Circadian Rhythm Gene PER3 Regulates Stemness Of Cancer Stem Cells And Its Underlying Mechanisms

Background & Aims: Malignant tumors have become the second leading cause of death in humans,and patients with advanced malignant tumors often suffer from chemotherapy resistance and relapse,resulting in treatment failure.In this study,two malignant tumors(oral squamous cell carcinoma(OSCC)and prostate cancer(PCa)),which are often resistant to treatment in clinic,were used as the research objects.Molecular mechanistic commonalities and differences of treatment resistance in OSCC and PCa was compared,exploring the source of therapeutic resistance of malignant tumors.Cancer stem cells(CSCs)are a small subset of cells with stem cell characteristics in tumor tissues.Studies have shown that CSCs are closely related to tumor recurrence,metastasis and drug resistance.Circadian rhythm disturbances have been shown to be closely related to various diseases such as cardiovascular diseases,metabolic disorders and tumors.At the same time,circadian rhythm-related genes(including PER3)have been reported to be involved in the occurrence and development of malignant tumors.Tumor chronotherapy designed according to the body’s circadian rhythm has important therapeutic value.However,the circadian properties of CSCs and the molecular mechanism of the circadian rhythm gene PER3 regulated stemness of CSCs are still unclear.Therefore,this study aimed to explore the circadian rhythm characteristics of CSCs and preliminarily elucidate the molecular mechanism of PER3 regulated self-renewal of CSCs,and it is expected to provide fractional supplement for the chronological treatment of malignant tumors.Methods:Fluorescence activated cell sorting(FACS)was used to sort out CD44 and ALDH1 double positive(DP)expression cell subsets and CD44 and ALDH1 double negative(DN)expression cell subsets.The characteristics of CSCs of DP and DN cells were verified by experiments of colony formation,serial spheres formatting,and limiting dilution tumorigenesis.RNA-seq was used to analyze the enrichment of DP and DN cells in chemoresistance,castration resistance,circadian rhythm pathways,and verified by experiments of colony formation,serial spheres formatting,and limiting dilution tumorigenesis under chemotherapy and castration conditions.After lentivirus silencing and overexpression of PER3,the characteristics of CSCs in DP and DN cells were detected.The molecular mechanisms in stemness of CSCs regulation by PER3 via experiments of RNA-seq,RT-q PCR,WB,immunoprecipitation,immunofluorescence,and limiting dilution tumorigenesis.In addition,the expression of PER3 in OSCC and PCa and its relationship with prognosis were analyzed by TCGA database and immunohistochemical assays.Results: 1.Compared to DN cells,DP cells had more prominent CSCs characteristics in vitro and in vivo,and DP cells were more resistant to paclitaxel chemotherapy and castration treatment,and DP cells significantly down-regulated circadian signaling pathway and circadian rhythm gene PER3.2.Circadian rhythm gene PER3 could participate in the stemness regulation of CSCs through Wnt/β-catenin signaling pathway.3.Compared to normal tissues,the expression of PER3 was decreased in OSCC and PCa tumor tissues,and the expression of PER3 was negatively correlated with the clinical prognosis of OSCC and PCa patients.Conclusion: 1.The method of sorting DP and DN cells by FACS can stably enrich CSCs.2.Compared to DN cells,DP cells are more resistant to chemotherapy and castration.3.The circadian rhythm pathway and related genes of DP cells were significantly down-regulated,of which PER3 was the most significantly down-regulated gene.4.PER3 can regulate the self-renewal of CSCs through negative feedback of Wnt/β-catenin signaling pathway.5.PER3 could be used as a prognostic indicator for OSCC and PCa patients.