MiR-1246b, a novel miRNA molecule of extracellular vesicles in bronchoalveolar lavage fluid, promotes nodule growth through FGF14 in patients with lung cancer

Abstract

With the widespread development of chest computed tomography (CT), the detection rate of pulmonary nodules has increased; therefore, the classification of benign vs. malignant nodules has become a common problem in the clinic. MicroRNA, a potential tool, is expected to become a good choice for diagnosing and studying the occurrence and development of diseases through the vector of bronchoalveolar lavage fluid extracellular vesicles (BALF-EVs). In this study, radial endobronchial ultrasound (R-EBUS) was used to locate pulmonary nodules in patients. BALF was obtained, EVs were isolated, and small RNA sequencing was performed to screen differentially expressed miRNAs between benign and malignant pulmonary nodules. The binding targets and underlying mechanisms of the differentially expressed miRNAs were verified by in vitro and in vivo experiments. R-EBUS localization and sampling was used to obtain BALF, and EVs were successfully isolated and characterized. Differentially expressed miRNAs in BALF-EVs of patients with benign vs. malignant pulmonary nodules were screened by high-throughput small RNA sequencing. A new miRNA, miR-1246b, was identified. We found that FGF14 was the binding target of miR-1246b by luciferase assay. Subsequent mechanistic studies showed that miR-1246b inhibited the expression of FGF14 in lung cancer cells, further leading to ERK phosphorylation and epithelial-to-mesenchymal transition (EMT), which ultimately contributed to lung cancer cell proliferation, migration and invasion. In summary, our study demonstrates that the detection of miRNAs in BALF-EVs, a means of liquid biopsy, could assist in distinguishing malignant nodules from benign nodules. miR-1246b, which was extracted from BALF-EVs, targets FGF14 to promote lung cancer cell proliferation, migration and invasion.

Fig. 1. The collection, characterization and effect of BALF-EVs on the proliferation and invasion of lung cancer cells.

A Chest CT showing a right lower pulmonary nodule (~2.5 cm), as indicated by the red arrow. The R-EBUS image of the right lower pulmonary nodule shows an abnormal echo area, indicating that the ultrasound probe has reached the lesion. B Morphology of BALF-EVs in BALF samples under an electron microscope (80 kV, ×10.0 K), Bar = 100 nm. C ZetaView measurement of EV PSD in BALF samples. D western blot detection of EV markers. E Malignant BALF-EV uptake by H1299 cells was evaluated under a laser confocal microscope (600×) in H1299 cells. Blue indicates the nucleus, and green indicates EVs. F, G EV effect on proliferation according to CCK-8 (F) and EdU assays (G) in H1299 cells. H A wound healing assay was performed on H1299 cells treated as indicated. I Transwell assays were performed on H1299 cells treated as indicated. In (F–I), a group without EVs, including culture medium only, was used as a negative control. In (B–I), the results are representative of three independent experiments. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01 (Student’s t test).

Fig. 2. High-throughput sequencing and verification of small RNAs in BALF-EVs.

A Cluster analysis of EV samples. The log10 values of miRNAs in the samples were used for cluster analysis. B The differential gene volcano plot showing the overall differential gene distribution. The threshold was set to |logFC | >1 and FDR < 0.05. The genes of interest are marked with arrows and accompanied by their locus labels/gene names. C Hierarchical clustering diagram of differentially expressed miRNAs. The vertical column represents the sample, each row represents a miRNA, the right side is marked with the miRNA name, and the expression level gradually decreases from red to blue. D GO enrichment differences for the target genes of newly identified miRNAs. E KEGG enrichment analysis of the target genes of newly identified miRNAs. The top 10 positions were selected for pathway enrichment analysis. F Chr5_14653 expression assessed by qPCR in BALF-EV samples. The results showed that the level of chr5_14653 was higher in malignant nodules than in benign nodules. G ROC analysis indicated that the AUC for chr5_14653 in the diagnosis of benign and malignant nodules was 0.743. H Schematic diagram of the chr5_14653 (miR-1246b) structure and its FGF14 binding site. I, J Histograms and dot plots of signaling pathways predicted by the miRanda and PITA databases. K The dual-luciferase assay results indicated that the LUC/RLUC values for FGF14-WT+miRNA mimic were significantly lower than those for the other groups, suggesting that FGF-14 is the target gene of miR-1246b. L, M FGF14 expression examined by immunohistochemistry. The left picture shows a typical pathological image from stage IA adenocarcinoma, and the right picture is from an inflammatory nodule. M shows the quantitative analysis of the immunohistochemistry results. In (K), the results are representative of three independent experiments (One-way ANOVA); In (F and M), number of samples = 20 (Student’s t test). The data are presented as the means ± SDs. *P < 0.05.

Fig. 3. miR-1246b may promote cell proliferation, migration and invasion by inhibiting FGF14.

A miR-1246b and FGF14 mRNA levels were determined by qPCR in lung adenocarcinoma cells. The results indicated that the levels of miR-1246b were low and the levels of FGF14 mRNA were high in H1299 cells and that the expression of miR-1246b was high and the levels of the FGF14 gene were low in H1975 cells. B, C Cell proliferation was detected by CCK-8 (B) and EdU (C) assays in H1299 and H1975 cells following miR-1246b inhibitor or mimic treatment. D The cell migration ability was evaluated by wound healing assay. E Cells were tested for their invasion ability using Transwell assays. F qPCR verified the expression of FGF14 mRNA. G The expression of FGF14 protein was examined by western blotting. H qPCR verified the expression of E-cadherin, N-cadherin, Vimentin, and ERK mRNA in lung cancer cells. I The expression of E-cadherin, N-cadherin, Vimentin, p-ERK, and ERK protein was examined by western blotting in lung cancer cells. All the results are representative of three independent experiments. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01 (Student’s t test).

Fig. 4. FGF14 downregulation promoted cell proliferation, migration and invasion in H1299 and H1975 cells.

The FGF14 gene was silenced in H1299 and H1975 cells. A FGF14 mRNA expression examined by qPCR in 2 cell lines. B FGF14 protein expression examined by western blotting. C, D Cell proliferation was assessed by CCK-8 assay (C) and EdU assay (D). E The cell migration ability was examined by wound healing assay. F H1299 and H1975 cells were assessed for their migration ability using a Transwell assay. G qPCR was performed to determine E-cadherin, N-cadherin, Vimentin, and ERK mRNA levels in H1299 and H1975 cells. H The expression of E-cadherin, N-cadherin, Vimentin, p-ERK, and ERK protein was analyzed by western blotting in H1299 and H1975 cells. All the results are representative of three independent experiments. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01 (Student’s t test).

Fig. 5. miR-1246b and FGF14 expression rescue could reverse the effect of inhibition of these factors on the malignant behavior in H1299 cells.

A H1299 cells transfected with miR-1246b inhibitors+siFGF14 or miR-1246b mimics+ovFGF14 were tested for viability using the EdU assay at the indicated time points. B A wound healing assay was performed on H1299 cells transfected and treated as indicated. C Transwell assays were performed on H1299 cells transfected as indicated. D Results of qPCR to measure ERK mRNA levels in H1299 cells treated as indicated. E Western blotting was used to measure p-ERK/ERK protein levels in H1299 cells. All the results are representative of three independent experiments. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01 (Student’s t test).

Fig. 6. The effect of miR-1246b on H1299 cell NSG mouse xenograft tumors.

A Samples from mouse xenografts injected with miR-1246b agomir or antagomir. B, C Tumor volume (B) and weight (C) in mouse xenografts. D, E FGF14 expression examined by immunohistochemical staining in tumor tissues. D Shows the quantitative analyses based on IOD values obtained from immunohistochemical staining of FGF14 in tumor tissues. All the results are representative of five independent experiments. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01 (Student’s t test).

Fig. 7. Schematic diagram of the molecular mechanisms by which miR-1246b promotes lung cancer progression.

miR-1246b delivered by BALF-EVs promotes lung cancer progression by inhibiting FGF14 and activating ERK.