miR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma.

Based on the microRNA (miRNA) expression signatures of hypopharyngeal and esophageal squamous cell carcinoma, we found that miR-1 was significantly down-regulated in cancer cells. In this study, we investigated the functional significance of miR-1 in head and neck squamous cell carcinoma (HNSCC) cells and identified miR-1-regulated novel cancer pathways. Gain-of-function studies using miR-1 revealed significant decreases in HNSCC cell proliferation, invasion, and migration. In addition, the promotion of cell apoptosis and cell cycle arrest was demonstrated following miR-1e transfection of cancer cells. A search for the targets of miR-1 revealed that transgelin 2 (TAGLN2) was directly regulated by miR-1. Silencing of TAGLN2 significantly inhibited cell proliferation and invasion in HNSCC cells. Down-regulation of miR-1 and up-regulation of TAGLN2 were confirmed in HNSCC clinical specimens. Our data indicate that TAGLN2 may have an oncogenic function and may be regulated by miR-1, a tumor suppressive miRNA in HNSCC. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC carcinogenesis.


IntroductIon
Head and neck squamous cell carcinoma (HNSCC) constitutes the sixth most common malignancy worldwide [1]. In spite of considerable advances in multimodality therapy including surgery, radiotherapy, and chemotherapy, the overall five year survival rate for patients with this type of cancer is among the lowest of all major cancer types and has not improved during the last decade [2]. Local tumor recurrence and distant metastasis after conventional therapy appear to be major contributing factors for restricted survival of HNSCC patients. Therefore, understanding the molecular oncogenic pathways underlying HNSCC would help to improve diagnosis, approaches to therapy, and prevention of the disease.
MicroRNAs (miRNAs) are endogenous, short, noncoding RNA molecules which regulate gene expression by translational repression or degradation of mRNA in a sequence-specific manner [3]. Bioinformatic prediction indicates that miRNAs regulate more than 30% of the protein coding genes [4]. It is estimated that approximately 1,000 miRNAs exist in the vertebrate genome. At this time, 1,048 human miRNAs are registered at miRBase release 16.0 (http://microrna.sanger.ac.uk/). A growing body of evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression www.impactjournals.com/oncotarget [5]. miRNAs can be divided into two classes: those which are oncogenic miRNAs and those which are tumor suppressive miRNAs. Up-regulated miRNAs could act as oncogenes by negatively regulating tumor suppressor genes, while down-regulated miRNAs could function as tumor suppressors by repressing oncogenes [6,7].
Studies of tumor suppressive miRNAs and searches for their target genes are important for our understanding of miRNA-regulated cancer pathways, including those specific miRNAs altered in HNSCC [8][9][10]. Recently, our miRNA profiles showed that miR-133a was downregulated in cancer cells and that miR-133a had tumor suppressive functions [11][12][13]. Interestingly, miR-1 and miR-133a are located on the same chromosomal locus, forming a so called cluster. miR-1 and miR-133a are expressed in muscle and might be the most studied miRNAs in skeletal and cardiac muscle development [14,15].
miR-1 was also down-regulated in our miRNA screening of hypopharyngeal and esophageal squamous cell carcinoma [13,16]. However, the functional significance of miR-1 has not been clarified in HNSCC. The aim of this study was to investigate the function of miR-1 in HNSCC cell lines and to identify miR-1-regulated cancer pathways. For target genes searches of miR-1 in HNSCC cells, we performed genome-wide gene expression analysis. We focused on transgelin 2 (TAGLN2) as a candidate target of miR-1, as it was among the most down-regulated genes. Insight into the association between tumor suppressive miRNA and their target oncogene networks could enhance our understanding of the molecular mechanism of HNSCC carcinogenesis.

transfection on cell proliferation, migration, and invasion in Hnscc cell lines
To determine the function of miR-1, we performed gain-of-function analysis using miR-1 transfectants. The XTT assay showed statistically significant inhibition of cell proliferation in miR-1 transfectants in comparison with miRNA controls after 72 hr and 96 hr. For example, after 72 hr, both miR-1 transfected HSC3 and FaDu cultures grew only ~42% as much as control cultures (both P < 0.05, Figure 1A, top), while after 96 hr, proliferation fell to 23% and 9% of controls, respectively, (both P < 0.05, Figure 1A, bottom). Wound healing assays of miR-1-transfected HSC3 demonstrated that cell migration was significantly inhibited to 5% that of control (P < 0.05; Figure 1B).The Matrigel invasion assay showed that the number of invading cells was significantly decreased in miR-1 transfectants. Relative to control values (100%), the percentages of invading HSC3 and FaDu cells were only 45% and 27.1%, respectively (both P < 0.05; Figure  1C). Cell apoptosis in miR-1 transfected cells was assessed by flow cytometry. The fraction of early apoptotic cells significantly increased in miR-1 transfectants approximately 3-fold in HSC3 and 12-fold in FaDu compared with controls (both P < 0.05, Figure 2A). We also confirmed induction of apoptosis approximately 4-fold in FaDu by ectopic miR-1 performing TUNEL assay (data not shown). As for cell cycle distribution, cells in G0/G1 phase were significantly greater in miR-1 transfectant than those in the control ( Figure 2B). These results suggest that ectopic miR-1 expression induces G0/ G1 arrest in both HNSCC cell lines

Gene expression profiling identifies downregulated genes in mir-1 transfectants
To investigate candidate molecular targets of miR-1 in HNSCC cells, we examined the effect of miR-1 on protein coding genes. Mature miR-1 was transiently transfected into HSC3 and FaDu cells, with negative miRNA transfection used as a control. Comprehensive gene expression analysis (see Methods) clearly showed changes in gene expression patterns between miR-1 and negative-control transfectants. To identify candidate miR-1 target genes, a cut-off of value less than -2.00-fold was applied to the array data. This filter resulted in the identification of 59 genes that were significantly downregulated upon miR-1 transfection in both HSC3 and FaDu cells (top 20 genes are shown in Table 1). Entries from the microarray data were approved by the Gene Expression Omnibus (GEO), and were assigned GEO accession number GSE24782. The 3' UTR of these downregulated genes were examined for miR-1 target sites using the TargetScan database. Of the top 20 putative gene targets, 17 genes contained miR-1 target sites.
tAGln2 is a target of post-transcriptional repression by mir-1 TAGLN2 was the second ranked candidate gene in the genome-wide gene expression analysis. We focused on TAGLN2 and not the top ranked gene (SERP1), because the latter was not reported to be associated with carcinoma. The expression level of TAGLN2 mRNA was significantly decreased in both HNSCC cell lines (HSC3 and FaDu) transfected with miR-1 (Figure 3, upper). The protein expression levels were also markedly reduced in miR-1 transfectants (Figure 3, lower). HSC3 cells were used to determine the mechanism of miR-1 suppression of TAGLN2 expression. The TargetScan database identified three putative target sites in the 3'UTR of TAGLN2 (Figure 4, upper). A luciferase reporter assay confirmed the 3'UTR of TAGLN2 as the actual target of miR-1.
In this study, our data revealed that restoration of miR-1 expression suppressed cell proliferation, migration, and invasion and promoted apoptosis and cell cycle arrest in HNSCC cells. Our results and those of past reports indicate that miR-1 frequently functions as a tumor suppressor in human cancer. miRNAs control the expression of target genes which contribute to cancer development and progression. Because it is important to identify novel miRNA-mediated cancer pathways, we investigated miR-1-regulated oncogenic targets. We adopted a method of genome-wide gene expression analysis in two HNSCC cell lines, using miR-1 transfectants to identify targets. This strategy has led to the identification of tumor suppressive miRNAs targets [12,13,16,21,23,25]. Published articles revealed that miR-1 mediates cell apoptosis, targeting BCL2 in cardiac muscles [30]. In cancer, miR-1 induced apoptosis through repression of Mcl-1 in lung cancer [19]. miR-1 also targets c-Met in rhabdomyosarcoma [20]. However, to our knowledge, the target gene of miR-1 in HNSCC was unknown. TAGLN2 was significantly down-regulated The luciferase reporter assay revealed that TAGLN2 contains three sites that actually bind miR-1. This is the first report demonstrating that tumor suppressive miR-1 directly regulates TAGLN2 in HNSCC cells. TAGLN2 is a member of the calponin family of actin-binding proteins. TAGLN2 is a homolog of the protein TAGLN [31]. Though over-expression of TAGLN2 was observed in hepatocellular carcinoma, lung adenocarcinoma, and pancreatic cancer [32][33][34][35], analysis of TAGLN2 in HNSCC has not yet been reported. Here, we demonstrated significant up-regulation of TAGLN2 expression in HNSCC clinical specimens. It was demonstrated that increasing TAGLN2 expression was correlated with lymph node metastasis, distant metastasis, and the TNM classification in colorectal cancer [36]. Those results support the present loss-of-function analysis with si-TAGLN2, confirming that TAGLN2 mediates cell migration and invasion, suggesting that this gene may have an oncogenic function. However, it is still unknown how TAGLN2, an actin-binding protein, contributes to cell apoptosis, which should be clarified by further analysis.
In conclusion, our results show that restoration of miR-1 in cancer cells inhibits cell proliferation, invasion, and migration, supporting the hypothesis that miR-1 functions as a tumor suppressor in HNSCC. Our data also indicate that TAGLN2 may have an oncogenic function which is directly regulated by miR-1. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms and applications to diagnosis, therapy, and prevention of the disease.

Hnscc cell culture
Human HNSCC cell lines (HSC3, derived from a lymph node metastasis of tongue squamous cell carcinoma, and FaDu, derived from a primary lesion of hypopharyngeal squamous cell carcinoma) were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA). Both cell lines were grown in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C.

rnA isolation
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA concentrations were determined spectrophotometrically, and molecular integrity was checked by gel electrophoresis. RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

cell proliferation, migration and invasion assays
Cells were transfected with 10 nM miRNA and siRNA by reverse transfection and plated in 96 well plates at 3 x 10 3 cells per well. After 72 hr or 96 hr, cell proliferation was determined by the XTT assay, using the Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany) [13,16]. Triplicate wells were measured for cell viability in each treatment group.
Cell migration activity was evaluated using a wound-healing assay. HSC3 was plated in six well plates at 2 x 10 5 cells per well, and the cell monolayers were scraped using a micropipette tip. The initial gap length (0 hr) and the residual gap length (24 hr after wounding) were calculated from photomicrographs [12]. FaDu was not suitable for the wound healing assay because the cell monolayer tended to peel off during scraping.
A cell invasion assay was carried out using modified Boyden chambers containing transwell-precoated Matrigel membrane filter inserts with eight μm pores in 24 well tissue culture plates at 1 x 10 5 cells per well (BD Biosciences, Bedford, MA, USA) [12]. Triplicate wells were measured for cell invasion in each treatment group.

Flow cytometry
HSC3 and FaDu cells transiently transfected with miRNA-control, miR-1, siRNA-control and si-TAGLN2 were harvested 72 hr after transfection by trypsinization. After the double staining with FITC-Annexin V and Propidium iodide (PI) was done using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's recommendations, the cells were analyzed with a flow cytometry (FACScan®; BD Biosciences) equipped with a CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells, and then the relative ratio of early apoptotic cells to miRNAcontrol transfectant from each experiment were compared. Cells for cell cycle analysis were stained with PI using the CycleTEST™ PLUS DNA Reagent Kit (BD Biosciences) following the protocol and analyzed by FACScan. The percentage of the cells in G0/G1, S, and G2/M phase were counted and compared. Experiments were done in triplicate.

target gene search for mir-1
A genome-wide screen using miR-1 transfectants was performed to identify target genes of miR-1 in two HNSCC cell lines, HSC3 and FaDu. Oligo-microarray human 44K (Agilent Technologies) was used for expression profiling of the transfectants in comparison with a miRNA-negative-control transfectant [12, 13,16]. Hybridization and wash steps were performed as previously described [37]. The arrays were scanned using a Packard GSI Lumonics ScanArray 4000 (Perkin Elmer, Boston, MA, USA). The data were analyzed by means of DNASIS array software (Hitachi Software Engineering, Tokyo, Japan), which converted the signal intensity for each spot into text format. The log2 ratios of the mediansubtracted background intensities were analyzed. Data from each microarray study were normalized by a global normalization method [37].

real-time quantitative rt-Pcr
First-strand cDNA was synthesized from one μg of total RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Gene-specific PCR products were assayed continuously using a 7900-HT Real-Time PCR System according to the manufacturer's protocol. The initial PCR step consisted of a ten min hold at 95°C, followed by 40 cycles consisting of a 15 sec denaturation at 95°C and a one min annealing/extension at 63°C. TaqMan® probes and primers for TAGLN2 (P/N: Hs00761239_s1) and the GAPDH (A/N: NM_002046) internal control were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products). The expression levels of miR-1 (P/N: PM10617) were analyzed by TaqMan quantitative real-time PCR (TaqMan® MicroRNA Assay; Applied Biosystems) and normalized to RNU48 (A/N: X96648). All reactions were performed in triplicate, and included negative control reactions that lacked cDNA.