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Clinical specimens and cell culture

Tissue specimens for miRNA screening using a low-density array (LDA) were from 11 BC patients who had undergone cystectomy or transurethral resection of bladder tumours (TUR-BT) at Kagoshima University Hospital between 2007 and 2008. The tissue specimens for quantitative RT–PCR were from 23 BC patients who had received cystectomy or TUR-BT at Kagoshima University Hospital between 2006 and 2009. The patients’ backgrounds and clinicopathological characteristics are summarised in Supplementary Table 1. Normal bladder epitheliums (NBEs) were derived from patients with noncancerous disease. These specimens were staged according to the American Joint Committee on Cancer/Union Internationale Contre le Cancer tumour-node-metastasis classification and histologically graded (Sobin and Wittekind, 2002). Our study was approved by the Bioethics Committee of Kagoshima University; written previous informed consent and approval were given by these patients.

We used two human BC cell lines: BOY, which was established in our laboratory from an Asian male patient aged 66 years who was diagnosed with stage III BC with lung metastasis; and T24, which was invasive and obtained from the American Type Culture Collection. These cell lines were maintained in a minimum essential medium (MEM) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37 °C.

Tissue collection and RNA extraction

Tissues were immersed in RNAlater (QIAGEN, Valencia, CA, USA) and stored at −20 °C until the RNA extraction. Total RNA including miRNA was extracted using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA) following the manufacturer's protocol. The integrity of the RNA was checked with RNA 6000 Nano Assay Kit and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

MiRNA expression signatures and data normalisation

MicroRNA expression patterns were evaluated using the TaqMan LDA Human microRNA Panel v2.0 (Applied Biosystems, Foster City, CA, USA). The assay was composed of two steps: generation of cDNA by reverse transcription and a TaqMan real-time PCR assay. The description of real-time PCR and the list of human miRNAs can be found on the company’s website (http://www.appliedbiosystems.com). An analysis of relative miRNA expression data was performed using GeneSpring GX version 7.3.1 software (Agilent Technologies) according to the manufacturer’s instructions. A cutoff P-value of <0.05 was used to narrow down the candidates after global normalisation of the raw data. After global normalisation, additional normalisation was done by RNU48 and MammU6.

Quantitative real-time RT–PCR

TaqMan probes and primers for TAGLN2 (P/N: Hs00761239_m1; Applied Biosystems) were assay-on-demand gene expression products. All reactions were performed in duplicate and a negative control lacking cDNA was included. We followed the manufacturer's protocol for PCR conditions. Stem-loop RT–PCR (TaqMan MicroRNA Assays; P/N: PM10617 for miR-1, and PM10413 for miR-133a; Applied Biosystems) was used to quantitate miRNAs according to the earlier published conditions (Ichimi et al, 2009). To normalise the data for quantification of TAGLN2 mRNA and the miRNAs, we used human GUSB (P/N: Hs99999908_m1; Applied Biosystems) and RNU48 (P/N: 001006; Applied Biosystems), respectively, and the ΔΔCt method was employed to calculate the fold change. As a control RNA, we used Premium Total RNA from normal human bladder (AM7990; Applied Biosystems).

Mature miRNA and siRNA transfection

As described elsewhere (Ichimi et al, 2009), the BC cell lines were transfected with Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) and Opti-MEM (Invitrogen) with 10 nM of mature miRNA molecules. Pre-miR and negative-control miRNA (Applied Biosystems) were used in the gain-of-function experiments, whereas TAGLN2 siRNA (Cat no. HSS144745 and HSS144746; Invitrogen) and negative-control siRNA (D-001810-10; Thermo Fisher Scientific, Waltham, MA, USA) were used in the loss-of-function experiments. Cells were seeded in a 10-cm dish for protein extraction (8 × 105 per dish), in a six-well plate for apoptosis (10 × 104 per well) and for wound healing assay (20 × 104 per well), in a 24-well plate for mRNA extraction and luciferase reporter assay (5 × 104 per well), and in a 96-well plate for XTT assay (3000 per well).

Cell proliferation, migration, and invasion assays

Cell proliferation was determined using an XTT assay (Roche Applied Sciences, Tokyo, Japan) performed according to the manufacturer’s instructions. Cell migration activity was evaluated by wound healing assay. Cells were plated in six-well dishes, and 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. A cell invasion assay was carried out using modified Boyden Chambers consisting of transwell-precoated matrigel membrane filter inserts with 8-mm pores in 24-well tissue culture plates (BD Biosciences, Bedfold, MA, USA). Minimum essential medium containing 10% fetal bovine serum in the lower chamber served as the chemoattractant as described previously (Chiyomaru et al, 2010a). All experiments were performed in triplicate.

Apoptosis analysis

The BC cell lines transiently transfected with transfection reagent only (mock), si-control, si-TAGLN2, miR-control, miR-1, or miR-133a in six-well tissue culture plates as described earlier were harvested 72 h after transfection by trypsinisation and washed in cold PBS. Double staining with FITC-Annexin V and propidium iodide (PI) was carried out using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's recommendations and immediately analysed within an hour by flow cytometry (FACScan; BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells by the CellQuest software (BD Biosciences), and then the percentages of early apoptotic and apoptotic cells from each experiment were compared. Experiments were done in triplicate.

Target gene search for miR-1

Oligo-microarray Human 44K (Agilent) was used for expression profiling in miR-1-transfected BC cell lines (BOY and T24) in comparison with miR-negative control transfectant, as previously described (Chiyomaru et al, 2010a). Briefly, hybridisation and washing steps were performed in accordance with the manufacturer’s instructions. The arrays were scanned using a Packard GSI Lumonics ScanArray 4000 (PerkinElmer, Boston, MA, USA). The data obtained were analysed with DNASIS array software (Hitachi Software Engineering, Tokyo, Japan), which converted the signal intensity for each spot into text format. The Log2 ratios of the median subtracted background intensity were analysed. Data from each microarray study were normalised by global normalisation.

The predicted target genes and their miRNA binding site seed regions were investigated using TargetScan (release 5.1, http://www.targetscan.org/). The sequences of the predicted mature miRNAs were confirmed using miRBase (release 16.0, September 2010; http://microrna.sanger.ac.uk/).

Western blots

After 3 days of transfection, protein lysate (20 μg) was separated by NuPAGE on 4–12% bis-tris gel (Invitrogen) and transferred into a polyvinylidene fluoride membrane. Immunoblotting was done with diluted (1 : 150) polyclonal TAGLN2 antibody (HPA001925; Sigma-Aldrich, St Louis, MO, USA) and GAPDH antibody (MAB374; Chemicon, Temecula, CA, USA). The membrane was washed and then incubated with goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad, Hercules, CA, USA). Specific complexes were visualised with an echochemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK), and the expression level of these genes was evaluated using ImageJ software (ver. 1.43; http://rsbweb.nih.gov/ij/index.html).

Plasmid construction and dual-luciferase reporter assay

The miRNA target sequences were inserted between the XhoI–PmeI restriction sites in the 3′UTR of the hRluc gene in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). Primer sequences for full-length 3′UTR of TAGLN2 mRNA (5′-IndexTermATCGCTCGAGACAGATGGGCACCAACCGCG-3′ and 5′-IndexTermCTCTAGGTTTAAACATCTTCCTCAAGCCCCAGAC-3′) were designed. Specific miRNA target sequences (40 bp length, Supplementary Table 2) for miR-1 and miR-133a were artificially synthesised and inserted in the vector. Following that, T24 cells were transfected with 15 ng of vector, 10 nM of miRNA, and 1 μl of Lipofectamine 2000 (Invitrogen) in 100 μl of Opti-MEM (Invitrogen). The activities of firefly and Renilla luciferases in cell lysates were determined with a dual-luciferase assay system (E1910; Promega). Normalised data were calculated as the quotient of Renilla/firefly luciferase activities.

Immunohistochemistry

A tissue microarray of 47 urothelial carcinomas and 8 normal bladders was obtained from US Biomax, Inc. (BL208; Rockville, MD, USA). Detailed information on all tumour specimens can be found at http://www.biomax.us/index.php. Immunostaining was done on the tissue microarray following the manufacturer's protocol. The primary rabbit polyclonal antibodies against TAGLN2 (Sigma-Aldrich) were diluted by 1 : 25. The slides were treated with biotinylated anti-rabbit IgG (H+L) made in goat (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine-hydrogen peroxide (Sigma-Aldrich) was the chromogen, and the counterstaining was done with 0.5% haematoxylin. The positivity of endothelia served as an inner positive control. Immunostaining was evaluated according to a scoring method as described previously (Zhang et al, 2010). Each case was scored on the basis of the intensity and area of staining. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; and 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, <30% of cells stained positive; 2+, 30–60% stained positive; and 3+, >60% stained positive. A combined staining score (intensity+extension) of ⩽2 was low expression, a score between 3 and 4 was moderate expression, and a score between 5 and 6 was high expression.

Statistical analysis

The relationship between two variables and the numerical values obtained by real-time RT–PCR was analysed using the Mann–Whitney U-test. The relationship among three variables and the numerical values was analysed using the Bonferroni-adjusted Mann–Whitney U-test. The χ2-test was used to evaluate the relationships between the immunohistochemical score of TAGLN2 expression and clinicopathological factors. Expert StatView analysis software (version 4; SAS Institute Inc., Cary, NC, USA) was used in both cases. In the comparison among three variables, a nonadjusted statistical level of significance of P<0.05 corresponds to a Bonferroni-adjusted level of P<0.0167.