Introduction

Lung cancer remains the most frequent cause of cancer-related death in developed countries.1 Approximately 80% of lung cancers are classified histopathologically as non-small cell lung cancers. Patients with non-small cell lung cancers in advanced stages rarely survive more than 5 years despite aggressive chemotherapy, molecularly targeted therapy or chemoradiotherapy.2 Non-small cell lung cancer is subdivided into four major histological subtypes with distinct pathological characteristics: adenocarcinoma, squamous cell carcinoma (SCC), large cell carcinoma and neuroendocrine cancer.3 Molecular therapies such as gefitinib and erlotinib, which are specific for mutations in the epidermal growth factor receptor (EGFR; also known as ERBB1) gene and crizotinib, which is targeted against the EML4-ALK fusion gene, have been developed only for adenocarcinoma, but not for SCC in lung cancer (lung-SCC).4, 5, 6 For patients with lung-SCC, the standard therapeutic approach remains to be chemotherapy. Therefore, there is a need for additional treatment options based on current RNA network analysis of lung-SCC.

The discovery of non-coding RNAs in the human genome was an important conceptual breakthrough in the post-genome sequencing era.7 Improved understanding of non-coding RNAs is necessary for continued progress in cancer research. MicroRNAs (miRNAs) repress gene expression by inhibiting mRNA translation or by promoting mRNA degradation. Aberrant expression of miRNAs can contribute to cancer development, metastasis and drug resistance.8, 9, 10 Currently, 2588 human mature miRNAs are registered at miRBase release 20.0 (http://microrna.sanger.ac.uk/). miRNAs are unique in their ability to regulate multiple protein-coding genes. Bioinformatic predictions indicate that miRNAs regulate ~30–60% of the protein-coding genes in the human genome.11, 12

Our miRNA expression signatures of human cancers, including lung-SCC, revealed that expression of the miR-1/133a cluster is significantly reduced in several types of cancer tissues. Based on the signatures, we sequentially identified novel miR-1/133a cluster-regulated cancer pathways in prostate cancer, head and neck cancer, and urothelial carcinoma.13, 14, 15 However, the functional role and molecular targets of the miR-1/133a cluster in lung-SCC is unclear. The aim of the present study was to investigate the functional significance of the miR-1/133a cluster and to identify the molecular targets regulated by these clustered miRNAs in lung-SCC cells.

We found that restoration of either mature miR-1 or miR-133a inhibited cancer cell proliferation, migration and invasion. Gene expression data and in silico database analysis showed that coronin-1C (CORO1C) was a potential target of miR-1/133a cluster regulation. The CORO1C gene encodes a member of the WD repeat protein family and is involved in a variety of cellular processes.16, 17 Downregulating the CORO1C gene significantly inhibited cancer cell proliferation, migration and invasion. Furthermore, CORO1C-regulated molecular pathways were categorized by using si-CORO1C transfected cells. Further analysis of tumor-suppressive miR-1/133a–CORO1C-mediated cancer pathways might provides new insights into the potential mechanisms of lung-SCC oncogenesis and metastasis.

Materials and methods

Clinical specimens and RNA extraction

A total of 32 lung-SCCs and 22 normal lung specimens were collected from patients who underwent pneumonectomy at the Kagoshima University Hospital (Kagoshima, Japan) from 2010 to 2013. Archival formalin-fixed paraffin-embedded samples were used for quantitative real-time reverse transcription–PCR (qRT–PCR) analysis.

Samples were staged according to the International Association for the Study of Lung Cancer TNM classification and were histologically graded.18 Our study was approved by the Institutional Review Board for Clinical Research of the Kagoshima University School of Medicine. Prior written informed consent and approval were provided by each patient.

Formalin-fixed paraffin-embedded tissues were sectioned to 10-μm thickness, and eight tissue sections were used for RNA extraction. Total RNA (including miRNA) was extracted using Recover All Total Nucleic Acid Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocols. The integrity of the RNA was checked with the RNA 6000 Nano Assay Kit and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Cell culture

We used a human lung-SCC cell line (EBC-1) obtained from the Japanese Cancer Research Resources Bank (JCRB, Osaka, Japan). Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and maintained in a humidified incubator (5% CO2) at 37 °C.

Quantitative real-time reverse transcription–PCR

The procedure for PCR quantification was previously described.13, 19, 20 TaqMan probes and primers were assay-on-demand gene expression products: CORO1C (assay ID: Hs00902568_m1, Applied Biosystems, Foster City, CA, USA), synaptotagmin-1 as an integral membrane proteins of synaptic vesicles (SYT1) (assay ID: Hs00194572_m1, Applied Biosystems), STC2 (assay ID: Hs01063215_m1, Applied Biosystems) and cathepsin V as a group of lysosomal protease (CTSV) (assay ID: Hs00952036_m1, Applied Biosystems). Stem-loop RT–PCR for miR-1 (assay ID: 002222, Applied Biosystems) and miR-133a (assay ID: 002246, Applied Biosystems) was used to quantify the expression levels of miRNAs according to the manufacturer’s protocol. To normalize the data for quantification of mRNA and miRNAs, we used human GUSB (assay ID: Hs99999908_m1; Applied Biosystems) and RNU48 (assay ID: 001006; Applied Biosystems), and the delta–delta Ct method was employed to calculate the fold change.

Transfection of EBC-1 cells with mature miRNA and small interfering RNA (siRNA)

The following mature miRNA species and siRNA species were used in the present study: Pre-miR miRNA precursors (P/N: AM 17100; Applied Biosystems), negative-control miRNA (P/N: AM 17111; Applied), Stealth Select RNAi siRNA, si-CORO1C (cat no. HSS119044, cat no. HS119045; Invitrogen, Carlsbad, CA, USA) and negative-control siRNA (D-001810-10; Thermo Fisher Scientific, Waltham, MA, USA). RNAs were incubated with Lipofectamine RNAiMAX reagent (Invitrogen) and OPTI-MEM (Invitrogen) as described previously.13, 19, 20

Treatment of EBC-1 cells with 5-azacitidine (an inhibitor of DNA methyltransferase) or trichostatin A (a histone deacetylase inhibitor)

Cells were treated with 5 μM of 5-azacitidine or 300 nM of trichostatin A. After 96 or 24 h of treatment, total RNA was extracted and subjected to qRT–PCR.

Cell proliferation, migration and invasion assays

Cells were transfected with 10 nM miRNAs or siRNA by reverse transfection and plated in 96-well plates at 8 × 103 cells per well. After 72 h, cell proliferation was determined with the XTT assay using a Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany) as described previously.13, 19, 20

Cell migration activity was evaluated with wound healing assays. Cells were plated in six-well plates at 8 × 105 cells per well, and after 48 h of transfection the cell monolayer was scraped using a P-20 micropipette tip. The initial gap length (0 h) and the residual gap length 24 h after wounding were calculated from photomicrographs as described previously.13, 19, 20

Cell invasion assays were performed using modified Boyden chambers, consisting of transwell-precoated matrigel membrane filter inserts with eight micron pores in 24-well tissue culture plates (BD Biosciences, Bedford, MA, USA). RPMI 1640 medium containing 10% fetal bovine serum in the lower chamber served as the chemoattractant as described previously.13, 19, 20 All experiments were performed in triplicate.

Identification of putative target genes of miR-1/miR-133a and CORO1C

Genes regulated by miR-1/miR-133a were obtained from the TargetScan database (http://www.targetscan.org). To investigate the expression status of candidate miR-1/miR-133a target genes in lung-SCC clinical specimens, we examined gene expression profiles in the gene expression omnibus database (accession number: GSE11117). In addition, we performed gene expression analysis using miR-1 or miR-133a-transfected EBC-1 cells and si-CORO1C-transfected EBC-1 cells in comparison with control-transfected cells. Oligo-microarray human 60Kv (Agilent Technologies) was used for gene expression studies. Microarray procedures and data mining methods were described in previous studies.13, 15, 20

To identify molecular pathways regulated by CORO1C in EBC-1 cells, gene expression data were categorized according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the GENECODIS program (http://genecodis.dacya.ucm.es). The strategy behind this analysis procedure was previously described.14, 20

Western blot analysis

Cells were harvested after 72 h of transfection, protein lysates (100 μg) were separated by NuPAGE on 4–12% bis-tris gels (Invitrogen) and transferred to polyvinylidene fluoride membranes. Immunoblotting was done with diluted (1:1000) monoclonal anti-coronin 3 antibody (ab77203; Abcam, Cambridge, UK) and GAPDH antibody (MAB374; Chemicon, Temecula, CA, USA). The membrane was washed and then incubated with horse radish peroxide-linked anti-rabbit immunoglobulin-G antibody (#7074; Cell Signaling, Danvers, MA, USA). Specific complexes were visualized with an echochemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK) as described previously.13, 15, 20

Plasmid construction and dual-luciferase reporter assays

Partial wild-type sequences of the CORO1C 3′-untranslated region (UTR) or those with a mutated miR-1 target site (positions 111–118, 147–154 of the CORO1C 3′-UTR) and miR-133a target site (positions 147–154 and 371–377 of the CORO1C 3′-UTR) were inserted between the XhoI and PmeI restriction sites in the 3′-UTR of the hRluc gene in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA).

The synthesized DNA was cloned into the psiCHECK-2 vector. EBC-1 cells were transfected with 50 ng of the vector and 10 nM miR-1 or miR-133a using Lipofectamine 2000 (Invitrogen). The activities of firefly and renilla luciferases in cell lysates were determined with a dual-luciferase assay system (E1960; Promega). Normalized data were calculated as the ratio of renilla/firefly luciferase activities.

Statistical analysis

Relationships between two or more variables and numerical values were analyzed using the Mann–Whitney U-test or Bonferroni-adjusted Mann–Whitney U-test. In comparison among three or four variables, a non-adjusted statistical level of significance of P<0.05 corresponds to a Bonferroni-adjusted level of P<0.0167 or P<0.0125, respectively. Expert StatView software, version 4 (SAS Institute, Cary, NC, USA), was used in these analyses.

Results

Expression levels of miR-1 and miR-133a in lung-SCC specimens and cell line

To validate our past miRNA signature of lung-SCC results, we evaluated the expression of miR-1 and miR-133a in lung-SCC tissues (n=32) and normal lung tissues (n=22). The background of patients and clinicopathological characteristics are summarized in Table 1. Representative formalin-fixed paraffin-embedded specimens that were used for RNA extraction and expression analysis in this study are shown in Supplementary Figure 1.

Table 1 Characteristic of patients
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The expression levels of miR-1 and miR-133a were significantly reduced in tumor tissues compared WITH corresponding non-cancerous tissues (P<0.0001; Figures 1a and b, respectively). Spearman’s rank test showed a positive correlation between the expression of miR-1 and that of miR-133a (r=0.956 and P<0.0001, Figure 1c).

Figure 1
figure 1

The expression levels of microRNA-1 (miR-1) and miR-133a in lung-squamous cell carcinoma (lung-SCC) clinical specimens and EBC-1 cells, and effects of restoring the miRNAs on cell viabilities. (a, b) The expression levels of miR-1 (a) and miR-133a (b) in clinical specimens (lung-SCC and normal lung) and a lung-SCC cell line (EBC-1) were measured by quantitative real-time reverse transcription–PCR. RNU48 was used as an internal control. Significances were determined by Mann–Whitney U-test. (c) Spearman's rank test showed a positive correlation between the expression of miR-1 and that of miR-133a (r=0.956 and P<0.0001). (d) Cell proliferation was determined with XTT assays 72 h after transfection with 10 nM of miR-1 or miR-133a. **P<0.0001 (Bonferroni-adjusted Mann–Whitney U-test). (e) Cell migration activity was determined by migration assay 48 h after transfection. **P<0.0001 (Bonferroni). (f) Cell invasion activity was determined by matrigel invasion assay 48 h after transfection. **P<0.0001, *P=0.0004 (Bonferroni).

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There was a significant relationship between the expression level of miR-1 or miR-133a and venous invasion. However, there was no significant correlation with other clinicopathological parameters (stage, grade and infiltration). In lung-SCC cell line, EBC-1 cells also exhibited low expression levels of miR-1 and miR-133a compared with normal lung tissues (Figures 1a and b).

Treatment with trichostatin A significantly increased miR-1 and miR-133a expression levels in EBC-1 cells, whereas 5-azacitidine had no effect on their expression levels (Supplementary Figure 2).

Effects of restoring miR-1 and miR-133a on cell proliferation, migration and invasion activities of EBC-1 cells

To examine the functional roles of miR-1 and miR-133a, we performed gain-of-function studies using miRNA transfection into EBC-1 cells. XTT assays revealed significant inhibition of cell proliferation in EBC-1 cells transfected with miR-1 and miR-133a in comparison with mock-transfected cells and control transfectants (both P<0.0001, Figure 1d).

Moreover, wound healing assays revealed significant inhibition of cell migration in EBC-1 cells transfected with miR-1 and miR-133a (both P<0.0001, Figure 1e, Supplementary Figure 3a). Similarly, matrigel invasion assays revealed that transfection with miR-1 and miR-133a reduced cell invasion. Indeed, the number of invading cells was significantly decreased in EBC-1 cells transfected with miR-1 and miR-133a (both P<0.001, Figure 1f Supplementary Figure 3b).

It is plausible the miR-1/133a clusters may have important roles as tumor suppressors by targeting several oncogenic genes. However, we found no simultaneous effect of cell viability inhibition by miR-1 and miR-133a co-transfection on EBC-1 cells (data not shown). A similar phenomenon has been shown in our previous articles on miR-1/133a and miR-143/145 clusters in prostate cancer.13, 21

Identification of candidate genes targeted by the miR-1/133a cluster

The TargetScan database indicated that a total of 3117 and 2216 genes were putative targets of miR-1 and miR-133a, respectively. Moreover, 697 genes were common targets of both miR-1 and miR-133a. Among the 697 common genes, 12 genes were upregulated in lung-SCC clinical specimens according to the gene expression omnibus database (accession number GSE11117). When we examined all the putative target sites of miR-1 and miR-133a, four genes (STC2, SYT1, CTSV and CORO1C) had three conserved sites in total within their 3′-UTRs (Table 2). To gain further insight on which genes were affected by miR-1 and miR-133a transfection, we performed microarray analysis of miR-1 and miR-133a transfectants (EBC-1). Among the four genes, CORO1C was the most downregulated gene (log2 ratio <−1.5 in both miR-1 and miR-133a transfectants) (Table 2). Therefore, we focused on CORO1C for further studies.

Table 2 Common target genes of miR-1 and miR-133a highly expressed in lung-SCC
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CORO1C was a direct target of the miR-1/133a cluster in EBC-1 cells

We performed qRT–PCR and Western blotting to confirm CORO1C gene and CORO1C protein downregulation by restoration of miR-1 or miR-133a in EBC-1. The messenger RNA (mRNA) and protein expression levels of the CORO1C gene and its protein were significantly repressed in miR-1 and miR-133a transfectants in comparison with mock or miR-control transfectants (both P<0.001, Figure 2).

Figure 2
figure 2

Direct regulation of CORO1C by microRNA-1 (miR-1) and miR-133a in EBC-1 cells. (a) CORO1C messenger RNA (mRNA) expression was evaluated by quantitative real-time reverse transcription–PCR 72 h after transfection with miR-1 or miR-133a. GUSB was used as an internal control. **P<0.0001, *P=0.0002 (Bonferroni). (b) CORO1C protein expression was evaluated by Western blotting 72 h after transfection with miR-1 or miR-133a. GAPDH was used as a loading control.

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We then performed luciferase reporter assays in EBC-1 cells to determine whether CORO1C was directly regulated by miR-1 and miR-133a. The TargetScan database predicted that there were two binding sites for miR-1 in the 3′-UTR of CORO1C (positions 111–118 and 147–154; Figure 3a), and one binding site for miR-133a in the 3′-UTR of CORO1C (positions 371–377; Figure 3b). We used vectors encoding the partial wild-type sequence of the 3′-UTR of CORO1C mRNA, including the predicted miR-1 or miR-133a target sites. We found that the luminescence intensity was significantly reduced by co-transfection with miR-1 or miR-133a and the vector carrying the wild-type 3′-UTR of CORO1C, whereas transfection with the mutant vector (positions 111–118, 147–154 or 371–377 had been changed) blocked the decrease in luminescence (P<0.0001, Figures 3a and b). These data suggested that miR-1 and miR-133a bound directly to specific sites in the 3′-UTR of CORO1C mRNA.

Figure 3
figure 3

MicroRNA-1 (miR-1) and miR-133a binding sites in the 3′-untranslated region (3′-UTR) of CORO1C. (a, b) Luciferase reporter assays using vectors encoding putative miR-1 and miR-133a target sites at positions 111–118, 147–154 and 371–377 for both wild type and mutants. Renilla luciferase values were normalized to firefly luciferase values. *P<0.0001 (Bonferroni). (c) The clusters miR-1-1/miR-133a-2 and miR-1-2/miR-133a-1 are located in two different chromosomal regions 20q13.33 and 18q11.2, respectively.

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Downregulation of CORO1C expression in EBC-1 cells affected proliferation, migration and invasion activities

To investigate the functional role of CORO1C, we performed loss-of-function studies using si-CORO1C transfectants. First, we evaluated the knockdown efficiency of si-CORO1C transfection in EBC-1 cells. Western blotting and qRT–PCR indicated that si-CORO1C effectively downregulated CORO1C expression in EBC-1 cells (P<0.0001, Figures 4a and b).

Figure 4
figure 4

Silencing of CORO1C messenger RNA (mRNA) and protein expression by si-CORO1C transfection and effects of silencing of CORO1C on EBC-1 cell activities. (a) CORO1C mRNA expression was evaluated by quantitative real-time reverse transcription–PCR 72 h after transfection with miR-1 or miR-133a. GUSB was used as an internal control. **P<0.0001 (Bonferroni). (b) CORO1C protein expression was evaluated by Western blotting 72 h after transfection. GAPDH was used as a loading control. (c) EBC-1 cell proliferation was determined with XTT assays. **P<0.0001, *P=0.0025 (Bonferroni). (d) EBC-1 cell migration activity was determined by migration assay. **P<0.0001 (Bonferroni). (e) EBC-1 cell invasion activity was determined by matrigel invasion assay. **P<0.0001 (Bonferroni).

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XTT assays demonstrated that cell proliferation was significantly inhibited in si-CORO1C transfectants in comparison with mock or si-control transfectants in EBC-1 cells (P<0.005, Figure 4c). Moreover, wound healing assays revealed significant inhibition of cell migration in si-CORO1C transfectants in comparison with mock or si-control transfectants in EBC-1 cells (P<0.0001, Figure 4d, Supplementary Figure 5a). Similarly, matrigel invasion assays revealed that the number of invading cells was significantly decreased when EBC-1 cells were transfected with si-CORO1C (P<0.0001, Figure 4e,Supplementary Figure 5b).

Identification of molecular pathways regulated by CORO1C in EBC-1 cells

To investigate the molecular pathways regulated by CORO1C, a genome-wide gene expression analysis was performed in EBC-1 cells transfected with si-CORO1C. A total of 967 genes were identified as being downregulated in si-CORO1C transfected cells compared with the control (log2 ratio<−0.2). We categorized the downregulated genes according to KEGG pathways, and a total of 29 pathways were identified as significantly enriched (Table 3). Among these pathways, we focused on ‘pathways in cancer’, ‘focal adhesion’, ‘tight junction’ and ‘regulation of actin cytoskeleton’ pathways because si-CORO1C significantly inhibited cancer cell migration and invasion. Genes involved in these pathways are listed in Supplementary Table 1.

Table 3 Significantly enriched KEGG pathways regulated by si-CORO1C in lung-SCC cell
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Discussion

Accumulating evidence has suggested that aberrantly expressed miRNAs disrupt tightly regulated RNA networks in cancer cells. These events are believed to initiate cancer cell development and metastasis.8, 9 Therefore, identification of differentially expressed miRNAs in cancer cells is the first step to elucidating novel miRNA-regulated molecular mechanisms in cancer cells. Expression signatures of various types of cancer tissues are important sources for the study of miRNAs in cancer. Based on these points, our group has determined the miRNA expression signatures in various cancers and thereby identified tumor-suppressive miRNAs.15, 22, 23

Recent genome-wide miRNA expression technologies have clarified that miR-1/133a and miR-206/133b clusters are to be altered in several types of human cancers.24 In this study, we focused on miR-1/133a-clustered miRNAs because these miRNAs were frequently downregulated in various types of cancers, including lung-SCC.24, 25 Our present study showed that miR-1 and miR-133a were significantly reduced in lung-SCC tissues and that they function as tumor suppressors by modulating cancer cell migration and invasion in lung-SCC. Our studies and other reports strongly suggest that the miR-1/133a-clustered miRNAs might act as tumor suppressors in various human cancers.13, 14, 15, 26

The chromosomal location of the miR-1/133a cluster in the human genome is of significant interest. The miR-1-1/miR-133a-2, the miR-1-2/miR-133a-1 and the miR-206/miR-133b clusters are located in three different chromosomal regions, that is, 20q13.33, 18q11.2 and 6p12.1, respectively. The mature sequence of miR-1 differs from that of miR-206 by four nucleotides.24 The mature sequences of miR-133a-1 and miR-133a-2 possess identical sequences. The miR-133b sequence differs from miR-133a by a single nucleotide at the 3′-end.24 Silencing epigenetic mechanisms of these clustered miRNAs have been investigated, DNA methylation-mediated miR-1 downregulation was suggested in hepatocellular carcinoma and colorectal cancer.27, 28 In lung cancer cells, previous study and our data (Supplementary Figure 2) indicated that miR-1 and miR-133a silencing might be caused by hypoacetylation of histones.29 Detailed analyses will be necessary about epigenetic mechanism of miR-1/133a cluster in lung-SCC cells in future.

We also investigated the expression levels of miR-206/133b cluster in lung-SCC specimens. Our data indicated that these clustered miRNAs were downregulated in lung-SCC tissues compared with non-cancerous tissues (Supplementary Figure 6). Interestingly, our recent study showed that expression of mature miR-206 also suppressed cancer cell proliferation, migration and invasion in lung-SCC cells by targeting both mRNA and protein levels of MET and EGFR (under submission).

The genes regulated by miRNA in cancer cells vary according to cancer types. Thus, identification of the targets regulated by the tumor-suppressive miR-1/133a cluster is important for clarifying our understanding of lung-SCC oncogenesis and metastasis. In this study, we identified common target genes of the miR-1/133a cluster using a combination of in silico analysis and gene expression analysis with miR-1 and miR-133a transfectants. Using this strategy, we succeeded in proving that the tumor-suppressive miR-1/133a cluster-regulated oncogenic genes such as TAGLN2 in bladder cancer, renal cell carcinoma and maxillary sinus SCC, and PNP in prostate cancer.13, 14, 15 Moreover, we showed that restoration of miR-133a led to a reorganization of the actin cytoskeleton and a subsequent change in cell morphology, inhibitions of cell migration and invasion by direct targeting of actin-related protein 2/3 complex subunit 5 (ARPC5) in head and neck SCC.30 Identification of these target genes might contribute to elucidation of novel cancer networks in lung-SCC.

In this study, we focused on the CORO1C gene. We evaluated the expression levels of four candidate genes (STC2, SYT1, CTSV and CORO1C) using miR-1 or miR-133a transfectants by qRT–PCR methods. Among them, three genes (SYT1, CTSV and CORO1C) were significantly downregulated by both restoration of miR-1 and miR-133a in EBC-1 cell (Supplementary Figure 4). CORO1C is a member of coronin protein family, a WD-repeat-containing, actin-binding protein that regulates cell motility by coordination of actin filament turnover.31, 32 Other candidate genes, such as SYT1 and CTSV were excluded in this study because they were well investigated in previous studies. Our present data clearly showed that CORO1C was directly regulated by both miR-1 and miR-133a in lung-SCC cells. Furthermore, silencing of CORO1C gene expression significantly inhibited cancer cell migration and invasion. These results are consistent with past reports of gastric cancer and diffuse glioma.33, 34 According to the previous reports, silencing of CORO1C expression significantly inhibited cancer cell migration and invasion in human gastric and glioblastoma cell lines.33, 34 Furthermore, CORO1C was remarkably higher in tissues of lymph node metastases than primary gastric cancer tissues, and overexpression of CORO1C was associated with tumor-grade and lymph node metastasis.33 More recently, it was reported that miR-206 repressed cancer cell migration through direct regulation of CORO1C in triple-negative breast cancer cells.35 These results indicated that downregulation of miR-1/133a cluster or miR-206/133b cluster in cancer cells elevated the expression level of CORO1C, and these events might promote cancer cell migration and invasion.

For decades, the molecular mechanisms of cancer cell metastasis involving the regulation of the actin cytoskeleton have been intensively studied. Expression of CORO1C is localized at the leading edge of lamellipodia and invadopodia, and it physically interacts with the Arp2/3 complex.31, 32, 36, 37 Lamellipodia are thin, sheet-like membrane protrusions found at the leading edge of motile cells, and they are regulated by the Arp2/3 complex and cofilin.37, 38, 39 Invadopodia are actin-rich membrane protrusions that are formed by invasive cells, involving several proteins such as the Arp2/3 complex, WASP, cofilin and coronins.37, 38, 39 These results suggest that regulation of CORO1C by the miR-1/133a cluster might be an important mechanism for cancer cell migration and invasion.

To approach further functional roles of CORO1C, we performed genome-wide gene expression analysis using si-CORO1C-transfected EBC-1 cells to investigate CORO1C-regulated molecular pathways and targets. Several pathways that significantly contributed to cell migration and invasion were enriched downstream from CORO1C, such as ‘pathway in cancer’, ‘focal adhesion’, ‘tight junction’ and ‘regulation of actin cytoskeleton’ pathways. Several genes involved in these pathways were downregulated by both si-CORO1C and miR-1/133a transfectants, suggesting that these genes are involved in tumor-suppressive miR-1/133a-CORO1C pathway in lung-SCC cells. For example in ‘pathways in cancer’, several well-known oncogenes, such as BIRC5, HIF1A, MYC, RUNX1, VEGFC and VEGFA, might be involved in this network in lung-SCC cells (Supplementary Table 1). To better understand oncogenesis and metastasis in lung-SCC, it will be important to analyze genes involved in these pathways and their relationship with the miR-1/133a-CORO1C-mediated signal transduction.

In conclusion, miR-1/133a-clustered miRNAs were significantly downregulated in clinical lung-SCC specimens, and appeared to function as tumor suppressors through regulation of oncogenic CORO1C. Molecular pathways regulated by the tumor-suppressive miR-1/133a cluster might control actin cytoskeleton networks in cancer cells. Elucidation of oncogenic CORO1C pathways targeted by the tumor-suppressive miR-1/133a cluster may reveal novel effective therapeutic strategies for the treatment of lung-SCC.