Knockdown of SIRT1 Suppresses Bladder Cancer Cell Proliferation and Migration and Induces Cell Cycle Arrest and Antioxidant Response through FOXO3a-Mediated Pathways

Bladder cancer (BCa) is one of the most common tumors, but its underlying mechanism has not been fully clarified. Our transcriptome analysis suggested a close link of Sirtuins, Peroxisome Proliferator-Activated Receptor (PPAR), cell cycle regulation, reactive oxygen species (ROS) metabolism, and Forkhead Box Class O (FOXO) signaling pathway in BCa. SIRT1 is a key member of Sirtuins, playing important roles in aging and energy metabolism, which has been reported to be involved in various metabolic diseases and tumors. We observed that SIRT1 was upregulated in BCa tissues at both mRNA and protein levels. By establishing a SIRT1-knockdown BCa cell model, our results suggested that proliferation and viability were suppressed. Moreover, migration rate was inhibited as well, possibly via reduction of epithelial-mesenchymal transition (EMT). In addition, cell cycle arrest was significantly induced, consisting with strongly decreased proteins involved (CDK2/4/6). Furthermore, ROS production was slightly reduced, accompanied by increasing of antioxidant enzymes and total/acetylated FOXO3a. Consistently with our Path-net analysis, we observed no significant alteration of apoptosis in the SIRT1-knockdown BCa cells. Taken together, our results suggested that SIRT1 deficiency in BCa cells could suppress cell viability by activating antioxidant response and inducing cell cycle arrest possibly via FOXO3a-related pathways.


Introduction
Bladder cancer (BCa), also named urinary bladder cancer, is one of the most common tumors ranking as the 9th leading cause of death worldwide [1,2]. A horrible threat as it is to human health, its underlying mechanism, especially its metabolic alterations, has not been fully clarified yet [3][4][5].
Sirtuins, also known as Sir2-like proteins, are a family of NAD + -dependent deacetylases and ADP-ribosyltransferases [6], playing a vital role in aging [7]. Sir2 was first discovered in yeast via a model of replicative lifespan [8], and later it was shown that addition of an extra copy of the SIR2 gene could extend replicative lifespan by 40% while deleting SIR2 shortened lifespan [9].
Recent studies of our group based on a transcriptome analysis using human bladder cancer tissues compared with normal bladder tissues [37][38][39], indicating a close correlation of Sirtuin family, PPAR signaling pathway, cell cycle regulation, ROS metabolism, and FOXO signaling pathway in BCa. Therefore, this study aims to investigate the effect of SIRT1 in BCa and the underlying mechanism.

Ethical Statement for Human Bladder Tissue Samples.
Human bladder cancer tissue samples ( = 19) and human paracancerous tissues ( = 19) were all collected from patients suffering bladder cancer in surgery. And all normal bladder tissue samples ( = 4) were obtained from donors who died from accident. The samples were either stored in liquid nitrogen for later RNA isolation or fixed in 4% concentration paraformaldehyde (PFA) for the followed immunofluorescence staining analysis. The Ethics Committee at Zhongnan Hospital of Wuhan University approved the experiments using human bladder tissue samples for RNA and immunofluorescence staining analyses (approval number: 2015029, see Supplementary Material available online at https://doi.org/10.1155/2017/3781904). All methods used for human bladder tissue samples were performed in accordance with the approved guidelines and regulations. Written informed consent was obtained from all subjects.

Total RNA Isolation from Bladder Cells and Tissues.
We isolated total RNA from cultured bladder cells and collected bladder tissues using Qiagen RNeasy Mini Kit (Cat. #74101, Qiagen Ltd., Germany), combined with QIAshredder from Qiagen (Cat. #79654, Qiagen Ltd., Germany), using a centrifuge (Cat. #5424, Eppendorf Ltd., Germany) according to the manufacturer's protocol. DNase I (RNase-Free DNase Set, Cat. #79254, Qiagen Ltd., Germany) was used to remove contamination of gDNA from the RNA samples. Concentration of isolated RNA was measured with NanoPhotometer (Cat. #N60, Implen Ltd., Germany).

Microarray Analysis of mRNA Isolated from Human
Bladder Tissues. As previously reported by Cao et al. [37], Wang et al. [38], and Qian et al. [39] from our group, a transcriptome analysis was established by using three human BCa versus three normal bladder tissues. Briefly, biotinylated cDNA were prepared from 250 ng total RNA using the Ambion5 WT Expression Kit. Then, 5.5 g cDNA were hybridized on GeneChip Human Transcriptome Array 2.0 (16 h at 45 ∘ C) in Hybridization Oven 645. GeneChips were then washed and stained in the Affymetrix Fluidics Station 450 and scanned by Affymetrix5 GeneChip Command Console (AGCC), installed in GeneChip5 Scanner 3000 (7G). Data were analyzed by Robust Multichip Analysis (RMA). BCa related genes and pathways were analyzed by using Gene ontology (GO) and Go-map network analysis based on the Gene Cloud of Biotechnology Information software (GCBI System, Shanghai, China) (https://www.gcbi.com.cn) [40]. Thereafter, the gene list was subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID) [41] for annotation and overrepresentation analysis of the genes involved FOXO signaling pathway (map04068, KEGG pathway image, Kanehisa Laboratories, Japan [42,43]). The microarray data was uploaded to the Gene Expression Omnibus (GEO) database with accession number: GSE76211. All data are MIAME compliant.

Reverse Transcription and Quantitative Real-Time PCR (qRT-PCR).
Reverse transcription was conducted on an iCycler (Cat. #CFX Connect, Bio-Rad Ltd., USA) with ReverTra Ace qPCR RT Kit (Toyobo Ltd., China) using 1 g of total RNA mentioned above as template strand. Realtime polymerase chain reactions (qRT-PCR) were performed with iQTM SYBR5 Green Supermix (Bio-Rad Ltd., China) in a reaction system of 20 l total volume using 1 g of cDNA. Before qRT-PCR all primers had been tested for optimal annealing temperatures with gradient PCRs. Primer sequences and annealing temperatures used in qRT-PCR are listed in Table 1. Values of GAPDH were used for normalizing amplification. To be more scientific, we used relative gene abundance for further statistical analyses: Δct = ct target gene − ct GAPDH , for BCa cells ΔΔct = Δct siRNA-treated − Δct siRNA-untreated , for bladder tissues ΔΔct = Δct BCa patients − Δct paracancerous tissues , and relative gene abundance = 2 −ΔΔct (ct = threshold cycle).  Table 2. These RNA were used to transfect BCa cell lines with LipoJet6 (SignaGen Ltd., China), according to the

Western Blot and Immunoprecipitation Analysis.
For the following Western blot analysis, total protein was separated by electrophoresis in 10-12.5% SDS-PAGE and then transferred to the PVDF membrane (Millipore Ltd., USA). Membranes were then blocked in 5% fat-free milk and continuously incubated with primary antibodies (Table 3) at 4 ∘ C for overnight and secondary antibodies (Table 4) for 2 h at room temperature. Bands were visualized using an enhanced chemiluminescence (ECL) kit (Bio-Rad Ltd., USA) and detected by ChemiDoc XRS + Imaging System (Bio-Rad Ltd., USA). The immunoprecipitation analysis for acetylation level of FOXO3a was done according to Frazzi et al. [44]. Briefly, total cell lysates were prepared with lysis buffer and cleared by centrifugation. Then, 200 l of cell lysates was incubated with anti-FOXO3a antibody and rotated at 4 ∘ C for overnight. The samples were continually incubated with protein A agarose for 2 hours. After washing the immunoprecipitated BioMed Research International 5 produce was eluted, loaded on 10% polyacrylamide gels, and performed by SDS-PAGE, as well as detected by the ECL kit. The intensity of bands was measured with the ancillary software of ChemiDoc XRS + Imaging System (Bio-Rad Ltd., USA), named Image Lab (version 5.1, build 8). To compare the acetylation level of FOXO3a, we calculated the relative acetylation rate of FOXO3a: the relative acetylation rate = intensity of anti-acetyl Lysine/intensity of total FOXO3a band. The intensity of total FOXO3a was used as a loading control and its rate of NC group was normalized to 1.

Immunofluorescence Staining for Human Bladder Tissue
Samples. The bladder tissue samples were fixed by 4% PFA containing 2% sucrose in PBS at 4 ∘ C for overnight and embedded into paraffin (Paraplast, Sigma-Aldrich Ltd., USA) using a tissue processor (Cat. #STP 120, Thermo Fisher Scientific Ltd., UK). Paraffin sections (4 m) were cut with a rotation microtome (Cat. #HM325, Thermo Fisher Scientific Ltd., Germany). The sections were serially incubated with primary antibody and Cy3-labeled or FITC-labeled secondary antibody in humidified atmosphere (Tables 3 and 4). Nuclei were labeled with DAPI (2 g/ml). Immunofluorescence staining images for paraffin sections were analyzed by the fluorescence microscope.

Immunofluorescence Analysis for BCa Cells.
Coverslips with BCa cells were washed three times with ice-cold PBS and fixed by 4% PFA for 30 min. Cells were then treated with 0.1% Triton X-100 solution and blocked using normal goat serum for 30 min at room temperature. Afterwards, the cells were incubated with the indicated primary antibody (Table 3) at the proper dilution for 2 h at room temperature, washed with PBS for three times, and incubated with Cy3-labeled or FITC labeled secondary antibody (Table 4) for 1 h. Nuclei were stained by 1 mM TOTO-3 iodide for 10 min at room temperature. Immunofluorescence staining was analyzed by the fluorescence microscope.
2.6. Statistical Analyses. All data described as mean ± SD form is from three or more independent experiments. Twotailed Student's paired and unpaired -tests were used to evaluate the statistical significance of the data. All of the statistical analyses were performed with SPSS 16.0. Statistical significance was set at probability values of < 0.05.

Upregulation of SIRT1 in BCa Tissues Compared with
Paracancerous Tissues and Normal Bladder Tissues. qRT-PCR analysis was performed to evaluate the expression of SIRT1 gene, indicating significant upregulation in the BCa tissues compared with the paired paracancerous tissues ( = 16, < 0.05, Figure 1(a)). Double immunofluorescence staining showed that, in the BCa tissues, SIRT1 protein was strongly increased in the OCT4-positive cells (Figure 1(b)), which has been suggested to be a potential biomarker for BCa [37], whereas the corresponding paracancerous bladder tissues and the normal bladder tissues exhibited slight staining of both OCT4 and SIRT1 protein (Figure 1(b)), consisting with the qRT-PCR result. Overrepresentation analysis using microarray raw data [37][38][39] and DAVID database revealed that, in the BCa tissues, increased SIRT1 expression could affect oxidative stress resistance, cell cycle regulation, and energy metabolism, via FOXO signaling pathway (Figure 1(c)).

SIRT1 Deficiency Affected ROS Level in BCa Cells.
A cell model of SIRT1 deficiency was established by siRNAtransfection in the BCa cells. The BCa cells were transfected by three distinct SIRT1-target-specific-siRNA (si-1, si-2, and si-3) and negative-control-siRNA (NC) (sequences are listed in Table 1). Efficiency of knockdown was validated by qRT-PCR 48 h after transfection (Figure 2(a)). Considering qRT-PCR result in both cell lines overall, we used si-2 to perform the following experiments. Knockdown rate was 80.4% in EJ (contaminated by T24 as per "http://iclac.org/databases/cross-contaminations/") and 86.8% in T24 cells. We noticed a significant downregulation of SIRT1 protein abundance was observed by Western blot analysis (Figure 2 SIRT1 is reported to play a role in the metabolism of the reactive oxygen species (ROS) in cells [36][37][38], but how SIRT1 affected ROS metabolism in bladder cancer cells has not been reported yet. Therefore, we used DCFH-DA to stain the si-SIRT1 group and NC group before flow cytometry analysis. The results suggested that BCa cells lacking SIRT1 exhibited a reduced level of ROS production than the NC group (Figure 2(d)). Consistently, ROS staining using fluorescence staining showed a similar result (Figure 2(e)), accompanied by increasing of distinct antioxidant enzymes (Catalase and SOD2, Figure 2(f)). Moreover, our results suggested that the central transcription receptors (FOXO3a, PPAR , and acetylated p53) were strongly upregulated in the si-SIRT1 group (Figure 2(f)).

Acetylation Level of FOXO3a Was Induced in the BCa
Cells with Downregulated SIRT1. Since no effective antibody for acetylated FOXO3a is available, we used immunoprecipitation experiment to test the acetylation level of FOXO3a (Figure 2(g)). By using the intensity of total FOXO3a as a loading control, the relative acetylation rate of FOXO3a was statistically analyzed (Figure 2(h)). The results suggested that the acetylation level of FOXO3a in the downregulated SIRT1 group was significantly increased.

Knockdown of SIRT1 Inhibited Proliferation and Viability in BCa Cells and Triggered Cell Cycle Arrest at G0/G1 Phase.
Clonogenic survival assay revealed that the ability of clone formation was decreased in the si-SIRT1 cells by 36.5%, comparing with the NC group (Figures 3(a) and 3(b)). Moreover, MTT assay revealed that the si-SIRT1-treated BCa cells grew significantly slower than the negative-control-siRNA-treated group (Figure 3(c)), consisting with strongly reduced Ki-67 positive cells in the si-SIRT1 group (Figure 3(d)), which could be used as an indicator for proliferation [45].   Flow cytometry analysis of cell cycle indicated that there was higher percentage of cells at G0/G1 phase in si-SIRT1 group than in NC group while it was on the contrary at S phase (Figures 3(e) and 3(g)), which suggested that SIRT1 deficiency induced cell cycle arrest at G0/G1 phase. In addition, Western blot result suggested proteins involved in regulating G0/G1 phase (CDK2/4/6) were considerably decreased in si-SIRT1 group (Figure 3(f)).

Downregulation of SIRT1 Inhibited Migration in BCa Cells.
Cell migration was measured using transwell migration assay, and migration rate was significantly lower in si-SIRT1 group than the NC group both in EJ (contaminated by T24 as per "http://iclac.org/databases/cross-contaminations/") and in T24 (Figures 4(a) and 4(b)). Affected by the transfection of SIRT1-target-specific-siRNA, the migration rate declined by 67.5% ( < 0.05) in EJ (contaminated by T24 as per * * * * * * * * * *   "http://iclac.org/databases/cross-contaminations/") and 48.5% in T24 ( < 0.01). Furthermore, the protein abundances of E-cadherin and N-cadherin, involved in the epithelialmesenchymal transition (EMT) process, were affected by reduction of SIRT1. Our results showed that the E-cadherin level was obviously higher and the N-cadherin was lower in si-SIRT1 group (Figures 4(c) and 4(d)), suggesting that activity of EMT process was reduced by SIRT1 deficiency.

Lower Level of SIRT1 Expression Had No Significant
Effect on Apoptosis in BCa Cells. We also performed flow cytometry analysis to investigate apoptosis alternation of SIRT1 deficiency. But after multiple repeated tests, the result yet suggested no significant alteration between si-SIRT1 group and NC group ( Figure 5(a)). To make further affirmation, we conducted TUNEL assay, and the result suggested no significant alteration either ( Figure 5(b)). Consistently, in immunofluorescence staining, no significant (n.s.) alteration of the apoptosis related proteins cleaved-Caspases 3, 7, and 9 in si-SIRT1 group was observed (Figures 5(d)-5(f)).

Discussion
In our previous studies [37][38][39] based on comprehensive transcriptome analysis using human bladder cancer tissues compared with normal bladder tissues, we found that SIRT1 played a vital role in tumorigenesis and further development of human bladder cancer (BCa). SIRT1 is a crucial gene in process of aging [9], energy metabolism, and autophagy [11], but its role in BCA remains largely unknown. Our further investigation in human bladder tissue samples at a larger scale revealed that SIRT1 possessed an overexpression in human bladder cancer tissues than in paracancerous tissues or normal bladder tissues, at both transcriptional and protein levels ( Figure 1). Therefore, overrepresentation analysis and DAVID database (Figure 1) revealed that SIRT1 could interfere FOXO signaling pathway to affect oxidative stress resistance, cell cycle regulation, and energy metabolism in the BCa tissues. FOXO proteins constitute a family of transcription factors that play important role in regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity. FOXO3a is a key submember in the FOXO family involved in AKT/FOXO3a/ -catenin pathway [39], regulating cell cycle, oxidative stress response, and apoptosis [46][47][48]. In the downregulated SIRT1 BCa cells, total and acetylated FOXO3a were strongly induced (Figure 2), suggesting the correlated cell cycle regulation and antioxidant response were affected. These results were similar as reported by Frazzi et al. [44], which indicated that by using an inhibitor of SIRT1 (resveratrol) the acetylation level of FOXO3a was strongly upregulated in the Hodgkin lymphoma cells. Indeed, in our SIRT1 deficiency cell model, we observed a significantly reduced ROS production ( Figure 2). As we know, the role that ROS plays in cancer biology is quite complicated, which can be seen as a double-edged sword. A modest level of ROS is essential for tumorigenesis, whereas an excessive level would suppress tumors [49,50]. ROS can serve as a signal of either apoptosis or survival, which is determined by dosage, type, duration, and site of ROS production [51]. As Lin et al. reported, ROS produced in mitochondria could be the major source of ROS production and therefore lead to BCa cells apoptosis [52]. Therefore, the alteration of mitochondrial SOD2 was analyzed, which clears mitochondrial ROS, thus protecting cells from cell death [53]. Consistently, our result revealed an obvious upregulation of SOD2 protein (Figure 2)    which may be responsible for no significant increase of BCa cells apoptosis observed ( Figure 5). Another key enzyme involved in antioxidant response is Catalase, which has been reported to protect chromosomes against ionizing radiation [53] or oxidative damage [54]; therefore it suppresses cell death. And it also showed an increase in our study (Figure 2(f)), which may be another reason to explain that no significant increase of apoptosis occurred in the si-SIRT1 treated BCa cells ( Figure 5). Moreover, we observed a significantly induced cell cycle arrest at G0/G1 phase (Figure 3), followed by downregulation of proteins involved in G0/G1 to S phase progression (CDK2/4/6). Cyclin-dependent kinases (CDKs) are a family of protein kinases playing vital roles in regulating cell cycle [55]. Among them CDK4 and CDK6 are responsible for helping cells getting out from G0 phase and into G1 phase [56,57]; meanwhile CDK2 plays its role in G1 phase [58]. Downregulation of CDK2/4/6 could lead to cell cycle arrest at G0/G1 phase [59], which may explain our result of flow cytometry analysis (Figure 2).
We highly suspect that the highly activated antioxidant response may play a vital role in the process avoiding the BCa cells with SIRT1 deficiency from apoptosis. However, further studies are needed for the detailed mechanism.
For further discussion, it is reported that the overexpression of Thromboxane-A2 isoform-receptor (TP ) plays an important role in the process of human bladder cancer, and TP agonist decreased acetylation of FOXO3 via upregulation of SIRT1 in the bladder cancer cell line UMUC3 [60]. However, no significant alteration of TP between human normal bladder and bladder cancer tissue was found in our microarray. It may suggest that there are other pathways regulating SIRT1 expression in bladder cancer. Moreover, as reported by Lin et al. [52], capsaicin inhibits tumor-associated NADH oxidase (tNOX) and SIRT1, thus changing multiple phenotypes of bladder cancer cells including apoptosis, cell cycle progression, and cell migration. In contrast, in our study we directly knocked down SIRT1, which could help us focus on the effect of SIRT1 downregulation, avoiding the interference of other factors.

Conclusions
Our study for the first time suggested that SIRT1 deficiency in bladder cancer cells could suppress proliferation and ROS production, as well as induce cell cycle arrest, possibly via the FOXO3a-mediated pathways.

Disclosure
All the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.