The Important Tumor Suppressor Role Of Per1 In Regulating The Cyclins-CDKs-CKIs Network In SCC15 Human Oral Squamous Cell Carcinoma Cells

Objective: To determine the regulatory effect of the circadian clock gene Per1 on the cell cycle related genes, and the influence to cell proliferation, apoptosis, cell cycle and tumorigenicity in vivo in human oral squamous cell carcinoma SCC15 cells.Methods: Three groups short hairpin RNA(shRNA) of lentivirus recombinant plasmids were designed to against the RNA of Per1 gene, and then transfected the SCC15 cells. The best interference group was screen out as the experimental group with detection of Western-blot and real-time quantitative PCR(QRT-PCR). The transfected lentivirus plasmid which had no interference effect to any gene was set as the control group(Control-shRNA). The SCC15 cells without making any treatment was set as the blank group. The mRNA expressions of cell cycle-related genes p53, CyclinD1, CyclinE, CyclinA2, CyclinB1, CDK1, CDK2, CDK4, CDK6, p16, p21, Wee1, cdc25, E2 F, Rb1 in each group of cells were detected by QRT-PCR. Cell proliferation, apoptosis and cell cycle distribution in each group were detected using flow cytometry. The cells of experimental group and blank group were subcutaneously inoculated into nude mice to observe tumorigenesis, respectively.Results: Three groups of Per1-shRNA lentivirus plasmids were constructed successfully. The Per1-shRNA-I group was identified to have the best interference effect by RT-PCR and Western-blot analysis, set as the experimental group. The mRNA expression levels of CyclinD1, CyclinE, CyclinB1, CDK1 and Wee1 gene in Per1-shRNA-I group were significantly higher than Control-shRNA group and SCC15 group(P<0.05), but the mRNA expression levels of p53, CyclinA2, p16, p21 and cdc25 were significantly lower(P<0.05), the mRNA expression level of each gene between the Control-shRNA group and SCC15 group had no statistically difference(P>0.05). The mRNA expression level of CDK2、CDK4、CDK6、E2F and Rb1 had no statistically difference in three groups(P>0.05). Compared with the Control-shRNA group and SCC15 group, the proliferation index was significantly higher in Per1-shRNA-I group(P<0.05), the apoptosis index was significantly lower(P<0.05), the number of cells in S phase was significantly decreased(P<0.05), the number of cells in G2/M phase was significantly increased(P<0.05), while the proliferation index, apoptotic index and cell cycle distribution had no difference in Control-shRNA group and SCC15 group(P>0.05). The tumorigenic ability in vivo was significantly enhanced in Per1-shRNA-I group(P<0.05).Conclusion: The circadian clock gene Per1 was an important tumor suppressor gene. Perl could regulate a large number of downstream cell cycle genes. The alteration of its expression could affect the cell cycle progression, proliferation and apoptosis imbalance and the tumorigenic ability in vivo. Depth study of Per1 may further illuminate the mechanism of the initiation and development of cancer, to provide a new effective molecular target for cancer treatment.