THE MECHANISM OF WWP1 GENE IN ORAL SQUAMOUS CELL
CARCINOMA
古乃威 NAI-WEI KU
1#, 林如華 JU-HWA LIN
1*1DEPARTMENT OF BIOLOGICAL SCIENCE AND TECHNOLOGY, CHINA MEDICAL UNIVERSITY, TAICHUNG, TAIWAN
Abstract
The mortality of oral squamous cell carcinoma (OSCC) is one of the ten leading causes of cancer deaths in Taiwan. Environmental carcinogens such as betel quid chewing, tobacco smoking and alcohol drinking have been identified as major risk factors for OSCC. Our laboratory has found that WW domain containing E3 ubiquitin protein ligase 1 (WWP1)
overexpressed in OSCC due to gene amplification. WWP1 belongs to the C2-WW-HECT type E3 family, and the involvement of the HECT-type E3s in crucial signaling pathways implicates in tumorigenesis. The amplification and overexpression of WWP1 was also found in prostate cancer and breast cancer. Knockdown of WWP1 suppressed cell proliferation and induced apoptosis. These finding suggest an oncogenic role of WWP1 in carcinogenesis. In this study, we established the LacO/IPTG controlled shRNA inducible cell system to investigate the functional roles of WWP1 in OSCC. To verify the shRNA effect, we examined WWP1 mRNA and protein expression by real time PCR and western blotting. Inhibition of cell growth was examined by trypan blue counting and MTT assay. The effects on cell cycle phase and apoptosis were determined by flow cytometry. We will prove that
knockdown of WWP1 would cause cell cycle arrest and induce apoptosis in OSCC cell line. These findings suggest that WWP1 could have an oncogenic role in OSCC. Key word: WWP1、shRNA、OSCC
Result
Fig1. WWP1 represented DNA copy number amplification and mRNA overexpression in clinical OSCC samples.(A) 23 of 59 (39%) OSCC
patients represented WWP1 DNA amplification.(B) 16 of 46 (35%) OSCC patients represented WWP1 mRNA overexpression.
Fig2. Identifying WWP1 DNA copy number change and WWP1 mRNA expression for OSCC cell lines by Q-PCR.
Fig3. Knockdown of WWP1 by shRNA.
Fig4. MTT assay. Interference of WWP1 expression suppresses the cell growth of OECM-1 and TW206.
Fig5. WWP1 knockdown induces apoptosis in OECM-1 and TW206 oral cancer cells.
Fig6. The effects of WWP1 Knockdown on the apoptosis-associated proteins in OECM-1 cells.
Fig7. Cloning shRNA sequence of interest into pLAS1.3xLacO .
Fig8.The effects of WWP1 knockdown on the viability from OECM-1 cells by IPTG inducing.
Inducible system establishment
DNA copy number mRNA level
Table1.Knockdown information. OECM-1 2.0% 9.3% 2.2% control WWP1sh3 WWP1sh 5 3.2% 10.4% 0.3% TW206 control WWP1sh3 WWP1sh5 2.8% 13.6% 7.3% 1.5% 9.3% 4.9% β-actin- 1.00 1.22 1.37 0.88 1.38 1.00 1.09 0.96 0.89 1.13 Caspase 3- Caspase 7- 1.00 0.88 0.78 1.01 0.61 1.00 0.84 0.91 0.67 0.85 -total -CL Caspase 8- 1.00 0.97 1.24 0.70 1.07 -CL -total -CL -total 1.00 0.72 1.00 0.56 0.86 Caspase 9- P53- PARP- -CL -total -CL -total 1.00 0.83 0.84 0.77 0.64 1.00 1.69 1.73 1.17 1.53 1.00 0.23 0.27 0.22 0.26 1.00 1.80 2.10 0.73 1.05 1.00 0.15 0.12 0.10 0.09
