The Mechanism In Circadian Rhythm Protein PER1 Inhibiting Proliferation Of Breast Cancer Cells

As the focus protein in this paper,PERIOD 1(PER1)is one of the circadian rhythm protein family members.Its rhythmic expression exhibiting both in cell and individual level plays an important role in molecular circadian oscillation and exerts its extensive functions in regulating melanin retention,blood pressure.Additionally,PER1 is closely related to oncogenesis of breast cancer evidenced by that PER1 is deregulated in breast cancer cells.Prior researches observed that the inhibited tumor cell proliferation and cell cycle arrestted at G0/G1 phase in breast cancer cells overexpressed with PER1.Nevertheless,the underlying molecular mechanism remains to be elucidated.The well-known tumor suppressor gene p53 is one of the genes that are highly related to human cancer.The loss,mutant,recombination of p53 gene are found in more than half kinds of human cancer.p53,modulated by multiple intracellular and extracellular factors,plays significant roles in maintaining genome stability,cell growth,cell cycle etc.Particularly,p53 protein enhances p21WAF1/CIP1 expression.p21WAF1/CIP1 inhibits CDK2/cyclinE activity and subsequently arrests cell cycle at G0/G1 phase.We started this research to explore the molecular mechanism underlying in PER1 inhibiting breast cancer cell proliferation.First,PER1 overexpressed in MCF-7,ZR-75-30 cells inhibited cell proliferation and arrested cell cycle at G0/G1 phase in accordance with previous studies.Secondly,PER1 overexpression enhanced p21-Luc activity in HeLa,MCF-7,ZR-75-30 cells with simultaneous p53 overexpression.Then p21 mRNA level was found upregulated in T47 D cells co-transfected with the Flag-Per1 and Flag-p53 and in ZR-75-30 cells solely transfected with PER1 expressing plasmids.Interestingly,this enhancement of p21-Luc activity disappeared in MCF-7,ZR-75-30 cells that co-transfected with p53 siRNAs and Flag-tagged Per1.These results implied that activation of p21 transcription by PER1 is in p53-dependent manner.Lastly,the interaction between exogenous PER1 and p53 was detected by CO-Immunoprecipitation(CO-IP)performed in HEK-293 T cells and mammalian two hybrid system performed in T47 D,MCF-7cells.Next,in MCF-7 cells,we confirmed interaction and co-localization of endogenous PER1 and p53 in MCF-7 cells by CO-IP and double-label immunefluorescence.Later,HEK-293 T cells were transfected with EGFP-tagged p53 together with Flag-tagged full-length PER1 or truncate PER1.It appears that PER1 interacted with p53 through its PAS domain.Finally,we determined that interaction between PER1 and p53 has no effect on p53 protein level.This section demonstrated that PER1 interacts with p53 to activate p21 transcription upregulating p21 mRNA level.Moreover,this interaction doesn't influence p53 protein level.Our research illustrates the possible molecular mechanism through which PER1 inhibits breast cancer cell proliferation and arrests cell cycle at G0/G1 phase.PER1 associates with p53 through its PAS domain and thereby activates p21 transcription.The increased p21WAF1/CIP1 protein level induced by p21 transcription supresses CDK2/cyclin E complex activity,further retarding cell cycle at G0/G1 phase.This provides new insights for the molecular explanation of circadian rhythm regulation in breast cancer oncogenesis and functional links between circadian rhythm and cell cycle.