Clock Gene Per2 Regulates Cell Proliferation, Apoptosis And Cell Cycle Progression In Human Oral Squamous Cell Carcinoma

Part one Cell culture of OSCC cell lines and oral mucosa epithelial cellsObjective: Culture oral mucosa epithelial cells and identify the purity of oral mucosa epithelial cells.Methods: Normal oral mucosa was collected from maxillofacial plastic surgery in Affiliated Hospital of Stomatology,Chongqing Medical University approved by the local ethics committee. The mucosa was keeped in 1.0 u/ml dispaseⅡ(Roche, USA) at 4℃ overnight. Complete epithelial layer was separated from mucosa under a microscope. The epithelial layer was digested in 0.25% trip LETM Expressat 37℃, then inculated in 6-well plate paved by rat tail collage using OKM at 37℃ in a humidified atmosphere of 95% air and 5% CO2. The Oral mucosa epithelial cells were continously passaged per 4 days. The third generation of cells was used for making cell climbing slices. Tca8113 cells and SCC15 cells(Chongqing key Laboratory of Oral Diseases and Biomedical Sciences, China) were cultured in RPMI 1640 and DMEM/F16, respectively, with 10% fetal bovine serumat 37℃ in an atmosphere of 95% humidity and 5% CO2. The slices were incubated firstly with the rabbit monoclonal anti-keratin antibody overnight at 4℃, and secondly with the rabbit SP kit for 1 h at 37℃ according to the manufacturer’s recommended protocol. Finally, slices were examined under a microscope.Results: Under microscope oral mucosa epithelial cells had paving stone appearance, and keratin was expressed in 100% of the cells, which identified the purity of oral mucosa epithelial cells.Conclusion: Pury oral mucosa epithelial cells can be harvested by screening without serum and complete epithelial layer separation.Part two The expressions of Per2 in oral mucosa epithelial cells, Tca8113 cells and SCC15 cellsObjective: Detect the expressions of Per2 in oral mucosa epithelial cells, different OSCC cells lines to prepare for further research.Methods: Detect the expressions of Per2 m RNA and protein in oral mucosa epithelial cells, Tca8113 cells and SCC15 cells using QPCR and WB, respectively. The SPSS 17.0 statistical software package was used to anlayse and calculate the mean and SD of the data. And One-way ANOVA was used to analyse the differences between different groups. A value of P<0.05 was considered statistically significant.Results: The expression of Per2 m RNA and protein in oral mucosa epithelial cells, Tca8113 cells and SCC15 cells: In oral mucosa epithelial cells, Tca8113 cells and SCC15 cells, the expression of Per2 m RNA was 2.41±0.21, 1.00±0.12 and 0.9±0.17 respectively; and the expression of Per2 protein was 2.87±0.26, 1.11±0.13 and 0.98±0.32, respectively.Conclusion: The expression of Per2 m RNA and protein in Tca8113 and SCC15 were significantly lower than those of oral mucosa epithelial cells(P<0.05), indicating Per2 expression is reduced in OSCC.Part three Down regulation of Per2 in Tca8113 cells by sh RNA plasmids.Objective: Down regulate Per2 in oral squamous cell carcinoma(OSCC) cells line Tca8113 cells using sh RNA, and then detected m RNA and protein expression of Per2. Set up Per2 interference model to prepare for further research.Methods: The plasmids p GPU6-Per2-sh RNA-I ~ III and p GPU6-Control-sh RNA were bought from Chengdu Biotechnology Co.,Ltd. Plasmids were transfected using Lipo2000 and OPI-MEM. The effect of Per2 down regulation was examined 36~72 h later. There were five groups in our experiment, these are Per2-sh RNA-I, Per2-sh RNA-II, Per2-sh RNA-II, control-sh RNA and Tca8113 group. Per2-sh RNA-I, Per2-sh RNA-II, Per2-sh RNA-III and control-sh RNA group were transfected with p GPU6-Per2-sh RNA-I, p GPU6- Per2-sh RNA-II, p GPU6-Per2-sh RNA-III and p GPU6-Control-sh RNA, respectively; and Tca8113 group didn’t accept any reagents. Detect the expressions of Per2 m RNA and protein in different groups using QPCR and WB, respectively. The SPSS 17.0 statistical software package was used to anlayse and calculate the mean and SD of the data. And One-way ANOVA was used to analyse the differences between the various groups transfected with different plasmids. A value of P<0.05 was considered statistically significant.Results: In Tca8113, Control-sh RNA, Per2-sh RNA-I, Per2-sh RNA-II and Per2-sh RNA- III group, the expression of Per2 m RNA was 3.20±0.52, 3.01±0.11, 1.67±0.30, 1.45±0.34, and 1.00±0.13, respectively; and the expression of Per2 protein was 3.21±0.42,3.18±0.52,1.52±0.11, 1.22±0.15 and 0.87±0.21, respectively. Per2 m RNA and protein expression showed no significant difference between Control- sh RNA and Tca8113 group(P>0.05). However, Per2 m RNA and protein expression were significantly lower in Per2-sh RNA-III group than those of Control-sh RNA and Tca8113 group(P<0.05).Conclusion: Per2 was effectively reduced in Per2-sh RNA-III group, and Per2-sh RNA-III group was adopted in subsequent experiment.Part four Per2 downregulation alters cell cycle, cell proliferation and apoptosis and regulates cell cycle Cyclins-CDKs-CKIs network in OSCCObjective: In this study, we down regulated Per2 in OSCC cells line Tca8113 cells, and then detected the alterations of cell cycle, cell proliferation and apoptosis and all the important genes in Cyclins-CDKs(Cyclin-dependent protein kinases)-CKIs(Cyclin-dependent kinase inhibitors) network to further illustrate the relationship of Per2 with the occurrence and development of cancers.Methods: We used sh RNA to down regulate Per2 in OSCC cells line Tca8113 cells, then detected the alterations of cell cycle, cell proliferation and apoptosis by flow cytometric analysis and the m RNA expression alterations of all the important genes( Cyclin A2, Cyclin B1, C-myc, Cyclin D1, Cyclin E, P53,CDK1、CDK2、CDK4、CDK6、Rb1、E2F1、Wee1、cdc25、p16、p21)in Cyclins-CDKs-CKIs cell cycle network by QPCR. The SPSS 17.0 statistical software package was used to anlayse and calculate the mean and SD of the data. And One-way ANOVA was used to analyse the differences between the various groups transfected with different plasmids. A value of P<0.05 was considered statistically significant.Results: Compared with those of Tca8113 and Control-sh RNA group, Per2-sh RNA-III group had a significantly decreased number of cells in G1/G0 phase(P<0.05), significantly increased PI(P<0.05), and significantly decreased AI(P<0.05). Number of cells in G1/G0 phase, PI and AI showed no significant difference between Tca8113 and Control-sh RNA group(P>0.05).Compared with those of Tca8113 and Control-sh RNA group, Per2-sh RNA-III group had a significantly decreased m RNA expression of p53, p16 and p21(P<0.05), significantly increased m RNA expression of Cyclin A2, Cyclin B1, Cyclin D1, CDK4, CDK6, E2F1(P<0.05), and a similar m RNA expression of C-myc, Cyclin E, CDK1, CDK2, cdc25, Wee1, Rb1(P>0.05). Between Tca8113 and Control-sh RNA group, there was no significant difference in m RNA expression of p53, p16, p21 Cyclin A2, Cyclin B1, Cyclin D1, CDK4, CDK6 and E2F1(P>0.05).Conclusion: In Tca8113 cells, Per2 down regulation significantly increased the m RNA expressions of Cyclin A2, Cyclin B1, Cyclin D1, CDK4, CDK6 and E2F1, while significantly decreased the m RNA expressions of p53, p16 and p21. Cell proliferation was significantly higher, apoptosis was significantly lower, and progression of cell cycle was changed. Our study represents the first demonstration that in OSCC clock gene Per2 plays an important role in G1/S checkpoint and the three aspects of Cyclins-CDKs-CKIs network at transcriptional level. On this basis,the further research of Per2 at protein level and modification level after protein translation may further define the interaction of circadian rhythm and cell cycle, and their relationship with the carcinogenesis, which may provide effectively new molecular targets for treatments of cancers.