferation with and without GDF15 treatment (Fig. 4c). The results indicated that miR-338 played an important role in fibroblast activation induced by GDF15.

Fig. 4

GDF15 reduced the expression of miR-338 in MRC5 cells. A, qRT-PCR results showed that the expression of miR-338 was decreased in response to GDF15 expression. B, Western blotting results showed that miR-338 mimics attenuated the expression of col1a and α-SMA regardless of treatment with GDF15, whereas miR-338 inhibitors promoted the expression of col1 and α-SMA regardless of treatment with GDF15. C, CCK-8 assay results showed that miR-338 mimics reduced the growth rate and the miR-338 inhibitor increased the growth rate of MRC5 cells. Data represent the mean ± SD from three independent experiments.*,**,♯, P < 0.05

GDF15 increased the expression of STAT1 in MRC5 cells

To determine the role of STAT1 in GDF15-induced fibroblast activation, we first assessed the expression of STAT1 in MRC5 cells with GDF15 treatment using western blotting. Our data showed that GDF15 significantly increased STAT1 expression in MRC5 cells (P < 0.05, Fig. 5a). Following this, STAT1 siRNA and cDNA were transfected into MRC5 cells. The data showed that STAT1 siRNA diminished the expression of col1a and α-SMA in MRC5 cells in the presence or absence of GDF15 treatment. However, STAT1 cDNA promoted the expression of col1a and α-SMA in MRC5 cells in the presence and absence of GDF15 treatment (Fig. 5b). CCK-8 assay data showed that STAT1 siRNA inhibited MRC5 cell proliferation in the presence and absence of GDF15 treatment. Meanwhile, STAT1 cDNA stimulated MRC5 cell proliferation in the presence and absence of GDF15 treatment (Fig. 5c). The data indicated that STAT1 played a significant role in GDF15-induced fibroblast activation.

Fig. 5

GDF15 increased STAT1 expression in MRC5 cells. A, Western blotting results showed that GDF15 induced STAT1 expression. B, Western blotting results showed that STAT1 siRNA attenuated the expression of col1a and α-SMA, whereas STAT1 cDNA promoted their expression in the presence or absence of GDF15 treatment. C, CCK-8 assay results showed that STAT1 siRNA reduced the growth rate, whereas STAT1 cDNA increased the growth rate of MRC5 cells. Data represent the mean ± SEM from three independent experiments.*,**,♯, P < 0.05

GDF15 activates MRC5 cells through miR-338/STAT1 pathway

According to bioinformatics analysis, we could speculate that miR-338 could bind to the 3’UTR of the STAT1 mRNA. To investigate the role miR-338 in STAT1 expression, the normal or mutant binding site of miR-338 in 3’UTR of the STAT1 was cloned into the pmirGLO dual-luciferase miRNA target expression vector. The upregulation of miR-338 weakened the luciferase activity of the wild-type STAT1 3’UTR reporter gene, while it did not alter the activity of the mutant reporter gene (Fig. 6a). Following this, the effect of miR-338 on STAT1 expression in MRC5 cells was detected using western blotting. Our data indicated that miR-338 mimics reduced STAT1 expression, whereas miR-338 inhibitors increased STAT1 expression (Fig. 6b). The findings indicated that STAT1 might be a direct target of miR-338.

Fig. 6

GDF15 activated MRC5 cells through the miR-338/STAT1 pathway. A, Using TargetScan (http://www.targetscan.org/), the conserved miR-338 binding site in the 3’-UTR of STAT1 mRNA was constructed in the pmirGLO dual-luciferase miRNA target expression vector. Luciferase activity was analyzed in the MRC5 cells. MRC5 cells were co-transfected with miR-30 mimics and a luciferase reporter. B, miR-338 regulated STAT1 protein expression. C, miR-338 mimics attenuated the expression of col1 and α-SMA protein induced by STAT1 overexpression upon co-treatment with GDF15. D, STAT1 knockdown attenuated col1 and α-SMA protein expression induced by the miR-338 inhibitor upon co-treatment with GDF15. Data represent the means ± SD from three independent experiments. *,**,♯, ♯♯,P < 0.05

MRC5 cells were co-transfected with miR-338 mimics and STAT1 cDNA. The western blotting results showed that miR-338 mimics reversed the expression of col1a and α-SMA induced by STAT1 upregulation (Fig. 6c). When MRC5 cells were co-transfected with miR-338 inhibitors and STAT1 siRNA, the miR-338 inhibitors was found to reverse col1a and α-SMA expression induced by decreased STAT1 expression (Fig. 6d). Thus, our data indicated that GDF15 might activate MRC5 cells via miR-338/STAT1.