Loss Of The Clock Gene Per1 Promomotes Oral Squamous Cell Carcinoma Progression By Inhibiting Cellautophagic Apoptosis And Enhancing Proliferation Via The AKT/mTOR Pathway

Background: Current studies have shown that the clock gene Per1 is downregulated in various tumors and plays an important role in promoting tumor progression.However,the biological functions and mechanism of Per1 in tumors remain largely unknown.Objection: To study the expression level and the clinical significance of Per1 in oral squamous cell carcinoma(OSCC)and verify the biological function of Per1 in vivo and in vitro.Methods: Eighty-six specimens of OSCC tissues and adjacent noncancerous tissues with complete clinical data and follow-up data were collected.The expression of Per1 in 86 specimens of OSCC was detected by immunohistochemistry.Kaplan-Meier survival analysis and log rank test were used to analyze the effect of different expression of Per1 on the prognosis of OSCC patients.Chi-Square test was used to analyze thecorrelation between the expression of Per1 and clinicopathological parameters of OSCC patients.Per1 overexpression lentivirus and Per1-shRNA lentivirus were used to overexpress or knock down Per1 in OSCC cells respectively,the formation of autophagosomes was observed under electron microscope,MTT assay and CCK8 assay were used to detect cell proliferation,Tunel assay and flow cytometry were used to detect apoptosis,and western blotting was used to detect the expressions of p-mTOR?mTOR?p-AKT?AKT?LC3B?Beclin1?P62 protein expression.autophagy inhibitor Autophinib and AKT activator SC79 were added to SCC15 cells with Per1 overexpression for recovery experiments.Lysosomal inhibitor chloroquine(CQ)was added to Per1 overexpressing SCC15 cells,and LC3 BII and P62 protein expressions were detected by western blotting.Per1 overexpressing SCC15 cells were used for in vivo tumor formation experiments,the volume,weight and growth rate of tumors were measured.RT-qPCR was used to detect the mRNA expression of BAX?LC3B and Ki67 in tumor tissues.Western blotting was used to detect the proteins expression of p-mTOR?mTOR?p-AKT?AKT?BAX?LC3B?P62 and Ki67.Results: Compared with normal oral mucosa cells and adjacent normal tissues,the expression of Per1 in OSCC cells and OSCC tissues decreased significantly(P<0.05).The expression level of Per1 was significantly correlated with the survival time,cervical lymph nodemetastasis,tumor size and TNM clinical stage in OSCC patients(P<0.05).Per1 overexpression in OSCC SCC15 cells(Per1-OE SCC15 cells)significantly promoted cell apoptosis and autophagy while inhibited cell proliferation(p<0.05),meanwhile,the protein expression level of P62?p-mTOR and p-AKT decreased significantly(P<0.05),and the ratio of LC3B?/LC3B? and Beclin1 expression level increased significantly(P<0.05).However,the results obtained in Per1-silenced OSCC TSCCA cells were opposite.The autophagy inhibitor autophinib and AKT activator SC79 were added to Per1-OE SCC15 cells respectively,it was found that the increased apoptosis decreased significantly with the addition of autophagy inhibitor(p<0.05),and the increased apoptosis and autophagy decreased significantly with the addition of AKT activator(p<0.05).The expression of LC3 BII and P62 in Per1-OE SCC15 cells increased significantly after addition of lysosomal inhibitor chloroquine(CQ)(p<0.05).In addition,tumor formation experiments in nude mice demonstrated that overexpression of Per1 could inhibit the growth of OSCC tumors.Conclusions: Our results demonstrate that the low expression of circadian clock gene Per1 promotes cell proliferation and inhibits autophagic apoptosis by activating the AKT/mTOR pathway to promote the development of OSCC.Per1 may be a valuable therapeutic target for OSCC patients.