Background
Bladder cancer is the most common malignancy of the urogenital system and is one of the major causes of cancer-related death among Chinese patients. Based on statistics by Chen et al., an estimated 80,500 new cases of urinary bladder cancer would be diagnosed and approximately 32,900 deaths from bladder cancer were anticipated in China in 2015 [
1]. Bladder cancer can be classified into two types according to the tumor invasion depth: non-muscle invasive tumor (70 ~ 80%) and muscle-invasive tumor (20 ~ 30%) [
2]. Compared with non-muscle invasive tumors, muscle-invasive tumors are a highly aggressive disease. The 5-year survival rate for patients with non-muscle invasive bladder cancer is nearly 90%, whereas that for patients with muscle-invasive bladder cancer is approximately 60% [
3]. Therefore, it is of great clinical significance to clarify the mechanisms underlying the aggressive progression of bladder cancer, which will help to identify specific molecular targets and develop more effective therapies for this disease.
Survivin (encoded by the gene BIRC5) is a key member of the inhibitor of apoptosis protein (IAP) family [
4]. Dysregulation of Survivin is a typical signature of many cancers. Survivin is not present in normal bladder urothelium but was expressed in a high percentage of patients with bladder cancer [
5]. A multicenter study found Survivin expression was associated with an elevated risk of bladder cancer recurrence and cancer-specific mortality [
6]. Survivin and E-cadherin could help identify patients in the initial pTa stage of bladder cancer who were at risk of developing progressive disease [
7]. The results above have led to the proposal of targeting Survivin as a promising alternative treatment for bladder cancer.
microRNAs (miRNAs) are endogenous small non-coding RNA molecules (19-22 nucleotides in length) that regulate protein coding gene expression by binding to the 3’ untranslated region (UTR) of mRNAs to yield an RNA-induced silencing complex [
8]. Increasing evidence suggests that miRNAs are aberrantly expressed in various human cancers and that they play significant roles in cancer initiation, development, and metastasis [
9]. Some highly expressed miRNAs could function as oncogenes by repressing tumor suppressors, whereas miRNAs expressed at lower levels could function as tumor suppressors by negatively regulating oncogenes [
10]. miRNAs potently influence cellular activities through the regulation of extensive gene expression networks [
11,
12]. Therapeutic modulation of a single miRNA may therefore simultaneously affect many pathways to achieve better clinical results. Survivin was reported to be post-transcriptionally regulated by several miRNAs in various tumors [
13]. The identification of miRNAs that target Survivin in the setting of bladder cancer will help in the development of Survivin-based therapies for bladder cancer [
14].
Although the dysregulation of miRNAs and Survivin plays important roles in the carcinogenesis of bladder cancer, there are limited reports about the correlation between Survivin and miRNAs in bladder cancer. In this study, we predicted that Survivin was a target of miR-138-5p. After measuring the expression levels of miR-138-5p and Survivin in human bladder cancer tissues and adjacent noncancerous tissue samples, we detected an inverse correlation between miR-138-5p expression and Survivin protein levels. Furthermore, we experimentally investigated the direct regulation of Survivin by miR-138-5p as well as the biological role of miR-138-5p targeting Survivin in human bladder cancer cell lines and in a mouse tumor xenograft model.
Methods
Cells and human tissues
The human bladder cancer cell lines T24 and J82 were purchased from the Shanghai Institute of Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) in a humidified atmosphere containing 5% CO2. The bladder cancer specimens and paired normal adjacent tissues were obtained from patients undergoing a surgical procedure at the Affiliated Drum Tower Hospital of Nanjing University, School of Medicine (Nanjing, China). All the patients provided written consent, and the Ethics Committee from Nanjing University approved all aspects of this study. Tissue fragments were immediately frozen in liquid nitrogen at the time of surgery and stored at -80 °C.
RNA isolation and quantitative RT-PCR
Total RNA was extracted from cultured cells and human tissues using TRIzol Reagent (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. Assays to quantify miRNA levels were performed using TaqMan miRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, 1 μg of total RNA was reverse-transcribed into cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and a stem-loop RT primer (Applied Biosystems). The reaction conditions were as follows: 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Real-time PCR was performed using a TaqMan PCR kit on an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). The reactions were incubated in a 96-well optical plate at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All of the reactions were performed in triplicate. After the reaction, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT was determined from the triplicate PCRs. A comparative CT method was used to compare each condition to the controls. The relative levels of miRNAs in the cells and tissues were normalized to the U6 levels. The amount of miRNA relative to the internal control U6 was calculated with the equation 2-△△CT, in which △△CT = (CT miR-138-5p - CT
U6) tumor - (CT
miR-138-5p - CT
U6) control. To quantify Survivin mRNA expression, 1 μg of total RNA was reverse-transcribed into cDNA using oligo dT and AMV reverse transcriptase (TaKaRa, Dalian, China) with the following cycling conditions: 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Next, real-time PCR was performed with the resulting RT product, SYBR Green Dye (Invitrogen) and specific primers for Survivin and GAPDH. The sequences of the primers were as follows: Survivin (sense), 5′- CAACCGGACGAATGCTTTT-3′; Survivin (antisense), 5′-AAGAACTGGCCCTTCTTGGA-3′; GAPDH (sense), 5′-CGAGCCACATCGCTCAGACA-3′; and GAPDH (antisense), 5’-GTGGTGAAGACGCCAGTGGA-3’. The reactions were incubated at 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. After the reactions were completed, the CT values were determined by setting a fixed threshold. The relative amount of Survivin mRNA was normalized to GAPDH using the △△CT method as described above.
Protein extraction and Western blotting
The cells and tissues were lysed in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Scientific 78440), incubated on ice for 30 min and then centrifuged for 10 min (12, 000 × g, 4 °C). The supernatant was collected, and the protein concentration was calculated using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The Survivin protein levels were analyzed by Western blotting with a monoclonal anti-human Survivin antibody (71G4B7, 2808 s. Cell Signaling Technology, MA, USA). The protein levels were normalized by probing the same blots with a GAPDH antibody (FL-335, sc-25778, Santa Cruz Biotechnology, CA, USA).
Overexpression or knockdown of miR-138-5p
miR-138-5p overexpression was achieved by transfecting bladder cancer cell lines with an miRNA mimic (a synthetic RNA oligonucleotide duplex mimicking the miRNA precursor), and knockdown was achieved by transfecting a miRNA inhibitor (a chemically modified single-stranded antisense oligonucleotide designed to specifically target the mature miRNA). Synthetic mim-miR-138-5p, anti-miR-138-5p and the corresponding negative control scrambled RNAs (mim-scramble and anti-scramble) were purchased from GenePharma (Shanghai, China). T24 and J82 cells were seeded into 6-well plates using RPMI 1640 media supplemented with 10% FBS. The following day when the cells reached 60-70% confluency, the cells were transfected with Lipofectamine 2000 (Invitrogen) using Opti-MEM Reduced Serum Medium (Gibco, Carlsbad, CA, USA). Equal amounts of either mim-miR-138-5p or mim-scramble were used in each well. For miRNA knockdown, equal amounts of anti-miR-138-5p or anti-scramble were used. After 6 hours, the media was replaced with RPMI 1640 supplemented with 2% FBS. The cells were harvested 24 hours after transfection and subjected to analysis by quantitative RT-PCR or Western blotting.
Plasmid construction and siRNA interference assay
A mammalian expression plasmid encoding the full-length human BIRC5 open reading frame (ORF) without the miR-138-5p-responsive 3′-UTR was purchased from Ribobio (Guangzhou, China). An empty plasmid served as the negative control. Three siRNA sequences targeting different sites of the human Survivin cDNA (si-Survivin) were designed and synthesized by Ribobio (Guangzhou, China). A scrambled siRNA that does not target the human Survivin cDNA was synthesized as a negative control. The siRNA sequences were as follows: si-Survivin #1, 5′-ACCGCATCTCTACATTCAA-3′ (sense); si-Survivin #2, 5′-TCCGGTTGCGCTTTCCTTT-3′ (sense); and si-Survivin #3, 5′-ACCACCGCATCTCTACATT-3′ (sense). Either the Survivin overexpression plasmid or the Survivin siRNAs were transfected into T24 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Total RNA and protein were isolated 24 hours after transfection. The Survivin mRNA and protein expression levels were assessed by quantitative RT-PCR and Western blotting, respectively. The siRNA sequence with the best interfering effect (si-Survivin#1) was selected and used for all subsequent experiments.
Luciferase reporter assay
To test the direct binding of miR-138-5p to the target gene BIRC5, a luciferase reporter assay was performed as previously described. [
15] A sequence containing the presumed miR-138-5p binding site was designed from the human BIRC5 3′-UTR. The sequence was inserted into the p-MIR-reporter plasmid (Ambion). The insertion was confirmed to be correct by sequencing. To test the binding specificity, the sequences that interacted with the miR-138-5p seed sequence were mutated (site 1 from CACCAGC to GTGGTCG, and site 2 from ACCAGCA to TGGTCGT), and the mutant BIRC5 3′-UTR was inserted into an equivalent luciferase reporter. For the luciferase reporter assays, HEK293 cells were cultured in 24-well plates, and each well was transfected with 0.4 μg of firefly luciferase reporter plasmid, 0.4 μg of a β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (20 pmol) of mim-miR-138-5p, anti-miR-138-5p, or the scrambled negative control RNAs using Lipofectamine2000 (Invitrogen). The β-gal plasmid was used as a transfection control. Twenty-four hours after transfection, the cells were assayed using a luciferase assay kit (Promega, Madison, WI, USA).
Cell proliferation assay
The proliferation of T24 cells was determined using the Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer’s instructions. T24 cells were plated at a density of 5 × 103 cells per well in 96-well plates and incubated overnight in RPMI 1640 medium supplemented with 10% FBS. The next day, the T24 cells were transfected with mim-miR-138-5p, anti-miR-138-5p, BIRC5 siRNA and/or the overexpression plasmid, and the medium was replaced with RPMI 1640 medium supplemented with 2% FBS. At 12, 24, 36 and 48 hours after transfection, 10 μL of CCK-8 reagent was added to the test well and incubated for 3 hours. The absorbance was measured at a wavelength of 450 nm.
Cell invasion assay
The invasive ability of T24 cells transfected with either mimics or inhibitors (mim-miR-138-5p or anti-miR-138-5p, respectively), BIRC5 siRNA and/or the overexpression plasmid was tested in a Transwell Boyden chamber (6.5 mm; Costar). The polycarbonate membranes (8-μm pore size) on the bottom of the upper compartment of the Transwells were coated with 1% human fibronectin (R&D Systems. catalog no. 1918-FN). The cells were harvested 24 hours after transfection and suspended in serum-free DMEM culture medium. Then, cells were added to the upper chamber (4 × 104 cells/well). At the same time, 0.5 mL of DMEM containing 10% FBS was added to the lower compartment, and the Transwell-containing plates were incubated for 12 hours in a humidified atmosphere containing 5% CO2 atmosphere. After the incubation, cells that had invaded to the lower surface of the filter membrane were fixed with 4% paraformaldehyde for 25 min at room temperature, washed three times with distilled water, and stained with 0.1% crystal violet in 0.1 M borate and 2% ethanol for 15 min at room temperature. Cells remaining on the upper surface of the filter membrane (non-migratory) were gently scraped off using a cotton swab. Images of the lower surfaces (with the migratory cells) were captured by using a photomicroscope (5 fields/chamber) (BX51 Olympus, Japan), and the cells in randomly selected fields were counted by a blinded investigator.
Construction of the miR-138-5p overexpression lentiviral vector
A lentiviral vector that overexpresses miR-138-5p was purchased from Invitrogen. When T24 cells reached 70% confluence in either 6-well plates or 100-mm dishes, lentivirus at a multiplicity of infection of 10 was added together with Polybrene (final concentration 5 μg/ml) according to the manufacturer’s instructions. Cells were then harvested for animal experiments.
Establishment of Tumor Xenografts in Mice
Six-week-old male nude mice were purchased from the Model Animal Research Center at Nanjing University (Nanjing, China) and maintained under specific pathogen-free conditions at Nanjing University. The mice were randomly divided into 4 groups and subcutaneously injected with T24 cells (2 × 10
6 cells per mouse, 5 mice per group, Fig.
4a) that were infected with either control lentivirus or a lentivirus that overexpressed miR-138-5p followed by co-transfection with a control plasmid or a BIRC5 overexpression plasmid. The needle was inserted into the left side of the armpit, midway down, at a depth of 5 mm deep an angle of 45°. The volume of the tumors was measured every 4 days after implantation. The tumor volume was calculated by the following formula: tumor volume [mm
3] = (length [mm]) × (width [mm])
2 × 0.52. The mice were sacrificed after 28 days. The tumors were excised, and the tumor weight was measured. Portions of the tumor samples were used for protein and total RNA extraction, and the remainder was fixed in 4% paraformaldehyde for 24 hours and then processed for hematoxylin and eosin (H&E) staining as well as immunohistochemical staining for Survivin and Ki-67.
Statistical analysis
All of the Western blotting and cell invasion assay results are representative of at least three independent experiments. Quantitative RT-PCR and the luciferase reporter and cell proliferation assays were performed in triplicate, and each experiment was repeated several times. The data shown are the mean ± SD or mean ± SE of at least three independent experiments.
Discussion
Survivin is an oncogene that regulates the apoptosis, proliferation, and invasion of many cancers, including bladder cancer [
16‐
19]. Survivin has been recognized as a highly specific biomarker for bladder cancer and its expression is relative to the presence, stage, progression and mortality of bladder cancer [
20]. As a tumor biomarker, Survivin protein is highly expressed in bladder tumors and either absent or weakly expressed in the normal adjacent bladder mucosa [
21]. Interestingly, we found that the Survivin mRNA was detectable in normal bladder tissue and did not differ as much as the protein levels between bladder cancer and normal adjacent bladder mucosa. The discordance between Survivin protein and mRNA in bladder cancer suggested that post-transcriptional regulation might be involved in Survivin protein expression. One essential mode of post-transcriptional regulation is the repression of mRNA transcripts by miRNA. miRNAs regulate gene expression by the sequence-selective targeting of mRNAs, leading to either translational repression or mRNA degradation [
8,
22].
It was reported that miRNAs related to post-transcriptional regulation play an important role in Survivin dysregulation in some human cancers [
13]. However, there is limited information about the miRNA regulation of Survivin expression in bladder cancer. In this study, we searched for miRNAs that can target Survivin and identified miR-138-5p as a candidate. We experimentally validated the direct inhibition of Survivin translation by miR-138-5p by overexpressing and knocking down miR-138-5p in bladder cancer cells. In addition, we showed that in cultured bladder cancer cells, miR-138-5p inhibited Survivin expression as well as cell proliferation and invasion; furthermore, miR-138-5p also slowed tumor growth in a xenograft mouse model. The results demonstrated a novel regulatory network involving miR-138-5p and Survivin to fine-tune the proliferation and invasion of bladder cancer.
miRNAs are aberrantly expressed during the carcinogenesis of bladder cancer [
23,
24]. Some microRNAs have been categorized as “oncomiRs” as opposed to “tumor suppressor miRs” [
25‐
27]. In this study, we found that the levels of miR-138-5p in bladder cancer were much lower than those in normal adjacent bladder mucosa. Down-regulation of miR-138-5p has also been reported in other cancers [
28‐
30]. All these results suggested that miR-138-5p may work as a tumor suppressor in bladder cancer. It is well known that a single miRNA can target multiple genes, whereas multiple miRNAs can target a single gene. For example, miR-138-5p could inhibit the translation of ZEB2 mRNA and suppress the ZEB2-mediated metastatic potential of bladder cancer [
31]. miR-138-5p could also suppress cell proliferation by targeting Bag-1 in gallbladder carcinoma [
32]. To investigate the role of this new pathway in the network of bladder carcinogenesis signaling, we overexpressed miR-138-5p in bladder cancer cells and found that proliferation and invasion of bladder cancer cells were inhibited, which mimic the function of Survivin reduction by targeted siRNA. Interestingly, we further observed that the restoration of Survivin expression by an overexpressing plasmid can successfully attenuate the anti-proliferative and anti-invasive effect of miR-138-5p on bladder cancer cells, although miR-138-5p may also have many other targets. In the
in vivo study, the Survivin overexpression plasmid significantly rescued the suppressed tumor growth induced by cells transduced with lentivirus-miR-138-5p. As is common knowledge, the effect of the plasmid did not last as long as that of the lentiviral vector, which was one limitation of our study. Therefore, the tumors in the rescue group did not grow to the level of the control group as expected. In the following supplemental animal experiment, we increased the dose of Survivin overexpression plasmid, and the tumors in rescue group almost reached the levels of tumors in control group (~80%) (Additional file
3: Figure S3). These results confirmed that targeting Survivin is an important mechanism of the tumor-suppressive function of miR-138-5p.
Many studies have shown that Survivin could serve as a molecular target for bladder cancer therapy [
33‐
35]. Knockdown of Survivin by using siRNA could dramatically suppress the proliferation and invasion of bladder cancer cells. As previously mentioned, post-transcriptional regulation by miRNA plays an important part in dysregulating Survivin signaling in bladder cancer [
13]. Modulating the miRNA activities may provide exciting opportunities for bladder cancer therapy. The rationale for using miRNAs as therapeutic agents is based on the two following criteria: (1) miRNA expression is dysregulated in cancer compared to that in normal tissues and (2) the cancer phenotype can be adjusted by targeting miRNA expression. Emerging evidence suggests that either inhibiting the overexpression of oncogenic miRNAs or introducing more tumor-suppressive miRNAs could become a novel treatment strategy in cancer therapy [
36]. Compared to other strategies, miRNA-based therapeutics have several advantages; for example, miRNAs as therapeutic agents have the ability to target multiple genes, frequently in the context of a network [
37]. In the present study, we found that elevated expression of Survivin was related to the decrease of tumor suppressor miR-138-5p. Treatment with miR-138-5p exhibited an anti-tumor effect both
in vitro and
in vivo by negatively regulating Survivin expression. Thus, it is hypothesized that a replacement treatment with miR-138-5p mimics could be a promising strategy for cancers characterized by down-regulation of miR-138-5p.
Acknowledgements
We would like to thank Dr. Wei Jiang for his hard work in preparing the clinical specimens.