The Mechanism In Circadian Rhythm Protein NPAS2 Inhibiting Proliferation Of Breast Cancer Cells

NPAS2 studied in the paper is a member of circadian rhythm protein family.Emerging data have demonstrated that NPAS2 has a substantial impact on the development of breast cancer,possibly through regulation and interfere of the expression of cancer-related genes,involved in cell cycle checkpoint and DNA repair and reply.We started this research to explore the molecular mechanism underlying in NPAS2 inhibiting breast cancer cell proliferation.First,NPAS2 overexpressed in MCF-7,ZR-75-30,T47 D cells inhibited cell proliferation and arrested cell cycle at G0/G1 phase.Secondly,the overexpression of NPAS2 inhibited CyclinD1-Luc activity in HeLa,MCF-7 cells in a depend on ER? manner.What's more,NPAS2 observily down-regulated the activity of ERE-Luc with simultaneous ER? overexpression.Quantitative Real-time PCR results showed that ER?'s downstream targets CyclinD1,pS2 and c-myc mRNA level was down-regulated by NPAS2 overexpression in MCF-7 cells.Besides,the protein level cyclinD1 and E2 F induced G0/G1 cell cycle arrest was down-regulated by overexpression of NPAS2 in MCF-7 cell.Next,the interaction between exogenous NPAS2 and ER? was detected by CO-Immunoprecipitation(CO-IP)performed in HEK-293 T cells.We certified interaction and co-localization of endogenous NPAS2 and ER? in MCF-7 cells by CO-IP and double-label immunefluorescence.Later,HEK-293 T cells were transfected with HA-NPAS2 together with Flag-tagged full-length ER? or truncate ER?.It appears that ER? interacted with NPAS2 through its LBD and DBD domain.Finally,we confirmed that interaction between NPAS2 and ER? has no effect on ER? protein level.Lastly,Co-immnoprecipitation experiments displayed that exogenous NPAS2 interact with A1B1 in HEK-293 T cell.CO-IP shown NPAS2,AIB1,ER? can interact each other forming three element complex in MCF-7 cell.Moreover,the direct interaction of ER? and AIB1 was inhibited in the overexpression of NPAS2 using mammaliain two hybrid system.and CO-IP assay.Besides,we also demonstrated that NPAS2 inhibits the rhythmic expression of AIB1 in transcriptional level by quantitative real-time PCR.This section display NPAS2 inhibits ER? function by inhibiting the interaction between AIB1 and ER? in the ER?-positive breast cancer cells.Besides,overexpression of NPAS2 inhibit the rhythmic expression and rhythm amplitude of AIB1,then decreasing ER? transcriptional activity.In this study,we shown NPAS2 inhibit the proliferation of breast cancer cell and induce G0/G1 arrest via modulating ER? transcriptional activity and downregulating expression of CyclinD1 and E2 F which induced G0/G1 arrested.What's more,we demonstrated the molecular mechanism of NPAS2 inhibited ER? function by inhibiting the interaction between AIB1 and ER?,which leading to the decreasing transcriptional activity of ER?.These findings not only support the concept that NPAS2 plays a tumor-suppressive role in breast cancer,but also provide a mechanism for how NPAS2 functions as a tumor suppressor in breast cancer.Moreover,this study provids a new molecular mechanism for understanding circadian rhythm protein function in the occurrence and development of breast cancer...