br Dys regulated cell cycle progression is an
Dys-regulated cell cycle progression is an important mechanism of Okadaic acid cancer development (15). CDKN1A-interacting zinc finger protein 1 (CIZ1) is primarily identified as regulator of G1/S cell cycle (16-18). It functions as a nuclear protein that directly regulates the intracellular localization of CDKN1A (17). It has also been found to play an important role in regulating apoptosis and cell cycle by interacting with PDRG1, a DNA damage-regulated protein (19). Aberrant expression of CIZ1 is observed in various cancers, including prostate cancer (20) and colon cancer (21). However, a previous study found that CIZ1 knockout mice was more sensitive to oncogene-induced cellular transformation (22).CIZ1 deletion mice developed different types of leukemia after retroviral insertion-mediated mutations (22). Still, the precise role of CIZ1 in BC is largely undetermined.
In this study, we identified CIZ1 as a potential oncogene in BC. CIZ1 was up-regulated in BC tissues and cells. Knockdown of CIZ1 resulted in suppressed proliferation and growth of BC cells. Apoptosis was induced after CIZ1 silencing in BC cells. In addition, apoptosis and cell cycle associated genes were dys-regulated after CIZ1 knockdown in BC cells.
2. Materials and Methods 2.1 Patient information
All the 20 bladder cancer patients were undergoing radical cystectomy or transurethral resection at the Affiliated Hospital of Inner Mongolia Medical University from 2016 to 2017. The patients were pathologically diagnosed as bladder urothelial carcinoma. The adjacent normal tissues, which were collected from the 6 cases among the 20 patients, were obtained from the tissues more than 2.0 cm away from the cancer areas. Fresh samples were fixed in 4% paraformaldehyde for more than 24 hours and then subjected to immunohistochemistry analysis. The information of the patients were presented in Table 1. The study was approved by the Ethics Committee of The Affiliated Hospital of Inner Mongolia Medical University. A written informed consent was obtained from all patients.
2.2 Immunohistochemical analysis of human bladder cancer and normal bladder tissues
Human bladder cancer or normal bladder tissues were fixed in 4% formalin. 5μm of paraffin-embedded (FFPE) were deparaffinized in xylene and hydrated in graded alcohol and were then subjected to immunohistochemical analysis of CIZ1. Citrate buffer (pH 6.0) was used for antigen retrieval. After blocking endogenous peroxidase with 3% hydrogen peroxide, the slides were incubated with 10% goat serum for 60 min. The sections were incubated with primary CIZ1 antibody at 4°C overnight and then were incubated with secondary antibodies at room temperature for 30min. AEC was used as chromogens. Antibody against CIZ1 was purchased from Atlas Antibodies (HPA020387). Zeiss microscope was used for photographing the staining.
Human Bladder cancer cells J28, T24, EJ and 5637, and Bladder epithelial cells SV-HUC-1 were obtained from ATCC. All the cells were cultured in Dulbecco modified Eagle’s medium (DMEM, Hyclone), supplemented with 10% fetal bovine serum (Corning) and 1% penicillin and streptomycin solution (Corning), at 37℃ containing 5% CO2.
2.4 Packaging of shCIZ1 lentivirus
Lentivirus system which is composed of pGCSIL-GFP, pHelper1.0 and Helper2.0 was used to knock down CIZ1 in T24 or 5637 cells. shRNA targeting sequences are as follow:shCIZ1,
5’-ACTTAGTGCTGCAACAGAA-3’, and the control shRNA, 5’-TTCTCCGAACGTGTCACGT-3’. Indicated shRNA was cloned into the pGCSIL-GFP vector and transfected into 293T cells with Lipofectamine TM 2000 (Invitrogen, Shanghai, China), accompanied with pHelper1.0 and Helper2.0. 48h later, viral supernatants were collected and filtered through a 0.45µm filter. The viral supernatants were subjected to infectingT24 or 5637 cells. The infection efficiency was detected by western blot and qRT-PCR assays
2.5 Total RNA isolation and quantitative real-time PCR
Total RNA was isolated from Bladder cancer and epithelial cells using Trizol reagent (Invitrogen) and Ultrapure RNA Kit (CWBIO, Beijing, China), following the manufacturer’s protocols. 1 μg of total RNA was subjected to reverse transcription using M-MLV reverse transcriptase (Promega). Quantification of the transcripts was analyzed by quantitative real-time PCR using SYBR master mixture (TransGen) on an IQ5 machine.
(Beyotime). The concentration of total protein was determined by BCA protein assay kit
sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then the protein on the gel was