Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2013 Apr 9;139(7):1189–1198. doi: 10.1007/s00432-013-1392-6

miR-708 promotes the development of bladder carcinoma via direct repression of Caspase-2

Tao Song 1,✉, Xu Zhang 1, Lei Zhang 1, Jun Dong 1, Wei Cai 1, Jiangping Gao 1, Baofa Hong 1
PMCID: PMC11824749  PMID: 23568547

Abstract

Purpose

Bladder cancer is one of the world’s top ten malignant tumors. The crucial role of microRNA in carcinogenesis has been well emphasized. Considering miRNA expression was tumor stage-, tissue-, or even development-specific, more experimental evidences about the functions of miRNAs in bladder cancer should be discovered to advance applying of miRNA in the diagnosis or therapy of cancer.

Methods

MiR-708 level in bladder carcinoma and adjacent noncancerous tissues was tested by real-time qPCR. Cell apoptosis was analyzed by using flow cytometry. The tumorigenicity of bladder carcinoma cells was evaluated in nude mice model. Luciferase reporter gene assays were performed to identify the interaction between miR-708 and 3′UTR of Caspase-2 mRNA. The protein level of Caspase-2 was determined by western blotting.

Results

In this study, we reported that miR-708 was frequently dysregulated in human bladder carcinoma tissues compared to normal tissues. In addition, we found that silencing of miR-708 could promote the T24 and 5637 cells to apoptosis and inhibit the bladder tumor growth in vivo. Also, Caspase-2 was proved to be one of direct targets of miR-708 in T24 and 5637 cells. Further results showed that Caspase-2 was involved in the miR-708 regulated cell apoptosis.

Conclusions

All together, these results suggest miR-708 may act as an oncogene and induce the carcinogenicity of bladder cancer by down-regulating Caspase-2 level.

Electronic supplementary material

The online version of this article (doi:10.1007/s00432-013-1392-6) contains supplementary material, which is available to authorized users.

Keywords: miR-708, Oncogene, Bladder carcinoma, Caspase-2

Introduction

Bladder carcinoma represents the seventh most common cancer and the ninth most common cause of cancer death for men (Ismaili et al. 2011). And in China, bladder cancer is the fifth most common cancer (Huang et al. 2010). Novel therapies have been shown to be promising (Ismaili et al. 2011). Unfortunately, patients with advanced bladder cancer face a 5-year survival rate of approximately 20–40 % (Noguchi et al. 2011; Wang et al. 2008; Sudarshan et al. 2005; Shelley et al. 2004; Herr 1997; Cookson et al. 1997). The development of bladder cancer biomarker has advanced significantly over the last decade, but has not yet been able to make a significant impact in the diagnosis and management of the disease (Alvarez and Lokeshwar 2007). The advance in understanding of the molecular events underlying urothelial neoplastic progression has spurred many investigations to look at comparative expression of microRNAs (miRNAs) in bladder carcinoma (Chen et al. 2011).

MiRNAs are post-transcriptional regulators that bind to the 3‘untranslated regions (UTRs) of target mRNAs, usually resulting in translational repression and gene silencing (Bartel 2009). It is widely accepted that miRNA expression is broadly altered in most forms of cancer (Wiklund et al. 2011; Esquela-Kerscher and Slack 2006) and miRNAs can act as oncogenes and/or tumor suppressors, playing an important role in oncogenesis and oncotherapy (Chen et al. 2011; Zhang et al. 2007; Lynam-Lennon et al. 2009; Papagiannakopoulos and Kosik 2008; Wang and Wu 2009). Meanwhile, miRNAs expression was tumor stage-, tissue-, or development-specific (Chen et al. 2011). The importance of miRNAs in the diagnosis and therapy of cancer has recently been emphasized (Trang et al. 2008; Li et al. 2009a). Our previous study proved that several miRNAs were dysregulated in the bladder carcinoma, including miR-708 (Song et al. 2010). In this study, we focused on the miR-708 for the reason that we found Caspase-2, which was involved in the carcinogenicity, was its predicted target.

Caspase 2, also known as CASP2, is an enzyme that plays a central role in the execution-phase of cell apoptosis (Imre et al. 2012; Tiwari et al. 2011; Kim et al. 2012). It belongs to a family of cysteine proteases called caspases that mediate cellular apoptosis through the proteolytic cleavage of specific protein substrates (Thornberry and Lazebnik 1998; Lamkanfi et al. 2007). More recently, Caspase-2 was found to suppress NF-kB activation, promote apoptosis, sustain G2/M checkpoint and impact tumor suppression in humans (Tang et al. 2011; Kitevska et al. 2009). In addition, Ren et al. (2012) found a significant down-regulation of Caspase-2 mRNA in many types of cancers. However, the mechanism about the expression regulation of Caspase-2 itself was barely reported.

Here, we firstly found that that up-regulation of miR-708 occurred very frequently in bladder carcinoma tissues. Furthermore, both gain- and loss-of-function studies revealed that miR-708 could block the apoptosis of cells. Moreover, we found that Caspase-2 was a direct target of miR-708. Meanwhile, the level of miR-708 is observed to be negatively associated with the mRNA level of Caspase-2 in bladder carcinoma tissue. Further investigation revealed that miR-708 blocked apoptosis dependent on directly down-regulating the level of Caspase-2. Our finding will help to elucidate the functions of miRNAs, which might provide insight into developing novel bladder tumor markers or new therapeutic strategies.

Methods

Tumor characteristics and cell lines

Bladder carcinoma and control tissue specimens were obtained from patients at General Hospital of the People’s Liberation Army (Beijing, China) after surgical resection with informed consent. The tumor tissues and adjacent normal tissues were frozen in liquid nitrogen after resection. No patient in the current study received chemotherapy or radiation therapy before the surgery. The institutional ethics committee of People’s Armed Police Corps General Hospital approved the study, and all patients gave written informed consent. Detailed information of bladder carcinoma patients is summarized in Table S1. The tumor-node-metastasis (TNM) classification system was used for staging the bladder carcinoma. The human bladder cancer cell lines T24 and 5637 were maintained in RPMI 1640 (Gibco). The medium was supplemented with 10 % fetal bovine serum (FBS) with 100 U/ml penicillin and 100 U/ml streptomycin. Cells were cultured at 37 °C in 5 % CO2.

Transfection

The miRNA mimic, miRNA inhibitor, interfering RNA complex (si-CASP2), and negative control RNA duplexes (denoted NC) were synthesized by Genepharma (Shanghai, China). Cells were transfected using Lipofetamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, mixture containing the miRNA, siRNA, NC, miRNA inhibitor or medium (mock group) and lipofectamine 2000 was prepared and added directly to cells at a final oligonucleotide concentration of 50 nM. Total RNA or protein was extracted for qRT-PCR or western blot analysis.

Quantitative reverse transcription PCR

Total RNA was extracted from cells or tissues using Trizol (Invitrogen) according to the manufacturer’s protocol. For cDNA synthesis, 1 μg of RNA was mixed with 500 ng of olig (dT) (Promega) or miRNA specific primers (invitrogen). Samples were reverse transcripted using M-MLV reverse transcriptase (Promega). The qPCR mixture contained 12.5 μl of 2 × SYBR green PCR mix (Fermetas), 0.3 μM of gene-specific forward and reverse primers, and 1 μl of cDNA template, made up to a final volume of 25 μl with distilled water. Cycling parameters were set as follows: initial activation step at 95 °C for 10 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 15 s. Melting curve analysis was performed at from 58 °C to 95 °C with stepwise fluorescence acquisition at every 1 °C s−1. The levels of gene expression were calculated by relative quantification using GAPDH or U6 snRNA as the endogenous reference genes. All samples were amplified in triplicate, and the data analysis was carried out using the MxPro qPCR system software (Stratagene).

Western blotting analysis

The cell pellets were lysed in RIPA Lysis Buffer (50 mM Tris-base, 1 mM EDTA, 150 mM NaCl, 0.1 % SDS, 1 % TritonX-100, 1 % Sodium deoxycholate) for 30 min on ice. Lysates were centrifuged (12,000g, 40 min, 4 °C). Proteins at the same amount were separated by 15 % SDS polyacrylamide gel electrophoresis and transferred electrophoretically to Hybond-ECL nitrocellulose membrane (Amersham Biosciences). Membranes were probed with mouse anti-Caspase-2 (Abcam) or Actin (Santa Cruz) in 5 % nonfat dry milk for 1 h at 37 °C. After washing in PBS with 0.5 % Tween 20 (PBST), the membrane was incubated in a 1:5,000 solution of HRP-conjugated goat anti-mouse secondary antibody at room temperature for 1 h. After further washing with PBST, the membrane was assayed by the enhanced chemiluminescence (ECL) western blotting detection system.

Vector construction and luciferase reporter assay

To create a luciferase reporter construct, 3′UTR fragment of Caspase-2 containing putative binding sites for miR-708 was inserted downstream of firefly luciferase in pGL3. Mutant 3′UTR, which carried the mutated sequence in the complementary site for miR-708, was generated using the fusion PCR method inserted downstream of firefly luciferase in pGL3. Cells were cotransfected with miRNA and 3′UTR or mutant 3′UTR luciferase reporter, using pRL-TK as control vector. At 48 h after transfection, Luciferase activity was measured using the Dual-Luciferase Assay kit (Promega) with a beta-counter luminometer. Relative luciferase activity was calculated as ratio of the raw firefly luciferase activity and the renilla luciferase activity.

Assessment of apoptosis

The transfected cells were collected by trypsinization and washed in PBS. Then cells were resuspended at a concentration of 1 × 106 cells/ml in Binding Buffer (0.01 M HEPES/NaOH, pH7.4, 14 mM NaCL, 0.25 mM CaCl2); 500 μl aliquots of cells were added into FACS tubes and mixed with 25 ng/ml fluorescein isothiocyanate–labeled annexin V and 10 mg/ml propidium iodide (PI) to incubation for 15 min at room temperature in the dark. Then, the cells were analyzed immediately by flow cytometry.

Tumorigenicity assay

The experimental procedure involving animals was performed in accordance with the Guide for the Care and Use of Laboratory Animals National Institutes of Health and according to the institutional ethical guidelines for animal experiments. MiR-708 inhibitor- or NC-transfected T24 cells (2 × 106) were suspended in 200 μl PBS and then injected subcutaneously into either side of the posterior flank of the male BALB/c athymic nude mouse at 6–8 weeks of age. Ten nude mice were included in each group, and tumor growth was examined every 10 days. Tumor volume (V) was monitored by measuring the length (L) and width (W) of the tumor with calipers and was calculated with the formula V = (L × W 2) × 0.5.

Statistical analysis

All quantitative data were analyzed using Student’s t tests. All tests performed were two-sided. P < 0.05 was considered to be statistically significant.

Results

MiR-708 is frequently up-regulated in bladder carcinoma

Our microarray result has found the level of miR-708 was up-regulated in bladder carcinoma tissue (Song et al. 2010). In this study, the tissue samples of 20 paired bladder carcinoma and adjacent noncancerous tissues were applied to identify the level of miR-708 by qRT-PCR. The result showed that among the 20 paired samples, miR-708 was significantly elevated (>twofold, P < 0.05) in 18 samples compared to their corresponding normal tissues (Fig. 1). These results suggested that up-regulation of miR-708 is a frequent event in human bladder carcinoma tissues and it might be involved in the tumourigenesis of bladder carcinoma.

Fig. 1.

Fig. 1

Open in a new tab

Expression of miR-708 is frequently up-regulated in human bladder carcinoma tissues. MiR-708 expression was analyzed in 20 paired bladder carcinoma tissues and adjacent nontumor tissues by qRT-PCR. The levels of miR-708 in bladder carcinoma tissues were significantly up-regulated compared with their corresponding normal tissues. *P < 0.05

Silencing of miR-708 promotes apoptosis and suppresses tumorigenicity

To further identify the potential role of miR-708 in the tumourigenesis of bladder carcinoma, the effect of miR-708 on the cell apoptosis was further detected. The level of miR-708 in bladder cancer cell lines was detected by northern blot. The result showed miR-708 in bladder cancer cell lines was at an obvious higher level compared with that in human normal bladder tissue and other cancer cell lines (Fig. S1). The miR-708 mimic and negative control (NC) were transfected to the bladder carcinoma cell lines (T24 and 5637), respectively, which have been upon the treatment of serum deprivation. The level of miR-708 was detected by qRT-PCR to confirm the efficiency of transfection (Fig. 2a). And then the apoptotic rate of cells was detected by using flow cytometry. The result demonstrated that transfection with miR-708 obviously decreased the apoptotic rates of T24 and 5637 cells (Fig. 2b). To further verify the finding of miR-708 blocking cell apoptosis, loss-of-function analysis was performed using inhibitor of miR-708. The level of miR-708 was detected by qRT-PCR to confirm the efficiency of transfection (Fig. 2a). Consistently, compared with NC, the transfection of miR-708 inhibitor induced obviously higher apoptotic rate (Fig. 2b). Those data suggested that miR-708 acted as an antiapoptotic factor in bladder carcinoma cells.

Fig. 2.

Fig. 2

Open in a new tab

miR-708 acts as an antiapoptotic factor in bladder carcinoma cells. NC (negative control), miR-708 mimic, and miR-708 inhibitor were transfected to T24 or 5637 cells, respectively. The level of miR-708 was detected by qRT-PCR to confirm the efficiency of transfection (a). After transfection for 48 h, the apoptotic rate of cells was analyzed by using flow cytometry (b). The result represents three independent experiments. **P < 0.01, *P < 0.05

To further evaluate the effect of miR-708 on tumorigenicity of bladder carcinoma cells, miR-708 inhibitor- and NC-transfected T24 cells in which the efficiency of transfection was verified by qRT-PCR (Fig. 3a) were injected subcutaneously into either posterior flank of the nude mice. A delayed tumor formation time (9/10 of NC versus 0/10 of miR-708 inhibitor in day 10) and the significant reduction of tumor volume (Fig. 3b, c) were found in miR-708 inhibitor group. The result suggested that miR-708 might have a tumor promoting effect in vivo.

Fig. 3.

Fig. 3

Open in a new tab

miR-708 inhibitor suppresses tumor growth in nude mice model. NC (negative control) and miR-708 inhibitor were transfected to T24. The level of miR-708 was detected by qRT-PCR to confirm the efficiency of transfection (a). MiR-708 inhibitor or NC-transfected T24 cells were suspended in 200 μl PBS and then injected subcutaneously into either side of the posterior flank of the male BALB/c athymic nude mouse at 6–8 weeks of age. Tumor growth was examined every 10 days. Tumor volume (V) was monitored by measuring the length (L) and width (W) of the tumor with calipers and was calculated with the formula V = (L × W2) × 0.5. Photographs of dissected tumors from nude mice (b) and the curve of tumor growth (c) are shown. *P < 0.05. Results are representative of three animals in every time point per group

Caspase-2 is a direct target of miR-708

It is generally accepted that miRNAs are post-transcriptional regulators that bind to the 3′untranslated regions (UTRs) of target mRNAs, usually resulting in translational repression and gene silencing (Bartel 2009). To further explore the mechanism of miR-708 blocking apoptosis and inducing tumorigenicity of bladder carcinoma cells, we analyzed the target candidate(s) of miR-708 by using the Targetscan bioinformatics tool. The result revealed the 3′UTR of Caspase-2 mRNA, which has been identified as an important and comprehensive apoptosis inducer and has displayed frequent down-regulation in cancers, harbored putative binding site for miR-708 (Fig. 4a). To verify whether Caspase-2 is a direct target of miR-708, a dual-luciferase reporter system was first employed. The 3′UTR of Caspase-2 mRNA was inserted downstream of the luciferase gene (pGL3-CASP2-3′UTR) and transfected into T24 and 5637 cells with miR-708 mimic or negative control (NC) and pRL-TK to normalize transfection. The result showed that miR-708 mimic could significantly down-regulate the luciferase activity of the pGL3-CASP2-3′UTR reporter in both T24 and 5637 cells (Fig. 4b), while NC did not influence the luciferase activity. In order to further prove its reliability, mutant of Caspase-2 mRNA 3′UTR was constructed by disrupting the miR-708 target sites through point substitutions (pGL3-CASP2-3′UTRm) (Fig. 4a) and cotransfected into T24 and 5637 cells together with miR-708 mimic or negative control (NC). It was shown that the luciferase expression of mutant 3′UTR of Caspase-2 mRNA (pGL3-CASP2-3′UTRm) was no longer controlled by miR-708 (Fig. 4b). In addition, inhibition of endogenous miR-708 by using the inhibitor of miR-708 led to increased luciferase activity of the Caspase-2 mRNA 3′UTR reporter but not that of the mutant in both T24 and 5637 cells (Fig. 4c). These results suggested that this site in the 3′UTR of Caspase-2 mRNA was the direct interaction site with miR-708.

Fig. 4.

Fig. 4

Open in a new tab

The 3′UTR of Caspase-2 mRNA is a direct target of miR-708. a Bioinformatic analysis of miR-708 predicted binding site in the Caspase-2 mRNA 3′UTR. There was a putative miR-708 target site located in the Caspase-2 mRNA 3′UTR (563-569). The mutant reporter vector was constructed with the point substitutions of the target site. b pGL3-CASP2-3′UTR reporter plasmid in which the luciferase coding sequence had been fused to the 3′UTR of Caspase-2 was cotransfected into T24 and 5637 cells with miR-708 or NC. It was found that the luciferase activities of pGL3-CASP2-3′UTR reporter in miR-708 transfected cells were significantly decreased compared with NC group. In contrast, the luciferase activities of pGL3-CASP2-3′UTR mutant reporter (pGL3-CASP2-3′UTRm) did not have difference after the cells transfected with miR-708 or NC. c pGL3-CASP2-3′UTR reporter was cotransfected into T24 and 5637 cells with miR-708 inhibitor or NC. It was found that the luciferase activities of pGL3-CASP2-3′UTR reporter in miR-708 inhibitor transfected cells were significantly increased compared with NC group. In contrast, the luciferase activities of pGL3-CASP2-3′UTRm did not have difference after the cells transfected with miR-708 inhibitor or NC. d miR-708 could decrease the level of Caspase-2 in T24 and 5637 cells. Cells were transfected with miR-708, si-CASP2 (siRNA targeting to Caspase-2), or NC. After 72 h, cells were harvested and loaded into SDS-PAGE. Actin was used as a loading control. The level of Caspase-2 protein was significant decreased in the cells transfected with miR-708, compared with NC. The value under each lane indicated the relative expression level of protein, which was represented by the intensity ratio between Caspase-2 and Actin fragments in each lane

To confirm the conclusion that Caspase-2 was a target of miR-708, the effect of miR-708 on the endogenous expression of Caspase-2 was further detected. MiR-708 mimic, si-CASP2 (siRNA targeting to Caspase-2), or NC were transfected into T24 or 5637 cells, respectively. The total protein was extracted from the cells at 72 h after the transfection. The western blot result showed the expression of Caspase-2 protein was significantly reduced after transfected with miR-708 mimic or si-CASP2, compared with NC (Fig. 4d). These results further confirmed that miR-708 could down-regulate the expression of Caspase-2 in bladder carcinoma cells.

Given the decreased level of Caspase-2 in many kinds of cancer, we here, respectively, analyzed the level of Caspase-2 mRNA in bladder carcinoma and adjacent noncancerous tissues by qRT-PCR. In comparison with noncancerous tissues, the level of Caspase-2 mRNA was decreased in adjacent bladder carcinoma tissues (>50 %, P < 0.05) in 16 samples (Fig. 5a). Moreover, it was found that the level of miR-708 is significantly negatively associated with the level of Caspase-2 mRNA (Fig. 5b, r = −0.4824, P < 0.05). These results further suggested that the elevated miR-708 might be responsible for the reduction of Caspase-2 mRNA in bladder carcinoma.

Fig. 5.

Fig. 5

Open in a new tab

Caspase-2 mRNA is frequently decreased and negatively associated with the level of miR-708 in bladder carcinoma tissues. a Caspase-2 expression was analyzed in 20 paired bladder carcinoma tissues and adjacent nontumor tissues by qRT-PCR. The levels of Caspase-2 mRNA in bladder carcinoma tissues were frequently down-regulated compared with their corresponding normal tissues. *P < 0.05. b The correlation between the level of miR-708 and Caspase-2 in bladder carcinoma tissues. Data were determined using GraphPad Prism 4

Caspase-2 is involved in miR-708 inhibited bladder carcinoma cells apoptosis

Considering Caspase-2 as an inducer of cell apoptosis, we further examine whether miR-708 inhibited apoptosis through regulating Caspase-2. The Caspase-2 expression level was over-expressed through cloning the entire Caspase-2 coding sequence to pcDNA3.1 vector. Then, we investigated whether increased Caspase-2 expression may change the apoptotic rate which has been attenuated by miR-708 mimic. The results revealed that over-expression of Caspase-2 could counteract the antiapoptotic function of miR-708 (Fig. 6a, b). This result suggested that Caspase-2 is involved in miR-708 regulated apoptosis in T24 and 5637 cells.

Fig. 6.

Fig. 6

Open in a new tab

miR-708 reduces cell apoptosis by targeting Caspase-2. T24 (a) and 5637 (b) cells were transfected and the apoptosis of cells was detected by using flow cytometry in the indicated time points. The results revealed that over-expression of Caspase-2 could counteract the antiapoptotic function of miR-708. NC, negative control RNA duplex; miR-708, miR-708 mimic; miR-708-inhibitor, the inhibitor of miR-708; si-CASP2, siRNA targeting to Caspase-2; miR-708/pcDNA3.1, cells were co-transfected with miR-708 mimic and blank pcDNA3.1; miR-708/CASP2-pcDNA3.1, cells were co-transfected with miR-708 mimic and Caspase-2 expression vector. The percentages of cell apoptosis were calculated by data from FACS analysis. Shown data are representative from three independent experiments

Discussion

miRNAs are short, noncoding RNAs that have been shown to play pivotal roles in carcinogenesis (Fendler et al. 2011). A recent study showed that approximately 50 % of annotated human miRNAs are located in areas of the genome which are associated with cancer (Li et al. 2009b).The increasing evidences suggested that targeting miRNAs is a valuable approach to cancer therapy, as has been shown recently for various types of cancer (Fendler et al. 2011). Our recent study has proved that miR-708 was significantly up-regulated in bladder carcinoma (Song et al. 2010). It suggested this miRNA might be involved in the pathogenesis of bladder cancer. In this study, to verify the role of miR-708 in bladder carcinoma, the level of miR-708 was both enhanced and inhibited in the bladder carcinoma cell lines, T24 and 5637. Knockdown of miR-708 by miR-708 inhibitor transfection in T24 and 5637 cells showed anti-cancer activity through inducing cell apoptosis and reduced the tumor growth in vivo. Our findings suggested that miR-708 had a critical role in bladder tumor tumorigenicity, making it as the potential candidate for diagnosis or therapeutics in bladder cancer.

The abnormal expression of miR-708 has been found in several cancers, such as nonsmall cell lung cancer (Jang et al. 2012), renal cell carcinoma (Saini et al. 2011), prostate cancer (Saini et al. 2012), colon cancer (Shenouda and Alahari 2009), and acute lymphoblastic leukemia (Han et al. 2011). However, there are conflicting results on the details of the role of miR-708 in the tumorigenicity of different cancer cells. For example, Jang et al. (2012) have demonstrated that miRNA-708 acts as an oncogene contributing to tumor growth and disease progression by directly down-regulating TMEM88, a negative regulator of the Wnt signaling pathway in lung cancer. Moreover, miR-708 was also verified to be induced in both colon tumors of APCMin/+ mice and CAC (colitis-associated colon tumor) samples (Shenouda and Alahari 2009). Notably, miR-708 was also found to be the most up-regulated miRNA in the acute lymphoblastic leukemia relapse samples compared with the samples of the patients at complete remission (CR) and associated with the in vivo glucocorticoid therapy response and with disease risk stratification (Han et al. 2011). However, the reduced miR-708 expression was found to lead to prostate cancer initiation, progression, and development by regulating the expression of CD44 as well as AKT2 (Saini et al. 2012), and intratumoral delivery of miR-708 was sufficient to trigger in vivo regression of established tumors in murine xenograft models of human RCC (Saini et al. 2011). Recently, the character of miRNA expression was tumor stage-, tissue-, or development-specific has been convinced by more and more data from different research groups. This particular character might partly explain the conflict roles in the different types of cancer. Our results about miR-708 up-regulation in bladder carcinoma tissues and its contribution to tumorigenicity of bladder carcinoma cells support the conclusion that miR-708 was, at least in bladder carcinoma cells, an oncogene.

Although oncogene role of miRNAs has been frequently observed in many tumor tissues, the knowledge about its molecular mechanisms by which miRNAs regulate the tumorigenesis is relatively less (Shenouda and Alahari 2009; Lee and Dutta 2006; Hammond 2006). In bladder carcinoma cell line T24 and 5637, using luciferase assay and western blotting assay, we found a direct link between miR-708 and its putative target Caspase-2, which has been extensively verified to be an apoptosis inducer.

Apoptosis is a form of cellular suicide that is essential for multiple biological events, and its deregulation in human can lead to diseases such as cancer (Yuan and Yankner 2000; Green and Evan 2000). Caspase-2 is ubiquitously expressed and the most evolutionarily conserved mammalian caspase. It can be activated by a range of death stimuli. In addition, Caspase-2 has also been reported to exert tumor suppressor function in vivo (Muppani et al. 2011) and may function in stress-induced cell death pathways, cell cycle maintenance, and the suppression of tumorigenesis (Tang et al. 2011). In our results, the knockdown of Caspase-2 in T24 and 5637 cells could phenocopy the elevated miR-708 in inhibiting cell apoptosis. Meanwhile, the over-expression of Caspase-2 reversed the apoptotic rate which was reduced by elevated miR-708. In addition, the mRNA level of Caspase-2 was demonstrated to negatively correlate with miR-708 level in bladder carcinoma tissues. Taken together, our findings defined that miR-708 suppressed apoptosis by directly inhibiting the expression of Caspase-2 in bladder carcinoma.

In summary, miR-708 works as an oncogene by directly targeting Caspase-2, through inhibiting cell apoptosis and inducing tumor growth. The identification of miR-708 as a critical oncogene of bladder tumor emphasizes an essential role of miR-708 in mediating bladder oncogenesis. Therefore, it implicates the potential application of miR-708 for treatment of bladder cancer. Further study is needed to examine the usefulness of miR-708 for clinical application.

Electronic supplementary material

Below is the link to the electronic supplementary material.

432_2013_1392_MOESM1_ESM.tif (813KB, tif)

Analysis of miR-708 expression in cancer cell lines and human bladder cancer tissue by northern blot. 1, adjacent noncancerous tissue. 2, bladder cancer tissue. 3, 5637 cells. 4, HepG2 cells. 5, Hela cells. 6,T24 cells. (TIFF 813 kb)

Supplementary material 2 (DOCX 13 kb) (13.6KB, docx)

Conflict of interest

The authors declare that they have no conflict of interest. We have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript.

References

  1. Alvarez A, Lokeshwar VB (2007) Bladder cancer biomarkers: current developments and future implementation. Curr Opin Urol 17(5):341–346 [DOI] [PubMed] [Google Scholar]
  2. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen YH, Wang SQ, Wu XL, Shen M, Chen ZG, Chen XG, Liu YX, Zhu XL, Guo F, Duan XZ, Han XC, Tao ZH (2011) Characterization of microRNAs expression profiling in one group of Chinese urothelial cell carcinoma identified by Solexa sequencing. Urol Oncol. doi:10.1016/j.bbr.2011.03.031 [DOI] [PubMed]
  4. Cookson MS, Herr HW, Zhang ZF, Soloway S, Sogani PC, Fair WR (1997) The treated natural history of high risk superficial bladder cancer: 15-year outcome. J Urol 158:62–67 [DOI] [PubMed] [Google Scholar]
  5. Esquela-Kerscher A, Slack FJ (2006) Oncomirs microRNAs with a role in cancer. Nat Rev Cancer 6:259–269 [DOI] [PubMed] [Google Scholar]
  6. Fendler A, Stephan C, Yousef GM, Jung K (2011) MicroRNAs as regulators of signal transduction in urological tumors. Clin Chem 57(7):954–968 [DOI] [PubMed] [Google Scholar]
  7. Green DR, Evan GI (2000) A matter of life and death. Nature 407(6805):802–809 [DOI] [PubMed] [Google Scholar]
  8. Hammond SM (2006) MicroRNAs as oncogenes. Curr Opin Genet Dev 16(1):4–9 [DOI] [PubMed] [Google Scholar]
  9. Han BW, Feng DD, Li ZG, Luo XQ, Zhang H, Li XJ, Zhang XJ, Zheng LL, Zeng CW, Lin KY, Zhang P, Xu L, Chen YQ (2011) A set of miRNAs that involve in the pathways of drug resistance and leukemic stem-cell differentiation is associated with the risk of relapse and glucocorticoid response in childhood ALL. Hum Mol Genet 20(24):4903–4915 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Herr HW (1997) Natural history of superficial bladder tumors: 10- to 20-year follow-up of treated patients. World J Urol 15:84–88 [DOI] [PubMed] [Google Scholar]
  11. Huang X, Yu HS, Chen Z, Li JL, Hu ZM, Gao JM (2010) A novel immunotherapy for superficial bladder cancer by the immobilization of streptavidin-tagged bioactive IL-2 on the biotinylated mucosal surface of the bladder wall. Chin J Cancer 29(6):611–616 [DOI] [PubMed] [Google Scholar]
  12. Imre G, Heering J, Takeda AN, Husmann M, Thiede B, zu Heringdorf DM, Green DR, van der Goot FG, Sinha B, Dötsch V, Rajalingam K (2012) Caspase-2 is an initiator caspase responsible for pore-forming toxin-mediated apoptosis.The. EMBO J 31:2615–2628 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ismaili N, Amzerin M, Flechon A (2011) Chemotherapy in advanced bladder cancer: current status and future. J Hematol Oncol 4:35 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jang JS, Jeon HS, Sun Z, Aubry MC, Tang H, Park CH, Rakhshan F, Schultz DA, Kolbert CP, Lupu R, Park JY, Harris CC, Yang P, Jen J (2012) Increased miR-708 expression in NSCLC and its association with poor survival in lung adenocarcinoma from never smokers. Clin Cancer Res 18(13):3658–3667 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim BM, Rode AB, Han EJ, Hong IS, Hong SH (2012) 5-Phenylselenyl- and 5-methylselenyl-methyl-2′-deoxyuridine induce oxidative stress, DNA damage, and caspase-2-dependent apoptosis in cancer cells. Apoptosis 17(2):200–216 [DOI] [PubMed] [Google Scholar]
  16. Kitevska T, Spencer DM, Hawkins CJ (2009) Caspase-2: controversial killer or checkpoint controller? Apoptosis 14(7):829–848 [DOI] [PubMed] [Google Scholar]
  17. Lamkanfi M, Festjens N, Declercq W, Vanden Berghe T, Vandenabeele P (2007) Caspases in cell survival, proliferation and differentiation. Cell Death Differ 14(1):44–55 [DOI] [PubMed] [Google Scholar]
  18. Lee YS, Dutta A (2006) MicroRNAs: small but potent oncogenes or tumor suppressors. Curr Opin Investig Drugs 7(6):560–564 [PubMed] [Google Scholar]
  19. Li C, Feng Y, Coukos G (2009a) Therapeutic microRNA strategies in human cancer. AAPS J 11(4):747–757 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Li S, Fu H, Wang Y, Tie Y, Xing R, Zhu J, Sun Z, Wei L, Zheng X (2009b) MicroRNA-101 regulates expression of the v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene in human hepatocellular carcinoma. Hepatology 49(4):1194–1202 [DOI] [PubMed] [Google Scholar]
  21. Lynam-Lennon N, Maher SG, Reynolds JV (2009) The roles of microRNA in cancer and apoptosis. Biol Rev Camb Philos Soc 84:55–71 [DOI] [PubMed] [Google Scholar]
  22. Muppani N, Nyman U, Joseph B (2011) TAp73alpha protects small cell lung carcinoma cells from caspase-2 induced mitochondrial mediated apoptotic cell death. Oncotarget 2(12):1145–1154 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Noguchi S, Mori T, Hoshino Y, Maruo K, Yamada N, Kitade Y, Naoe T, Akao Y (2011) MicroRNA-143 functions as a tumor suppressor in human bladder. Cancer Lett 307(2):211–220 [DOI] [PubMed] [Google Scholar]
  24. Papagiannakopoulos T, Kosik KS (2008) MicroRNAs: regulators of oncogenesis and stemness. BMC Med 6:15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ren K, Lu J, Porollo A, Du C (2012) Tumor-suppressing function of caspase-2 requires catalytic site Cys-320 and site Ser-139 in mice. J Biol Chem 287(18):14792–14802 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saini S, Yamamura S, Majid S, Shahryari V, Hirata H, Tanaka Y, Dahiya R (2011) MicroRNA-708 induces apoptosis and suppresses tumorigenicity in renal cancer cells. Cancer Res 71(19):6208–6219 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Saini S, Majid S, Shahryari V, Arora S, Yamamura S, Chang I, Zaman MS, Deng G, Tanaka Y, Dahiya R (2012) miRNA-708 control of CD44 + prostate cancer-initiating cells. Cancer Res 72(14):3618–3630 [DOI] [PubMed] [Google Scholar]
  28. Shelley MD, Wilt TJ, Barber J, Mason MD (2004) A meta-analysis of randomised trials suggests a survival benefit for combined radiotherapy and radical cystectomy compared with radical radiotherapy for invasive bladder cancer: are these data relevant to modern practice. Clin Oncol 16:166–171 [DOI] [PubMed] [Google Scholar]
  29. Shenouda SK, Alahari SK (2009) MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer Metastasis Rev 28(3–4):369–378 [DOI] [PubMed] [Google Scholar]
  30. Song T, Xia W, Shao N, Zhang X, Wang C, Wu Y, Dong J, Cai W, Li H (2010) Differential miRNA expression profiles in bladder urothelial carcinomas. Asian Pac J Cancer Prev 11(4):905–911 [PubMed] [Google Scholar]
  31. Sudarshan S, Holman DH, Hyer ML, Voelkel-Johnson C, Dong JY, Norris JS (2005) In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer. Cancer Gene Ther 12:12–18 [DOI] [PubMed] [Google Scholar]
  32. Tang Y, Wells JA, Arkin MR (2011) Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis. J Biol Chem 286(39):34147–34154 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281(5381):1312–1316 [DOI] [PubMed] [Google Scholar]
  34. Tiwari M, Lopez-Cruzan M, Morgan WW, Herman B (2011) Loss of caspase-2-dependent apoptosis induces autophagy after mitochondrial oxidative stress in primary cultures of young adult cortical neurons. J Biol Chem 286(10):8493–8506 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Trang P, Weidhaas JB, Slack FJ (2008) MicroRNAs as potential cancer therapeutics. Oncogene 27:S52–S57 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang V, Wu W (2009) MicroRNA-based therapeutics for cancer. BioDrugs 23:15–23 [DOI] [PubMed] [Google Scholar]
  37. Wang CC, Chiang YM, Kuo PL, Chang JK, Hsu YL (2008) Norsolorinic acid inhibits proliferation of T24 human bladder cancer cells by arresting the cell cycle at the G0/G1 phase and inducing a Fas/membrane-bound Fas ligand-mediated apoptotic pathway. Clin Exp Pharmacol Physiol 35:1301–1308 [DOI] [PubMed] [Google Scholar]
  38. Wiklund ED, Bramsen JB, Hulf T, Dyrskjøt L, Ramanathan R, Hansen TB, Villadsen SB, Gao S, Ostenfeld MS, Borre M, Peter ME, Ørntoft TF, Kjems J, Clark SJ (2011) Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer. Int J Cancer 128(6):1327–1334 [DOI] [PubMed] [Google Scholar]
  39. Yuan J, Yankner BA (2000) Apoptosis in the nervous system. Nature 407(6805):802–809 [DOI] [PubMed] [Google Scholar]
  40. Zhang B, Pan X, Cobb GP, Anderson TA (2007) MicroRNAs as oncogenes and tumor suppressors. Dev Biol 302:1–12 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

432_2013_1392_MOESM1_ESM.tif (813KB, tif)

Analysis of miR-708 expression in cancer cell lines and human bladder cancer tissue by northern blot. 1, adjacent noncancerous tissue. 2, bladder cancer tissue. 3, 5637 cells. 4, HepG2 cells. 5, Hela cells. 6,T24 cells. (TIFF 813 kb)

Supplementary material 2 (DOCX 13 kb) (13.6KB, docx)

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES