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
Bladder cancer (BCa), also named urinary bladder cancer, is one of the most common tumors ranking as the 9th leading cause of death worldwide [
Sirtuins, also known as Sir2-like proteins, are a family of NAD+-dependent deacetylases and ADP-ribosyltransferases [
Mammals contain seven Sirtuins (SIRT1–7), which are categorized by their highly conserved central NAD+-binding and catalytic domain, termed the Sirtuin core domain [
Recent studies of our group based on a transcriptome analysis using human bladder cancer tissues compared with normal bladder tissues [
Human bladder cancer tissue samples (
Human bladder cancer cell line EJ (contaminated by T24 as per “
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).
As previously reported by Cao et al. [
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
List of primers for qRT-PCR.
Gene | Symbol | Forward primer (5′-3′) | Reverse primer (5′-3′) | Annealing temperature (°C) | Length (bp) |
---|---|---|---|---|---|
Sirtuin 1 |
|
5′-TAGCCTTGTCAGATAAGGAAGGA-3′ | 5′-ACAGCTTCACAGTCAACTTTGT-3′ | 58 | 160 |
Glyceraldehyde-3-phosphate dehydrogenase |
|
5′-ACAACTTTGGTATCGTGGAAGG-3′ | 5′-GCCATCACGCCACAGTTTC-3′ | 56 | 101 |
List of
Mark name | Suppressed gene | Sense sequence (5′-3′) | Antisense sequence (5′-3′) | Supplier |
---|---|---|---|---|
NC | Negative control | UUCUCCGAACGUGUCACGUTT | ACGUGACACGUUCGGAGAATT | Viewsolid Biotech, Beijing |
|
SIRT1 | GGAAAUAUAUCCUGGACAATT | UUGUCCAGGAUAUAUUUCCTT | Viewsolid Biotech, Beijing |
|
SIRT1 | GCAACUAUACCCAGAACAUTT | AUGUUCUGGGUAUAGUUGCTT | Viewsolid Biotech, Beijing |
|
SIRT1 | GCUGAUGAACCGCUUGCUATT | UAGCAAGCGGUUCAUCAGCTT | Viewsolid Biotech, Beijing |
To measure the ROS level in the BCa cells, we used the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Sigma-Aldrich Ltd., USA) for staining. 48 h after transfection, the BCa cells were harvested and centrifuged; then 1 ml serum-free medium containing 10
24-well plate transwell chamber system with 8.0
48 h after transfection, the BCa cells were seeded in 96-well plates (3,000 cells per 200
After transfection for 48 h, distinct BCa cells were seeded in 6-well plates (800 cells per well) and grew into colonies for approximately 14 days. Then colonies were fixed by 4% PFA for 30 min, stained with crystal violet for 30 min, washed with PBS, naturally dried, counted, and photographed.
For cell cycle analysis, the BCa cells were transfected by
After 48 h transfection, coverslips with BCa cells were fixed by 4% PFA for 30 min at room temperature and washed three times by ice-cold PBS, continuously incubated with 0.1% Triton X-100 for 2 min, and washed by PBS for three times. Then, apoptotic cells were measured by the TdT-mediated dUTP-biotin nick end labeling test (TUNEL, Roche Applied Science Ltd., Germany), according to the manufacturer’s instructions. Nuclei were stained by 1
The RIPA buffer with protease inhibitor and phosphatase inhibitor (Sigma-Aldrich Ltd., USA) was used to lyse and sonicate BCa cells on ice for 30 min and then centrifuged at 12,00
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
List of primary antibodies.
Antigens | Species antibodies raised in | Dilution (IF) | Dilution (WB) | Supplier |
---|---|---|---|---|
Acetyllysine, acetylated KLH conjugates | Rabbit, polyclonal | — | 1 : 500 | Abcam, UK, Cat. #ab80178 |
Catalase, human | Rabbit, monoclonal | — | 1 : 2,000 | Abcam, UK, Cat. #ab76024 |
CDK2, human | Rabbit, monoclonal | — | 1 : 2,000 | Abcam, UK, Cat. #ab32147 |
CDK4, human | Rabbit, monoclonal | — | 1 : 2,000 | Abcam, UK, Cat. #ab124821 |
CDK6, human | Rabbit, monoclonal | — | 1 : 1,000 | Abcam, Cat. #ab124821 |
Cleaved caspase-3, human | Rabbit, monoclonal | 1 : 200 | — | Cell Signaling Technology, USA, Cat. #9664 |
Cleaved caspase-7, human | Rabbit, monoclonal | 1 : 200 | — | Cell Signaling Technology, USA, Cat. #8438 |
Cleaved caspase-9, human | Rabbit, monoclonal | 1 : 200 | — | Cell Signaling Technology, USA, Cat. #7237 |
FOXO3a, human | Rabbit, monoclonal | — | 1 : 1,000 | Abcam, UK, Cat. #ab53287 |
Ki-67, human | Rabbit, monoclonal | 1 : 200 | — | Novus Biologicals, USA, Cat. #NBP2-19012 |
OCT-4, human | Mouse, monoclonal | 1 : 200 | — | Novus Biologicals, USA, Cat. #NB110-90606 |
p53 (acetyl K370), human | Rabbit, monoclonal | — | 1 : 1,000 | Abcam, UK, Cat. #ab183544 |
PPAR |
Rabbit, monoclonal | — | 1 : 1,000 | Abcam, UK, Cat. #ab45036 |
SIRT1, human | Rabbit, monoclonal | 1 : 400 | 1 : 1,000 | Cell Signaling Technology, USA, Cat. #9475 |
SOD2, human | Rabbit, monoclonal | — | 1 : 1,000 | Abcam, UK, Cat. #ab68155 |
|
Mouse, monoclonal | — | 1 : 2,000 | Santa Cruz Biotechnology Inc., Dallas, TX., USA, Cat. #sc-47778 |
E-Cadherin, human | Rabbit, monoclonal | 1 : 200 | — | Cell Signaling Technology, USA, Cat. #3195 |
N-Cadherin, human | Rabbit, monoclonal | 1 : 200 | — | Cell Signaling Technology, USA, Cat. #13116 |
List of secondary antibodies and counterstaining of nuclei.
Secondary detection system used | Host | Method | Dilution | Supplier |
---|---|---|---|---|
Anti-mouse-IgG (H + L)-HRP | Goat | WB | 1 : 10,000 | Sungene Biotech, China, Cat. #LK2003 |
Anti-rabbit-IgG (H + L)-HRP | Goat | WB | 1 : 10,000 | Sungene Biotech, China, Cat. #LK2001 |
Anti-rabbit IgG (H + L), F(a |
Goat | IF | 1 : 50 | Cell Signaling Technology, USA, Cat. #4412 |
Anti-mouse IgG (H + L), F(a |
Goat | IF | 1 : 50 | Cell Signaling Technology, USA, Cat. #4408 |
Anti-goat IgG-FITC | Rabbit | IF | 1 : 100 | Boster Biological Technology, China, Cat. #BA1110 |
Anti-goat IgG-Cy3 | Rabbit | IF | 1 : 100 | Boster Biological Technology, China, Cat. #BA1034 |
Hoechst 33342 nucleic acid staining (DAPI) | — | IF | 1 : 750 | Molecular Probes/Invitrogen, USA, Cat. #A11007 |
The immunoprecipitation analysis for acetylation level of FOXO3a was done according to Frazzi et al. [
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
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
All data described as mean ± SD form is from three or more independent experiments. Two-tailed Student’s paired and unpaired
qRT-PCR analysis was performed to evaluate the expression of
A cell model of
SIRT1 is reported to play a role in the metabolism of the reactive oxygen species (ROS) in cells [
Since no effective antibody for acetylated FOXO3a is available, we used immunoprecipitation experiment to test the acetylation level of FOXO3a (Figure
Clonogenic survival assay revealed that the ability of clone formation was decreased in the
Flow cytometry analysis of cell cycle indicated that there was higher percentage of cells at G0/G1 phase in
Cell migration was measured using transwell migration assay, and migration rate was significantly lower in
We also performed flow cytometry analysis to investigate apoptosis alternation of
In our previous studies [
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/
Indeed, in our
Moreover, we observed a significantly induced cell cycle arrest at G0/G1 phase (Figure
We highly suspect that the highly activated antioxidant response may play a vital role in the process avoiding the BCa cells with
For further discussion, it is reported that the overexpression of Thromboxane-A2 isoform-
Our study for the first time suggested that
All the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The excellent technical assistance of Yuan Zhu, Shanshan Zhang, and Danni Shan is gratefully acknowledged. The authors would like to thank Dr. Adam J. Hsu at Mayo Clinic, Rochester, USA, for critical reading and editing this manuscript. They also would like to acknowledge the KEGG database developed by Kanehisa Laboratories for the original source of KEGG pathway images “FOXO signaling pathway (map04068).” This study was supported in part by grants from the Natural Sciences Foundation of Hubei Province (Grant no. 42014CFA006), Medical Science and Technology Project of Zhejiang Province (Grant no. 2016KYB082), and National Natural Science Foundation of China (Grant no. 81300578).