Lung cancer-derived exosomal miR-132-3p contributed to interstitial lung disease development

A549 cells and NHLF were purchased from the Chinese Academy of Science cell bank. A549 cells were cultured in RPMI-1640 medium (31,870,082, Gibco, CA, USA). NHLF were grown in DMEM (11,995,065, Gibco, CA, USA). All cultures were supplemented with 10% fetal bovine serum (FBS, 10100147C, Gibco, CA, USA) and 1% penicillin–streptomycin (15,140,122, Gibco, CA, USA). After the confluence of NHLF reached 70–80%, miR-NC, miR-132-3p mimic, and miR-132-3p inhibitor were synthesized by Ningbo Kangbeibio Biotech Ltd. and were transfected into NHLF using Lipo6000™ Transfection Reagent (C0526, Beyotime Biotechnology, Shanghai, China). The sequences such as miR-132-3p inhibitor/mimics in this study were listed: miR-132-3p inhibitor (5′-CGACCAUGGCUGUAGACUGUUA-3′), miR-132-3p mimics (sense: 5′-UAACAGUCUACAGCCAUGGUCG-3,′ and antisense: 5′-CGACCAUGGCUGUAGACUGUUA-3′).

The exosomes were isolated using Exosome Isolation Kit (UR52121; Umibio Science and Technology Group, Shanghai, China). Briefly, 20-ml supernatant of A549 cells culture was centrifuged for 20 min at 3000 g to remove the cell debris. Then, 5 ml of the exosome concentration solution was resuspended from the supernatant and incubated for 2.5 h at 4 °C. The mixture was centrifuged at 12,000 g for 60 min, resuspended in 100-μl PBS, and subsequently loaded into the exosomes purification filter to obtain the purified exosomes.

TEM was used for the morphology identification of the exosomes from A549 cells. Specifically, the exosome pellets were fixed by 2.5% glutaraldehyde, dehydrated with increasing alcohol concentrations, and observed under transmission electron microscopy (JEM-1400Flash, JEOL, Tokyo, Japan). A total of 100-μl exosomes were applied to ZetaView (Malvern Panalytical) to analyze the particle size.

To observe the exosome uptake for NHLF, the exosomes from A549 cells were labeled with PKH67-by-PKH67 Green Fluorescent Cell Linker Mini Kit (MINI67, Sigma, MO, USA) and were co-cultured with NHLF for 30 min. NHLF were incubated with Hoechst 33,342 staining solution (C1029, Beyotime Biotechnology, Shanghai, China) at 37 °C to obverse the nuclei. The fluorescence images were captured by the confocal microscope (Leica, Wetzlar, Germany).

MTT Cell Proliferation and Cytotoxicity Assay Kit (C0009S, Beyotime Biotechnology, Shanghai, China) were applied to determine the cell viability of NHLF. In brief, NHLF were plated into 96-well plates at the density of 1 × 10³ cells/well. Subsequently, each well of the plate was added with 10-μl MTT reagent for 4-h incubation, treated with the formazan solvent, and measured at the wavelength of 570 nm.

A total of 100-μl NHLF suspensions (1 × 10⁴ cells/ml) in different groups were seeded into a 6-well plate, and the NHLF were cultured in DMEM containing 10% FBS for 14 days. After culture for 14 days, the cells were rinsed with PBS, fixed, and stained. The cell clusters containing > 50 cells were counted and imaged by a CX41 light microscope (Olympus, Tokyo, Japan).

Flow cytometry was employed to examine the apoptosis of NHLF [15]. NHLF in different groups were stained with Annexin V-FITC/PI Cell Apoptosis Detection Kit (AC12L033; Shanghai Life iLab Biotech Ltd., Shanghai, China). NHLF were digested, centrifuged, collected, and stained with 5 μl of FITC-Annexin V and 5-μl PI for 10 min incubation under dark. Countstar Rigel S3 flow cytometer was used for analysis.

As previous study [16], the proteins were extracted from NHLF and the exosomes from A549 cells using RIPA buffer (high) (R0010, Solarbio, Shanghai, China) on ice for 20 min and quantified by BCA Kit (PC0020; Shanghai Acmec Biochemical Co., Ltd., Shanghai, China). All antibodies were purchased from Finetest Biotech Ltd. (Wuhan, China) or Abcam (Shanghai, China): anti-CD81 (1:500; FNab10448), anti-TSG101 (1:1000; FNab10812), anti-Alix (1:1000; ab275377), anti-tubulin (1:5000; ab6160), anti-Ki67 (1:200; FNab09788), anti-PCNA (1:5000; FNab06217), anti-Bcl-2 (1:1500, ab194583), anti-Bax (1:1500, ab32503), anti-Caspase-3 (1:2000, ab2302), anti-SPRY1 (1:1000; ab111523), and anti-β-actin (1:5000; ab8226). β-actin was used to normalize protein expression levels. Afterward, the horseradish peroxidase-labeled goat anti-rabbit HRP antibody (1:5000, ab97051) and goat anti-mouse HRP antibody (1:5000, ab205719) were purchased as the secondary antibody. Finally, protein bands were developed using ECL Plus Ultra-Sensitive Kit (PH0353, Phygene Life Sciences Company, Fuzhou, China).

Six-week-old male C57BL/6 J (B6) mice were purchased from Cavens Biogel (Suzhou, China), raised in a clean animal cabinet at 26 ℃, 12-h light/12-h dark period, and free to obtain water and full nutrition food. For the BLM group, the mice were treated with an intratracheal injection of 1 mg/ml BLM. After 10 days, the mice were treated with 100 μg exo, exo + miR-NC, or exo-miR-132-3p inhibitor via tail vein every 3 days for three times. After 25 days, mice were sacrificed, and lung tissues were separated to observe the histological changes.

The total RNA from NHLF and lung tissues were extracted by TsingZol Total RNA Extraction Reagent (TSP401, Tsingke, Nanjing, China) according to previous reports [15]. HiScript II 1st-Strand cDNA Synthesis Kit (+ gDNA wiper) and miRNA 1st-Strand cDNA Synthesis Kit (R212-01 and MR101-01, correspondingly) were utilized to achieve the mRNA and miRNA expressions by ChamQ Universal or miRNA SYBR Mix (Q711-02/03 and MQ101-01/02, correspondingly). These products were purchased from Vazyme (Nanjing, China). All primers were synthesized by MBL Beijing Biotech Co., Ltd., and GAPDH or U6 was used as the internal reference. The sequences of the primers were displayed in Table 1.

To validate further whether sprouty1 (SPRY1) was a direct target of miR-132-3p, the binding sites of wild-type (WT) and mutant (MUT) sequences of SPRY1 3′UTR with miR-132-3p were cloned into the pmirGLO vector to generate SPRY1-WT or SPRY1-MUT [17]. These reporter plasmids were co-transfected with miR-132-3p mimic or mimic NC into NHLF for 48 h. The luciferase activity was assessed by the Luciferase Reporter Gene Assay Kit (RG027, Beyotime Biotechnology, Shanghai, China).

Statistical analysis was performed using GraphPad Prism 7.0 software. Data were demonstrated as means ± standard deviation (SD). Two-tailed Student’s t-test and one-way ANOVA with Turkey’s test were performed to compare the difference between two or multiple groups, respectively. Data with P-values smaller than 0.05 was considered a significant difference.

In Fig. 1A and B, the prepared exosomes from A549 cells were identified by TEM and NTA, which indicated that the isolated exosomes from A549 cells had a 120-nm average diameter and typical two-layer membrane structure. In Fig. 1C, the exosomal positive indicators CD9, CD63, TSG101, Alix, and CD81, and one negative indicator calnexin, were used to determine the exosomes. There was a significant increase for Alix, TSG101, CD9, CD63, and CD81 protein levels in exo group compared with the corresponding cells. The tubulin and calnexin protein were expressed in cells but were absent in exosomes, which suggested that the isolated exosomes from A549 cells were successfully prepared.

To determine the function of lung cancer-derived exosomes, the prepared exosomes from A549 cells were labeled with PKH67 and co-cultured with NHLF, the normal human lung fibroblasts. In Fig. 2A, there was the strongest green fluorescence accumulation in the cytoplasm of NHLF, suggesting that the exosomes from A549 cells could be specifically up taken by NHLF. Functionally, clone formation, MTT, and Western blot assays showed that the exosomes from A549 cells promoted the viability and proliferation ability and the proliferation-related protein levels such as ki-67 and PCNA for NHLF. However, GW4869, an exosome biogenesis inhibitor, partly reversed the promotion effect of lung cancer-derived exosomes on the viability and proliferation of NHLF in Fig. 2B–D. Next, in Fig. 2E–F, flow cytometry, and Western blot assays, revealed that NHLF co-cultured with the A549 cells-derived exosomes inhibited the apoptosis rates and the pro-apoptosis proteins (Bax and Caspase-3) expressions and elevated the expression of the anti-apoptotic protein (Bcl-2). In the markers of fibrosis-associated genes such as alpha-smooth muscle actin (α-SMA), fibronectin (FN) and transforming growth factor β (TGF-β), collagen genes such as collagen type 1 (COL1A1) and collagen type 1 (COL3A1), the homeostatic chemokine (C-X-C motif chemokine ligand 12, CXCL12), and many pro-inflammatory mediators such as IL-1β, IL-6, and IL-8, their mRNA levels were significantly upregulated in exo group compared with the control group (NHLF without any treatments) in Fig. 2G–H. The promotion effect resulting from the lung cancer-derived exosomes was weakened by GW4869 treatment.

MiR-132-3p was reported as the non-small-cell lung tumor-derived exosomal miRNA biomarker [18]. We chose the miR-132-3p for further research in Fig. 3A. The result of qRT-PCR in Fig. 3B showed that miR-132-3p had a significantly high expression in exosomes from A549 cells compared with corresponding cells. To investigate the biological function of miR-132-3p, miR-132-3p mimic, miR-132-3p inhibitor, and miR-NC were transfected into NHLF. In Fig. 3C, the qRT-PCR results showed that the relative miR-132-3p expression was upregulated by miR-132-3p mimic. And there was an opposite trend in the miR-132-3p inhibitor group. Functionally, in Fig. 3D and E, MTT and clone formation assays showed that miR-132-3p mimic promoted the cell viability and proliferation of NHLF, whereas these phenotypes for NHLF transfected with miR-132-3p inhibitor were greatly inhibited. Next, flow cytometry assay revealed that the apoptosis rates of NHLF transfected with miR-132-3p mimic were decreased compared with miR-NC; however, in Fig. 3F, the miR-132-3p inhibitor group had a high apoptosis rate for NHLF. In Fig. 3G, miR-132-3p overexpression resulted in the downregulated expression of pro-apoptosis proteins and elevated levels of anti-apoptotic protein. In Fig. 3H and I, the miR-132-3p overexpression group had a high expression of α-SMA, FN, TGF-β, COL1A1, COL3A1, CXCL12, IL-1β, IL-6, and IL-8; however, miR-132-3p inhibitor brought about opposite effects.

To further explore the underlying mechanism of exosomal miR-132-3p in interstitial lung disease development, exo, miR-NC-exo, and miR-132-3p inhibitor-exo from A549 cells were used to treat the bleomycin (BLM)-induced fibrosing lung injury. In Fig. 4A, Masson’s trichrome, H&E staining, and IHC results showed that BLM-treated mice developed pulmonary fibrosis and had a high expression of α-SMA compared to the saline group. The degrees of pulmonary fibrosis in the exo and miR-NC-exo groups were markedly greater than in the BLM group. Interestingly, the treatment with miR-132-3p inhibitor-exo significantly inhibited the alveolar wall thickening, infiltration of inflammatory cells into the interstitium, and reduction in collagen deposition induced by BLM. In Fig. 4B, the fibrosis-associated genes (α-SMA and TGF-β), and collagen genes (COL1A1 and COL3A1) in BLM + exo and BLM + miR-NC-exo, had a remarkable upregulation compared with saline or BLM group. However, miR-132-3p inhibitor-exo effectively reduced the α-SMA, COL1A1, COL3A1, and TGF-β1 mRNA levels. Moreover, in Fig. 4C, the mRNA expression of IL-1β/6/8 was consistent with the fibrosis/collagen-associated genes.

We searched the potential binding genes of miR-132-3p in the TargetScan database and found that SPRY1 may be a target of miR-132-3p in Fig. 5A. In Fig. 5B, luciferase assay displayed that the luciferase activity of SPRY1-WT was diminished in response to miR-132-3p mimic, while there was no significant difference in the SPRY1-MUT group, indicating that SPRY1 was the target gene for miR-132-3p. In Fig. 5C, miR-132-3p overexpression inhibited the mRNA and protein levels of SPRY1. In addition, the mRNA of SPRY1 in the isolated exosomes had a lower expression compared with A549 cells (Fig. 5D).

We further examined the functional correlation between SPRY1 and exosomal miR-132-3p from A549 cells. The results in Fig. 6A showed that the mRNA and protein expression of SPRY1 markedly decreased in sh-SPRY1 group. Cell viability of NHLF markedly decreased by the inhibition of miR-132-3p, but knockdown of SPRY1 reversed the decrement induced by miR-132-3p inhibitor in Fig. 6B. As expected, the cell colony number of NHLF exhibited a similar variation trend with cell viability in Fig. 6C, whereas cell apoptosis rates and pro-apoptosis proteins showed the reverse results in Fig. 6D–E. In Fig. 6F–G, the knockdown of SPRY1 significantly reversed the inhibition effect of the miR-132-3p inhibitor on the mRNA levels of the fibrosis/collagen-associated genes (α-SMA, TGF-β1, COL1A1, and COL3A1) and many pro-inflammatory mediators (IL-1β/6/8). Collectively, these results demonstrated that miR-132-3p targeted SPRY1 to regulate the development of NHLF.