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. 2017 Jul 31;6(7):e366.
doi: 10.1038/oncsis.2017.66.

miR-151a Induces Partial EMT by Regulating E-cadherin in NSCLC Cells

Affiliations
Free PMC article

miR-151a Induces Partial EMT by Regulating E-cadherin in NSCLC Cells

I Daugaard et al. Oncogenesis. .
Free PMC article

Abstract

miR-151a and its host gene, focal adhesion kinase, FAK, are located in a region of chromosome 8q that is frequently amplified in solid tumors, including lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and metastasis remains the major challenge in battling lung cancer mortality. Here, we demonstrate that miR-151a is overexpressed in non-small cell lung cancer (NSCLC) patient specimens, as compared to healthy lung. In addition, miR-151a overexpression promotes proliferation, epithelial-to-mesenchymal transition (EMT) and induces tumor cell migration and invasion of NSCLC cells. Blocking miR-151a expression using anti-miR-151a approaches significantly reduced NCSLC cell proliferative and motility potential. Furthermore, we determined that miR-151a significantly regulates E-cadherin expression. Finally, functional rescue experiments determined that overexpression of E-cadherin in miR-151a NSCLC cell lines potently repressed miR-151a-induced partial EMT and cell migration of NSCLC cells. In conclusion, our findings suggest that miR-151a functions as an oncomiR in NSCLC by targeting E-cadherin mRNA and inducing proliferation, migration and partial EMT.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
miR-151a expression is induced in NSCLC. (a) miR-151a expression levels were characterized by miR-specific RT-qPCR analysis in a NSCLC (LAC) cohort comprising 52 primary LACs and 26 paired distant metastases (22 brain and 4 adrenal gland), as well as 10 tumor-adjacent normal lung samples. Relative expression is shown as a dot plot with mean±s.e.m. in each group indicated. (b) In situ hybridization using scrambled miR-control and miR-151a probes of normal, primary lung tumor (‘T’) and metastatic (‘M’) tissue. Areas with normal lung and brain cells are indicated with triangles and circles, respectively. High miR expression is shown as blue/purple, low expression is shown as light pink. One representative example shown of 3. (c) miR-151a expression in paired tumor and tumor-adjacent normal lung samples from 45 LAC patients. Expression data from all LAC patients with paired specimens available were downloaded from The Cancer Genome Atlas webpage (https://cancergenome.nih.gov/) and log2 transformed. Red or blue lines indicate paired samples with increased or decreased miR-151a expression in the tumor tissue, respectively. **P<0.01, ****P<0.0001; by two-tailed unpaired (a) or paired (c) Student’s t-test.
Figure 2
Figure 2
miR-151a enhances NSCLC cell growth. Cell proliferation of miR-modulated (miR-CTL, miR-151a and anti-miR-151a) (a) A549 cells, (b) H1299 cells and (c) H23 cells were analyzed by culturing cells at low density and counted for 4 days. (d) Colony Formation Assays of miR-modulated A549 cells. Representative images for each treatment are shown. Scale bar=1000 μm. (n=3, independent biological experiments, 3 technical replicates of each). (e) Growth analysis counting A549s transfected with miR-CTL, miR-151a or anti-miR-151a oligonucleotides (miR-mimics). (n=3 independent cell cultures and experiments, 3 technical replicates for each). (f) Growth analysis counting A549s stably expressing miR-CTL, anti-miR-151a or both miR-151a and anti-miR-151a (n=3 independent cell cultures and experiments, 3 technical replicates for each). (g) Growth analysis counting human lung endothelial cells (hLECs) stably expressing miR-CTL, miR-151a and anti-miR-151a. (n=1 biological replicate, 3 technical replicates of each). Throughout figure, all graphs are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by two-tailed Student’s t-test.
Figure 3
Figure 3
miR-151a enhances NSCLC cell migration and invasion and anti-miR-151a reduces cell motility. (a) Stably (Images and left graph) or transiently (right graph) miR-modulated A549 cells (miR-CTL, miR-151a or anti-miR-151a) were pre-treated with mitomycin c for 2 h and then migration was analyzed by scratch assay. Representative images are shown, scale bar=500 μm. (n=3 independent cell cultures and experiments). Scratch assays were also performed for stably miR-modulated (b) H23 cells and (c) H1299 cells. (d) Migration of mitomycin c pretreated miR-modulated A549 cells was analyzed by performing transwell migration assays for 6 h, at which point migrated cells were stained with DAPI and counted. Representative images are shown, scale bar=200 μm (n=3 independent cell cultures and experiments, 3 technical replicates of each). (e) Invasion assay were performed using stable miR-expressing A549s pre-treated with mitomycin c and allowed to migrate for 12 h. Representative images are shown, scale bar=200 μm (n=2 independent cell cultures and experiments, 3 technical replicates of each). Throughout figure, all graphs are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by two-tailed Student’s t-test.
Figure 4
Figure 4
miR-151a induces a mesenchymal-like phenotype and anti-miR-151a enhances an epithelial cell-like phenotype of NSCLC cells. Images (top panels) of (a) A549 cells and (b) H1299 cells untreated, treated with TGF-β, or stably transduced with miR-CTL, miR-151a or anti-miR-151a. Column 1: Bright field (Scale bar=400 μm), Column 2: Bright field (scale bar=100 μm). The relative proportion of mesenchymal, epithelial, or undefined cells were quantified and shown as stacked bar percentage plots (lower panels). The relative proportion of mesenchymal cells was compared between treatments using two-tailed Student’s t-test. One representative image shown of three independent cell cultures and experiments. Throughout figure, all graphs are shown as mean±s.e.m. **P<0.01; ***P<0.001; ****P<0.0001 by two-tailed Student’s t-test.
Figure 5
Figure 5
miR-151a reduces E-cadherin expression in NSCLC cells. (a) The relative expression levels of E-cadherin (left), fibronectin (middle) and Slug (right) were determined in stably miR-transduced A549 cells by RT-qPCR. (n=3 independent cell cultures and experiments, 3 technical replicates of each). (b) Representative confocal images of stably transduced A549s immunofluorescently stained with E-cadherin. Scale bar=10 μm. Relative fluorescence levels are quantified of the shown biological replicate (n=3 independent cell cultures and experiments, 12 technical replicates of each). (c) Immunoblot analysis of E-cadherin and tubulin protein levels in A549s transduced with miR constructs. Relative levels are quantified. One representative example of three is shown. (d) The expression levels of miR-151a was analyzed by in situ hybridization and compared with the expression of E-cadherin determined by immunohistochemical staining in three normal lung samples and 3 primary LACs (miR-151a expression: high=purple, low=light pink, E-cadherin expression: high=pink, low=blue). Cytokeratin 7 was included to identify cells of epithelial origin, for example, adenocarcinoma cells. Scale bar=50 μm. Throughout figure, all graphs are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by two-tailed Student’s t-test. Uncropped version of blot is shown in Supplementary Figure S8.
Figure 6
Figure 6
miR-151a interacts with E-cadherin mRNA. (a) Schematic representation of three potential 6-mer miR-151a-binding sites in the coding DNA sequence (CDS) of E-cadherin mRNA. (b) Relative luciferase levels in A549 cells transfected with constructs expressing the wild type (WT) binding sequences at site #1, #2 or #3, along with miR-CTL or miR-151a mimics, were determined 48 hr after transfection. (n=3 independent cell cultures and experiments, 3 technical replicates of each). (c) Schematic of miR-151a binding to WT or mutant E-cadherin mRNA. Relative luciferase levels of A549 cells transfected with constructs expressing WT or mutated (site #2) binding sequences and miR-CTL or miR-151a mimics were determined 48 hr after transfection. (n=3 independent cell cultures and experiments, 3 technical replicates of each). (d) Schematic of Argonaute immunopurification strategy for miR-RNA complexes (Ago-RIP). (e) A549 cells stably expressing either miR-151a or anti-miR-151a were generated. Relative levels of E-cadherin, RhoGDIA and GAPDH mRNA in input and Ago-RIP (IP) fractions were determined. ‘Corrected’ indicates mRNA levels in IP fractions when normalized to the mRNA level in corresponding input fractions. (n=3 independent cell cultures and experiments, 3 technical replicates of each). Throughout figure, all graphs are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by two-tailed Student’s t-test.
Figure 7
Figure 7
E-Cadherin is a functional target of miR-151a-induced partial EMT. (a) Schematic representation showing miR-151a binding to WT or mutant (silent mutant) E-cadherin mRNA (miR-151a resistant E-cadherin, left panel). Immunoblot analysis of E-cadherin and GAPDH protein levels in A549 cells transduced with miR-151a resistant E-cadherin or control constructs (middle panel). Relative levels are quantified (right panel). (b) Migration of mitomycin c pre-treated A549 cells (images and left graph), H23 cells (middle graph) and H1299 cells (right graph) transduced with miR-CTL or miR-151a and miR-151a resistant E-cadherin or control constructs were assessed by wound healing assay at time 0 and 6 h. Representative images (left panels), scale bar=500 μm. (n=3 independent cell cultures and experiments, 3 technical replicates of each). (c) Transwell migration assay using A549s transduced with miR-CTL or miR-151a and miR-151a resistant E-cadherin or control constructs. Cells were pre-treated with mitomycin c, plated at the top of a transwell and allowed to migrate for 6 h. Representative images are shown (left panels, scale bar=400 μm. (n=3 independent cell cultures and experiments, 3 technical replicates of each). (d) Images of A549 cells transduced with miR-CTL or miR-151a and miR-151a resistant E-cadherin or control constructs. Representative images are shown (left panel), scale bar=400 μm. The relative proportion of mesenchymal, epithelial, or undefined cells were quantified and shown as a stacked bar percentage plot (right panel). The relative proportion of mesenchymal cells was compared between treatments using two-tailed Student’s t-test. (n=3 independent cell cultures and experiments, three technical replicates of each). Throughout figure, all graphs are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by two-tailed Student’s t-test.

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