| Goal:Fam72a（family with sequence similarity 72 member A）is a gene of unknown function,expressing in almost all higher animal cells.Previous studies have shown that the expression level of Fam72a is closely related to tumor development,and bioinformatics analysis shows that Fam72a is highly expressed in tumor cells,and the expression of Fam72a is negatively correlated with the prognosis of tumor patients.This study aims to identify the roles of Fam72a in cell cycle,and to further explore the relationship between Fam72a and cell proliferation and tumorigenesis.In current research,we found that Fam72a could bind to PP2A.Therefore,we aim to further investigate the molecular mechanism and biological significance of the interaction between Fam72a and PP2A.Methods:（Ⅰ）Molecular mechanism of how Fam72a is regulated by cell cycle1.Using double thymidine block,flow cytometry,q RT-PCR and WB to investigate the m RNA and protein levels of Fam72a at different stages of the cell cycle.2.The transcription regulation of Fam72a is detected by using a dual-luciferase reporter gene experiment.3.The post-transcriptional regulation of Fam72a is explored through bioinformatics analysis,in vivo ubiquitination experiment,Flag-pulldown and other methods.（Ⅱ）Molecular mechanism of how Fam72a participating in cell cycle regulation through interaction with PP2A-B561.Using immunofluorescence and laser scanning confocal microscopy to determine the subcellular localization of Fam72a.2.Using Pulldown and immunoprecipitation to verify the interaction between Fam72a and PP2A-B56.3.Using immunofluorescence and WB to investigate the Fam72a regulates Hec1phosphorylation.Results:（Ⅰ）Fam72a is a novel cell cycle-regulated gene1.Fam72a is a periodically expressed gene in the cell cycle.Both the m RNA and protein levels began to increase in S phase,peaked at G2/M phase and declined at the end of M phase.2.Fam72a is transactivated by Foxm1.3.Fam72a is degraded via the ubiquitin-proteasome pathway in an APC/C-Cdh1dependent manner.（Ⅱ）Fam72a interacts with PP2A-B56 affects the function of the mitotic spindle1.Fam72a is required for mitotic spindle function.2.Fam72a interacts with PP2A-B56.3.Fam72a regulates Hec1 phosphorylation and mitotic progression.Conclusion:In this study,we found that Fam72a is a novel cell cycle-regulated gene.We showed that Fam72a is regulated by Foxm1 and APC/C-Cdh1 at the transcriptional and post-transcriptional levels.Functionally,Fam72a associates with the mitotic spindle,stabilizes kinetochore-spindle attachment and maintains chromosome alignment by supporting the PP2A-B56 complex to dephosphorylate Hec1 at the kinetochore-spindle interface. |
