The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer

Background: On the base of the microRNA (miRNA) expression signature of bladder cancer (BC), we found that miR-1 and miR-133a were significantly downregulated in BC. In this study, we focussed on the functional significance of miR-1 and miR-133a in BC cell lines and identified a molecular network of these miRNAs. Methods and results: We investigated the miRNA expression signature of BC clinical specimens and identified several downregulated miRNAs (miR-133a, miR-204, miR-1, miR-139-5p, and miR-370). MiR-1 and miR-133a showed potential role of tumour suppressors by functional analyses of BC cells such as cell proliferation, apoptosis, migration, and invasion assays. Molecular target searches of these miRNAs showed that transgelin 2 (TAGLN2) was directly regulated by both miR-1 and miR-133a. Silencing of TAGLN2 study demonstrated significant inhibitions of cell proliferation and increase of apoptosis in BC cell lines. The immunohistochemistry showed a positive correlation between TAGLN2 expression and tumour grade in clinical BC specimens. Conclusions: The downregulation of miR-1 and miR-133a was a frequent event in BC, and these miRNAs were recognised as tumour suppressive. TAGLN2 may be a target of both miRNAs and had a potential oncogenic function. Therefore, novel molecular networks provided by miRNAs may provide new insights into the underlying molecular mechanisms of BC.

Bladder cancer (BC) is the fourth most common tumour diagnosed and the second most common cause of death in patients with genitourinary tract malignancies worldwide (Parkin et al, 2005;Jemal et al, 2010). In Japan, the age-standardised mortality rate of BC has remained relatively stable in men but has increased slightly since 1993 in women (Qiu et al, 2009). There have been significant advances in treatment, including surgical techniques and adjuvant chemotherapy; however, BC continues to be a common disease with high mortality (Shirodkar and Lokeshwar, 2009). Therefore, new treatment modalities based on novel molecular networks in BC are desired.
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules of 20 -22 nucleotides that have a critical role in a variety of biological processes including development, differentiation, apoptosis, and cell proliferation. They regulate gene expression through translational repression and mRNA degradation. Although their biological functions remain largely unknown, recent studies suggest that miRNAs contribute to the development of various types of cancer (Ryan et al, 2010). A growing body of evidence indicates that miRNAs are aberrantly expressed in many human cancers, and they may function as oncogenes and tumour suppressors. Upregulated miRNAs could function as oncogenes by negatively regulating tumour-suppressor genes, whereas downregulated miRNAs could act as tumour suppressors, inhibiting cancers by regulating oncogenes (Lu et al, 2005;Calin and Croce, 2006;Ambs et al, 2008;Childs et al, 2009). Bioinformatic predictions indicate that miRNAs regulate 430% of the protein coding genes (Filipowicz et al, 2008). It is estimated that B1000 miRNAs exist in the vertebrate genome. So far, 1048 human miRNAs are registered at miRBase release 16.0 (http://microrna. sanger.ac.uk/).
MiR-1 and miR-133a were among the top five downregulated miRNAs in our screening. MiR-1 and miR-133a are muscleenriched miRNAs that inhibit proliferation of progenitor cells and promote myogenesis by targeting histone deacetylase (HDAC4) and serum response factor (SRF), respectively (Chen et al, 2006). Recently, miR-1 and miR-133a have been reported to be downregulated in various cancers and to have tumour-suppressive functions Nasser et al, 2008;Yan et al, 2009;Uchida et al, 2010;Chiyomaru et al, 2010a). We have reported that miR-133a directly regulated oncogenic FSCN1, LASP1, and GSTP1 genes in human BC (Uchida et al, 2010;Chiyomaru et al, 2010a, b). We recognised that miR-1 and miR-133a are located on the same chromosomal loci (18q11.2 and 20q13.33) (Chiyomaru et al, 2010b). Like this, several miRNAs are located on the same chromosomal region, in a so-called 'cluster'. Recent studies demonstrated that miR-17-92 cluster and miR-221/222 cluster, which harbour oncogenic miRNAs, had important roles in several human malignancies Di Leva et al, 2010). However, the functional roles of the target genes regulated by these miRNA clusters have not been thoroughly investigated.
The aim of this study was to investigate whether miR-1 has tumour-suppressive function in BC cell lines and to find common target genes of miR-1 and miR-133a. We focussed on transgelin 2 (TAGLN2), which was one of the most downregulated genes in oligo-microarray studies using an miR-1 transfectant and is a putative target gene of miR-1 and miR-133a as suggested by webbased software. The functional role of TAGLN2 has not yet been determined. Previous studies have reported that high expressions of TAGLN2 were observed in various human malignancies Shi et al, 2005;Huang et al, 2006;Rho et al, 2009;Zhang et al, 2010). We hypothesised that miR-1 and miR-133a directly regulate TAGLN2, which may have an oncogenic function in BC. We performed a luciferase reporter assay to determine whether TAGLN2 mRNA is actually targeted by miR-1 and miR-133a and a loss-of-function study using BC cell lines to investigate the functional roles of TAGLN2 in BC.

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% CO 2 and 95% air at 37 1C.

Tissue collection and RNA extraction
Tissues were immersed in RNAlater (QIAGEN, Valencia, CA, USA) and stored at À20 1C 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 o0.05 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 DDCt 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 Â 10 5 per dish), in a six-well plate for apoptosis (10 Â 10 4 per well) and for wound healing assay (20 Â 10 4 per well), in a 24-well plate for mRNA extraction and luciferase reporter assay (5 Â 10 4 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 miR-1 and miR-133a as tumour suppressors in BC H Yoshino et al 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.

Plasmid construction and dual-luciferase reporter assay
The miRNA target sequences were inserted between the XhoI-PmeI restriction sites in the 3 0 UTR of the hRluc gene in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). Primer sequences for full-length 3 0 UTR of TAGLN2 mRNA (5 0 -ATCGCTC GAGACAGATGGGCACCAACCGCG-3 0 and 5 0 -CTCTAGGTTTAAA CATCTTCCTCAAGCCCCAGAC-3 0 ) 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 ml of Lipofectamine 2000 (Invitrogen) in 100 ml 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 þ L) made in goat (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidinehydrogen 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 . 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 þ , mild staining; 2 þ , moderate staining; and 3 þ , intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1 þ , o30% of cells stained positive; 2 þ , 30 -60% stained positive; and 3 þ , 460% stained positive. A combined staining score (intensity þ extension) of p2 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 w 2 -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 Po0.05 corresponds to a Bonferroni-adjusted level of Po0.0167.

Identification of downregulated miRNAs in BC by miRNA expression signatures
We evaluated mature miRNA expression levels of 11 BC and 5 NBE specimens by miRNA expression signatures. A total of 41 and 19 downregulated miRNAs were selected after the normalisation using RNU48 and MammU6, respectively (Supplementary Tables 3  and 4). The 17 miRNAs were commonly downregulated with RNU48 and MammU6 normalisation ( Table 1). The top five miRNAs (miR-133a, miR-204, miR-1, miR-139-5p, and miR-370) in the list were subjected to further study.  Figure 1). These miRNA expressions were significantly lower in BC cell lines (BOY and T24) in comparison with the normal human bladder RNA (Po0.0001; Figure 2A).
Effect of the downregulated miRNA transfection on cell proliferation, migration activity, and invasion in BC cell lines To investigate the functional role of the five selected miRNAs, we performed gain-of-function studies using the miRNA transfectants. The XTT assay showed significant cell proliferation inhibitions in miR-1 and miR-133a transfectants in comparison with the miR-control transfectants (percentage of cell viability for BOY: 59.9±1.4, 64.1±1.4, and 100.0±1.2, respectively, Po0.0001; and for T24: 43.3 ± 0.6, 62.4 ± 0.7, and 100.0 ± 0.7, respectively, Po0.0001), but no significant inhibition was observed in other miRNA transfectants except the miR-204-transfected BOY cell line ( Figure 2B). The wound healing assay showed significant cell migration inhibitions in miR-1 and miR-133a transfectants in comparison with the controls (percentage of wound closure for BOY; 41.4±2.5, 71.3±4.4, and 100.0±3.2, respectively, Po0.0001; and for T24: 26.0 ± 2.4, 65.2 ± 3.9, and 100.0 ± 3.2, respectively, Po0.0001), but no significant inhibition was observed in other miRNA transfectants except miR-370-transfected BOY cell line ( Figure 2C). Matrigel invasion assay demonstrated that invading cell numbers were significantly decreased in both miR-1-and miR-133a-transfected BOY cell lines and miR-1-transfected T24 cell line in comparison with the controls (percentage of cell invasion for BOY: 23.4±3.0, 57.3±8.9, and 100.0±10.0, respectively, Po0.0001; and for T24: 43.4 ± 4.8, 96.5 ± 4.4, and 100.0 ± 3.8, respectively, Po0.0001; the miR-1 transfectant vs control), but no significant inhibition was observed in other miRNA transfectants except the miR-370-transfected BOY cell line ( Figure 2D). To evaluate the simultaneous effect of miR-1 and miR-133a, we evaluated another XTT assay by using miR-1 and miR-133a co-transfected BOY and T24 cell lines. We found similar effects of cell viability inhibition by the transfectants in comparison with that of each miR-1 or miR-133a transfectants (Supplementary Figure 1). Because miR-1 and miR-133a transfection had a significantly stronger tumour-suppressive effect among the five miRNAs, they were subjected to further analyses as the leading candidates for tumour-suppressive miRNAs in BC.

MiR-1 induced apoptosis in BC cell lines
Cell apoptosis in miR-1 transfectants was detected using flow cytometry. As shown by representative images in Figure 3A, the apoptotic cell fractions (early apoptotic and apoptotic; lower right and upper right, respectively) were greater in miR-1 transfectants than in miR-control transfectants (BOY and T24). As shown in Figure 3B, miR-1 transfection induced apoptosis in BC cell lines (BOY, 3.31 ± 0.51 and 1.00 ± 0.20; T24, 1.70 ± 0.08 and 1.00 ± 0.17, respectively, Po0.05). In terms of miR-133a, we previously demonstrated that miR-133a transfection also induced apoptosis in the same BC cell lines (Uchida et al, 2010).

Gene expression profile identifying downregulated genes in miR-1 transfectant
To gain further insight into which genes are affected by miR-1 transfection, we performed gene expression analysis with miR-1 transfectants and the controls (BOY and T24 cells). A total of 18 genes were downregulated less than -4.0-fold in miR-1 transfectants compared with the controls ( Table 2). The TargetScan program showed that 17 genes had putative target sites of miR-1 in their 3 0 UTR (Table 2). Previously, we had performed gene expression analysis with miR-133a transfectants (Uchida et al, 2010), and the TAGLN2 gene was commonly listed in the top 10 downregulated genes in current (miR-1) and former (miR-133a) signatures. Therefore, we focussed on the TAGLN2 gene as a promising candidate targeted by both miR-1 and miR-133a. Entries from the former and the current microarray data were approved by the Gene Expression Omnibus (GEO) and were assigned GEO accession numbers GSE19717 and GSE24782.

TAGLN2 expression in BC cell lines and TAGLN2 silencing by miR-1 and miR-133a transfection
The quantitative real-time RT -PCR analysis showed that the mRNA expression of TAGLN2 in the BOY and T24 cell lines was more than two-fold higher than that in the normal human bladder RNA ( Figure 4A). To examine the functional role of TAGLN2, we performed gain-of-function studies using miR-1 and miR-133a transfectant (BOY and T24) cell lines, and the mRNA and protein expression levels of TAGLN2 were markedly   Figure 4B).

TAGLN2 as a target of post-transcriptional repression by miR-1 and miR-133a
We performed a luciferase reporter assay to determine whether TAGLN2 mRNA has a target site for miR-1 and miR-133a. We used a vector encoding full-length 3 0 UTR of TAGLN2 mRNA and found that the luminescence intensity was significantly reduced in the miR-1 and miR-133a transfectant ( Figure 5A). Furthermore, the luminescence intensity significantly decreased at the three sites targeted by miR-1 (position 71 -77, 185 -191, and 348 -354) and two sites targeted by miR-133a (position 214 -220 and 242 -248) ( Figure 5B).

DISCUSSION
In this study, we identified 17 downregulated miRNAs that survived after three different normalisation methods. Among them, we tested miR-133a, miR-204, miR-1, miR-139-5p, and miR-370, which were the top five downregulated miRNAs. Investigators had demonstrated that miR-204 was a tumoursuppressive miRNA in head and neck tumour (Lee et al, 2010); miR-139-5p was downregulated in endometrial serous adenocarcinoma (Hiroki et al, 2010); and miR-370 expression was silenced by promoter hypermethylation in malignant human cholangiocytes (Meng et al, 2008). The expression levels of the five miRNAs were indeed downregulated in BC specimens as well as in BC cell lines. However, we found typical tumour-suppressive effects in miR-1-and miR-133a-transfected BC cell lines, and these miRNAs were plausible to be critical for BC development.
The miR-1 and miR-133 are cardiac and skeletal muscle-specific, bicistronic miRNAs transcriptionally controlled by some major regulators of muscle differentiation (Chen et al, 2006;Williams et al, 2009). These miRNAs have been reported to be  Nasser et al, 2008;Yan et al, 2009;Uchida et al, 2010;Chiyomaru et al, 2010a). Ectopic expression of miR-1 in HCC, lung cancer, prostate cancer, and rhabdomyosarcoma inhibited tumour cell growth (Ambs et al, 2008;Datta et al, 2008;Nasser et al, 2008;Yan et al, 2009). However, the function of miR-1 in BC remains to be elucidated. MiR-1 had an important role in the regulation of apoptosis, which is involved in post-transcriptional repression of BCL2 in cardiomyocyte (Tang et al, 2009). In cancer research fields, ectopic miR-1 induced apoptosis through enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of antiapoptotic Mcl-1 in lung cancer cells . Consistent with previous studies, re-expression of miR-1 in BC cell lines resulted in induction of apoptosis and reduced cell viability in this study. Regarding miR-133a, previous studies demonstrated that its expression was downregulated in pancreatic ductal adenocarcinoma, oesophageal squamous cell carcinoma, rhabdomyosarcoma, colorectal cancer, and squamous cell carcinoma of the tongue (Bandrés et al, 2006;Szafranska et al, 2007;Wong et al, 2008a, b;Arndt et al, 2009;Yan et al, 2009;Kano et al, 2010). MiR-133a inhibited proliferation and induced apoptosis and directly bound to pyruvate kinase type M2 expression, which are potent oncogenes (Wong et al, 2008b). However, there has been no study concerning genes targeted by both miR-1 and miR-133a, which are clustered on the same chromosomal loci in human malignancies. In BC, we have reported that miR-133a had a critical role in regulating oncogenic FSCN1, LASP1, and GSTP1 (Uchida et al, 2010;Chiyomaru et al, 2010a, b) and have demonstrated for the first time that LASP1 was the target of miR-1/miR-133a cluster (Chiyomaru et al, 2010b). In this study, the TAGLN2 gene was found to be another target of the miR-1/miR-133a cluster. It is plausible that the miR-1/miR-133a cluster may have important roles as tumour suppressors through downregulating these oncogenic genes. However, we found no simultaneous effect of cell viability inhibition by miR-1 and miR-133a co-transfection, suggesting that each miRNA may strongly repress same target  miR-1 and miR-133a as tumour suppressors in BC H Yoshino et al genes and no additional effect might be caused by the other miRNA. Further investigations are necessary to elucidate the simultaneous effect of the miR-1/miR-133a cluster. Our data suggest that retrieved expression of miR-1/miR-133a clusters could be a new therapeutic strategy for BC. TAGLN2 contains a conserved actin-binding domain also known as the calponin (a calcium-binding protein) homologue domain. TAGLN and TAGLN3 are homologues of TAGLN2, and TAGLN3 is a novel neuron-specific protein and has not been reported in cancer (Ito et al, 2005). The protein encoded by the TAGLN gene is an actin-binding protein like TAGLN2, found in fibroblasts and smooth muscle. Overexpression of the TAGLN protein has been observed in carcinomas of the stomach, liver, and oesophagus (Rho et al, 2009). Although the function of TAGLN2 is unknown, there have been a number of reports concerning the relationship between TAGLN2 expression and tumourigenesis Shi et al, 2005;Huang et al, 2006;Rho et al, 2009;Zhang et al, 2010). Overexpression of TAGLN2 was observed in HCC and pancreatic cancer Shi et al, 2005;Huang et al, 2006). Zhang et al (2010) demonstrated that increased TAGLN2 expression was correlated with lymph node metastasis, distant metastasis, and the TNM classification in colorectal cancer. We also found a significant correlation of TAGLN2 expression with metastasis region and tumour grade despite of no correlation with tumour stage. Our tissue microarray included no Ta tumour and only four metastasis-positive patients. Studies for larger number of samples with balanced pathological background are needed to elucidate the precise correlation between TAGLN2 expression and clinicopathological parameters. These studies have implied that TAGLN2 may represent a potential tumour biomarker. We found that cell viability was markedly decreased in TAGLN2 knockdown cells by inducing apoptosis, which suggests that this molecule may have oncogenic function. However, it is still unknown how TAGLN2, which is an actin-binding protein, interacts with apoptosis. Further examinations are necessary to elucidate this.
In summary, miR-1/miR-133a clusters may function as tumour suppressors through repression of oncogenic TAGLN2 in BC. MiR-1/miR-133a transfection and TAGLN2 knockdown resulted in decreased BC cell viability and induction of apoptosis. Novel molecular networks provided by miRNAs may provide new insights into the underlying molecular mechanisms of BC.