Role And Mechanism Of The Core Circadian Component BMAL1 In Pancreatic Cancer Growth And Progression

Background and ObjectiveThe circadian clock is an endogenous time-keeping as well as homeostasis-regulating system which exists in almost all living things.The circadian clock functions at the molecular,cellular and organic levels to modulate various biological and biochemical processes,and also it oscillates around 24 h to couple individual activities and behaviors.This strictly hierarchical clockwork includes a master clock that resides in the suprachiasmatic nucleus of the brain and multiple peripheral tissue oscillators.Molecularly,circadian oscillation is controlled by a series of interlocking transcription-translation feedback loops formed by certain circadian genes.Recently,more and more evidence has implicated circadian disruption/abnormality in a number of human diseases,including tumors.Pancreatic cancer(PC)is a highly lethal malignancy originated from the exocrine pancreas,which is characterized by an extremely poor prognosis.Currently,the dismal management of PC is attributed to its propensity for early local invasion and metastasis,profound resistance to chemotherapy,and a lack of effective diagnostic and therapeutic strategies.Early detection and targeted therapies by specific tumor markers offer the best hope for cure.Thus,a better understanding of the molecular events underlying the tumorigenic process of PC is highlighted for its prevention,diagnosis and treatment.On the basis of the close relationship between circadian disruption and tumor development,we aimed to investigate the role of the circadian clock,especially the core circadian gene BMAL1,in the formation and progression of PC.This study would provide new insights into the pathogenesis of human PC,as well as potential diagnostic,prognostic,and therapeutic targets for PC.Methods1.To study the expressin of core circadian components in PC and normal pancreatic tissues,we performed RT-PCR and immunochemistry analyses in surgically resected samples from patients.Bioinformatics analysis was also used to explore the circadian gene expressions in three independent GEO PC cohorts.2.To study the role of the core circadian gene BMAL1 in PC,BMAL1 stably knockdown/overexpressed human PC cell lines were established.Then High-throughput RNA-sequencing was used to determine the trancriptome change in Bx PC-3 cells after BMAL1 knockdown.In vitro studies including cell proliferation assay,colony formation assay,cell invasion assay,and flow cytometry analyses for apoptosis and cell cycle were performed.Subcutaneous tumor xenografts in nude mice were used for in vivo verification.Finally,the molecular mechanism by which BMAL1 regulates PC tumorigenesis was explored.3.To study the potential clinical significane of BMAL1,we correlated the BMAL1 expression level with clinicopathological features of PC patients;while the clinical data from the TCGA database was used to analyze the association between BMAL1 expression and patients' survival times.Results1.By analyzing four independent PC cohorts,we found that the expressions of core circadian genes including BMAL1,PER1,PER2,CRY1,CRY2,and RORA were all significantly downregulated in PC tissues compared with normal pancreatic tissues.Immunohistochemistry analysis confirmed that the protein levels of BMAL1,PER1,CRY1,and RORA were decresed in PC.2.RNA-seq analysis s detected an anti-apoptotic and pro-proliferative transcriptome profile after BMAL1 knockdown in Bx PC-3 cells.Systematic GO and pathway analyses revealed that the upregulated genes were mostly enriched in cell division-and cell cycle-related signaling;whereas the apoptotic signaling was suppressed.Meanwhile,the p53 signaling pathway,TNF signaling pathway,and apoptosis pathway were also downregulated by BMAL1 silencing.3.In vitro studies showed that BMAL1 upregulation significantly inhibited PC cell proliferation and invasion,and induced G2/M cell cycle arrest;whereas BMAL1 knockdown promoted PC growth,as demonstrated in Bmal1-manipulated As PC-1 and Bx PC-3 cell lines.4.Xenografts models confirmed that BMAL1 also held anti-oncogene functions in vivo.5.Mechanistic studies revealed that BMAL1 could directly bind to the p53 gene promoter(-515/-346 region)and thereby transcriptionally activate the downstream tumor suppressor pathway,leading to apoptosis and cell cycle arrest in a p53-dependent manner.6.Clinical investigation indicated that low BMAl1 expression was closely correlated with invasion depth,lymphatic metastasis,clinical stage,and histological grade of PC.Survival analysis using TCGA data showed that low BMAl1 level predicted poor cumulative survival times.ConclusionCicadian disruption is closely involved in human PC tumorigenesis.The core circadian component BMAL1 serves as an anti-oncogene by inhibiting PC cell proliferation and invasion,and promoting cell apoptosis.BMAL1 expression correlated with clinicopathological features and overall survival outcomes,and represented a predictive and progostic factor for patients with PC.