miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway

Abstract

Metastasis is the leading cause of high mortality in lung cancer patients, and metastatic lung cancer is difficult to treat. miRNAs are involved in various biological processes of cancer, including metastasis. Our previous studies revealed that miR-25 promoted non-small-cell lung cancer (NSCLC) cell proliferation and suppressed cell apoptosis by directly targeting TP53 and MOAP1. In this work, we further explored the miR-25 expression in NSCLC patients in the Cancer Genome Atlas (TCGA) database and measured the miR-25 expression levels in the tissues of NSCLC patients and cell lines. miR-25 was overexpressed in both NSCLC tissues and cell lines. NSCLC patients who expressed a higher level of miR-25 exhibited worse overall survival than those with a lower level of miR-25. Overexpression of miR-25 enhanced NSCLC cell migration and invasion, while the inhibition of miR-25 exhibited the opposite effects. We identified the large tumor suppressor homology 2 (LATS2) as a new target gene of miR-25 in lung cancer. The effects of miR-25 on promoting NSCLC cell migration and invasion were at least partially due to activation of the Hippo signaling pathway. Additionally, miR-25 antagomir inhibited xenograft tumor growth and metastasis by the upregulation of LATS2. Taken together, our findings demonstrate that miR-25 contribute to lung cancer cell proliferation and metastasis by targeting the LATS2/YAP signaling pathway, which implicate miR-25 as a promising therapeutic target for lung cancer metastasis. Given that oxidative stress induces the overexpression of miR-25 and plays a critical role in cancer progression, this study establishes miR-25 as an intermediate between oxidative stress and lung cancer metastasis.

Figure 1

The expression of miR-25 in lung cancer tissues and cell lines. (a) miR-25 expression in the tumor tissues of NSCLC patients as compared to the control, noncancerous lung tissues from the TCGA database. (b) miR-25 expression in tumor tissues of NSCLC patients with different stages from the TCGA database. (c) In the TCGA database, the overall survival rate curves of NSCLC patients with high and low expression of miR-25 were shown. (d) The expression levels of miR-25 in 15 pairs of NSCLC tissues and the adjacent normal tissues by qRT-PCR. (e) The expression levels of miR-25 in NSCLC cell lines and the noncancerous lung epithelial cell line BEAS-2B. Results were presented as the mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 2

Overexpression or inhibition of miR-25 on cell proliferation, migration, and invasion in both A549 and H1299 cells. (a, b) Colony formation of cells transfected with the miR-25 precursor or the miR-25 inhibitor or with respective controls. (c, d) Wound healing assays for cell migration with altered miR-25 expression. (e, f) Transwell assays for cell invasion with altered miR-25 expression. (g) Western blot analyses of the expression of metastasis-related proteins, E-cadherin, Vimentin, and MMP9 in A549 and H1299 cells with altered miR-25 expression. Results were presented as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 3

LATS2 is a target gene of miR-25 [21]. (a) The predicted binding site of miR-25 in the 3′UTR of LATS2 from miRBase. The mutated LATS2 3′UTR-binding site is indicated. (b) LATS2 expression in 15 pairs of NSCLC tissues and adjacent normal tissues by qRT-PCR. (c) LATS2 expression in NSCLC cell lines and the noncancerous lung epithelial cell line BEAS-2B. (d) Kaplan-Meier curve for overall survival in lung cancer patients with high or low LATS2 expression. Data were taken from TCGA. (e, f) Luciferase reporter assays in HEK-293 cells. (g, h) Luciferase reporter assays in H1299 cells. HEK-293T, A549, and H1299 cells were cotransfected with pRL-TK carrying a wild-type or mutant 3′UTR of LATS2 and the miR-25 precursor (60 ng) or the miR-25 inhibitor (10 pmol), and the luciferase activity was measured 48 h posttransfection. (i) Western blot assays for LATS2 in A549, H1299, and BEAS-2B cells. (j) Western blot analysis of LATS2 in A549 and H1299 cells transfected with miR-25 precursor or the miR-25 inhibitor. Experiments in this section were performed using our documented protocols [21]. Results were presented as the mean ± SD. ^∗P < 0.05 and ^∗∗∗P < 0.001.

Figure 4

The effects of LATS2 expression on A549 cell proliferation, migration, and invasion. (a) Colony formation of A549 cells transfected with LATS2, miR-25, or the combination of both miR-25 and LATS2 plasmids. (b, c) Wound healing and transwell assays for migration and invasion capacities of A549 cells transfected with miR-25 and/or LATS2. (d) Western blot assays for LATS2, downstream effectors of the Hippo pathway (phosphorylated YAP and YAP), and metastasis-related proteins (E-cadherin, Vimentin, and MMP9) in A549 cells transfected with miR-25 and/or LATS2. Results were presented as the mean ± SD. (e) Immunofluorescence assay for nuclear translocation of YAP protein. Representative immunofluorescent images of YAP staining were shown in the left panel (green: YAP; blue: nuclear; scale bar, 10 μm). The nuclear localization of YAP is quantified in the right panel by ImageJ software. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 5

miR-25 antagomir inhibits lung cancer growth and metastasis in mouse xenografts. (a) Time course of tumor volumes from immunodeficient mice treated with the miR-25 antagomir or a control. Results were presented as the mean ± SD. ^∗∗P < 0.01 and ^∗∗∗P < 0.001. (b, c) The image and the weight of xenograft tumors one month after the treatment. Mean ± SD was shown. ^∗∗P < 0.01. (d) Representative images of mouse lungs and livers. (e) Histological morphology of mouse lungs and livers by HE staining. The magnification of the upper row images was 40x, bar scale = 500 μm. The magnification of the bottom row images was 400x, bar scale = 50 μm. (f, h) The incidence of lung and liver metastasis in mice after tail intravenous injections with the miR-25 antagomir was shown in the table. (g) Quantification of the metastasis nodules in the lungs of each group (n = 10). ^∗P < 0.05. (i) Representative images of immunohistochemical analysis of LATS2, E-cadherin, Vimentin, and MMP9 in lung metastasis (magnification, 400x. bar scale = 50 μm). Brown represented the target protein in IHC, and blue represented nuclear staining.

Figure 6

Schematic presentation of a positive feedback loop involving miR-25/LATS2/YAP/TAZ/TEAD/MCM7/miR-25 in lung cancer.