Collection and identification of BALF-EVs in patients with pulmonary nodules
A total of 50 patients (25 with benign nodules and 25 with malignant nodules) were enrolled in this study. The patients ranged in age from 36 to 85 years, with 21 males and 28 females. R-EBUS was performed in each patient, and BALF was obtained after probing the abnormal pulmonary echo area (Fig. 1A). Among them, 25 malignant nodules include 23 cases of adenocarcinoma, 1 case of squamous cell carcinoma, and 1 case of small cell carcinoma. Six samples (3 for malignant and 3 for benign) were used for high-throughput sequencing, and 4 (2 for malignant and 2 for benign) were used for EV characterization. The general information of patients, nodule sizes, pathology, and tumor staging of the remaining 40 samples are shown in Table 2.
EVs were isolated from BALF by ultracentrifugation. To characterize EVs, transmission electron microscopy (TEM) was used to visualize the bilayer structure of EVs (Fig. 1B). The size of EVs was measured by ZetaView analysis, with an average size of approximately 160 nm and a concentration of 1.1 ⋅ 108 particles/ml (Fig. 1C). Western blot analysis revealed the presence of the marker proteins CD63 and ALIX in EVs from both groups. However, Calnexin, a typical negative marker was not detected in EVs (Fig. 1D). The above results indicated that EVs were successfully isolated from BALF.
The obtained EVs were cocultured with H1299 cells for 24 h to observe EV uptake by tumor cells (Fig. 1E). The results from CCK-8, EdU, wound healing and transwell assays indicated that the proliferation, migration and invasiveness of H1299 cells were significantly enhanced after the uptake of BALF-EVs derived from patients with malignant pulmonary nodules (Fig. 1 FJ)
Table 2. Characteristics of the patients
Item
|
n
|
Sex
|
n = 40
|
Male (%)
|
19 (38%)
|
Female (%)
|
21 (42%)
|
Age (years, mean±SD, range)
|
65.52±12.51, (36-85)
|
Malignant
|
n = 20
|
Histological type
|
|
Adenocarcinoma
|
18
|
Small cell carcinoma
|
1
|
Squamous cell carcinoma
|
1
|
TNM
|
|
Ⅰ
|
18
|
II
|
2
|
Benign
|
n = 20
|
Sarcoidosis
|
1
|
Inflammation
|
17
|
Aspergillosis
|
1
|
Mycobacteriosis avium
|
1
|
Abbreviation: SD, standard deviation.
miR-1246b was upregulated in BALF-EVs from patients with malignant pulmonary nodules, and FGF14 was a direct target of miR-1246b
To determine miRNA expression differences in BALF-EVs from patients with benign or malignant pulmonary nodules, we selected 3 benign and 3 malignant pulmonary nodules for high-throughput sequencing of small RNAs. Differential miRNAs were present in BALF-EVs from patients with benign and malignant samples (Fig. 2 A-E). On the basis of the cluster analysis results, 5 miRNAs that were upregulated in malignant nodules were selected, and qRT-PCR of 20 pairs of BALF-EV samples from patients with benign and malignant nodules was used for validation. The content of miR-1246b, a novel miRNA, was found to be higher in patients with benign nodules than in patients with malignant nodules. In the ROC analysis, the AUC was 0.741, and the sensitivity and specificity ratios were 80% and 60%, respectively (Fig. 2 F and G).
Analyses using the miRanda and PITA databases predicted FGF14 as a target of miR-1246b (Fig. 2 G-I). To confirm whether miR-1246b directly regulates FGF14, dual-luciferase reporter experiments were conducted. In 293T cells, miR-1246b expression significantly inhibited the luciferase activity of the wild-type (WT) FGF14 3'UTR but not the that of the mutant (MUT) FGF14 3'UTR; in contrast, inhibiting miR-1246b significantly increased the luciferase activity of the WT-FGF14 3'UTR, but the luciferase activity of the MUT-FGF14 3'UTR was unchanged (Fig. 2J), confirming the existence of a binding relationship between miR-1246b and FGF14. The expression of FGF14 protein was assessed in 20 pairs of benign and malignant lung nodule samples, and the results showed that FGF14 was highly expressed in malignant lung nodule tissue (Fig. 2 K, I).
miR-1246b promotes proliferation, migration and invasion in lung cancer cells
To further clarify the regulatory effect of miR-1246b on FGF14 in tumor cells, we detected the expression of miR-1246b and FGF14 in three lung adenocarcinoma cell lines, A549, H1299 and H1975. The expression of miR-1246b was the lowest in H1299 cells and highest in H1975 cells, while the expression of FGF14 was the opposite (Fig. 3A). The subsequent experiments were carried out using H1299 and H1975 cells.
miR-1246b mimics and inhibitors were transfected into H1299 and H1975 cells, cultured for 24 h, respectively. The results of the CCK-8 and EdU assays indicated that the overexpression of miR-1246b dramatically promoted the proliferation of lung cancer cells and that the knockdown of miR-1246b inhibited the proliferation of lung cancer cells (Fig. 3 B-D). Scratch assay results indicated that the overexpression of miR-1246b increased the migration rate of tumor cells. In contrast, the knockdown of miR-1246b reduced tumor cell migration (Fig. 3E, F). Transwell assays were used to assess the effect of miR-1246b on tumor cell invasion. As seen in Figure 3, compared with that in the control group, the number of cells in the miR-1246b mimic group that penetrated to the low chamber of the transwell obviously increased, and the miR-1246b inhibitor group showed the opposite results (Fig. 3G, H). These results suggest that miR-1246b promotes the proliferation, migration and invasion of lung cancer cells. To clarify the possible mechanism, we further performed western blotting. The data showed that miR-1246b downregulated the expression of FGF14 mRNA and protein in H1299 cells, and that the mRNA and protein expression levels of FGF14 increased after the downregulation of miR-1246b expression in H1975 cells. The overexpression of miR-1246b could enhance the expression of p-ERK/ERK, N-cadherin, and Vimentin protein and inhibit the expression of E-cadherin protein (Fig. 3 L-N). The tendency was observed by qPCR, but E-cadherin expression was inhibited. Therefore, we deduced that miR-1246b may promote tumor development by ERK and EMT alterations through FGF14 (Fig. 3 O, P).
FGF14 inhibits the proliferation, migration and invasion of lung cancer cells
Previous studies have found that FGF14 can inhibit the proliferation of lung cancer cells. In this experiment, plasmid transfection was used to downregulate the expression of FGF14 in H1299 and H1975 cells (Fig. 4 A-C). The results of CCK8 and EdU assays showed that after inhibiting the expression of FGF14 in lung cancer cells, the proliferation rate of tumor cells statistically increased (Fig. 4 D-F), and the scratch test results demonstrated that the migration ability of lung cancer cells enhanced (Fig. 4 G, H). Transwell experiments also suggested that compared with that in the control group, the number of tumor cells passing through the chamber was significantly increased after FGF14 expression was inhibited (Fig. 4 I, J). Further analyses found that with the decreased FGF14 expression in lung cancer cells, the expression of p-ERK/ERK, N-cadherin, and Vimentin increased, but the expression of E-cadherin decreased (Fig. 4 K-O). These results verified that FGF14 could promote the proliferation, migration and invasion of through regulating ERK pathway and EMT in lung cancer cells.
miR-1246b rescue experiment
The low expression of miR-1246b led to the increased expression of FGF14. We used a si-FGF14 plasmid to rescue the increased expression of the target gene; the overexpression of miR-1246b led to the decreased expression of FGF14, and we employed an FGF14 overexpression plasmid to rescue the reduced the expression of target genes. The EdU assay results showed recovery of the cell proliferation rate, and the data of wound healing and transwell experiments also suggested that the effect of miR-1246b on lung cancer cells migration and invasion abilities was inhibited. The expression of p-ERK/ERK protein was decreased in FGF14-overexpressing cells; however, in the si-FGF14 group, the above results were reversed. The qPCR analyses showed the same trend. The above data also suggest that in addition to ERK, there may be other pathways involved in the effect of miR-1246b-FGF14 on the malignant behaviours in lung cancer cells.
miR-1246b promotes the growth of lung cancer in vivo
Finally, we established a mouse subcutaneous xenograft model with H1299 cells to verify the underlying mechanisms of miR-1246b-induced tumor growth in vivo. With increasing time, the tumor volume and weight increased significantly in miR-1246b overexpressing xenograft tumors compared with those of the control group (Fig. 6A–D). Immunohistochemical results indicated that the expression of FGF14 was reduced in tumor tissues with high miR-1246b expression, indicating that miR-1246b could promote tumor proliferation by downregulating the expression of FGF14 (Fig. 6E, F).