Identification Of Circadian Rhythm-related Subtypes,the Development Of A Prognosis Model,and The Mechanism Of Melatonin Inhibition Of Colon Cancer Growth And Metastasis

Objective:Colorectal cancer has become the most prevalent malignancy of the digestive system today,with its high incidence,difficulty in early diagnosis and treatment,and ease of recurrence and metastasis after surgery,posing a great danger to human health,while also raising the economic burden on the public health system in China and the world at large.Worldwide,colorectal cancer has the third highest incidence rate among various malignancies and is the second leading cause of cancer-related deaths.In recent years,the incidence of colorectal cancer has been increasing in China,and most patients presenting with symptoms and signs of colorectal cancer are diagnosed with distant metastases and have poor prognoses.Therefore,it is important to explore the causes and pathogenesis of colorectal cancer in depth in order to prolong patient survival and reduce tumor recurrence.Circadian rhythm disorders are associated with a variety of human diseases,and a growing number of studies have found a strong correlation between circadian rhythm and the development of cancer.However,the specific mechanism of action of circadian rhythm disorders in regulating colon carcinogenesis and progression remains unclear.In this study,we used a bioinformatics approach to identify circadian rhythm-associated subtypes of colon cancer,explore the effects of circadian rhythm-related genes on the tumor microenvironment of colon cancer,construct a circadian rhythm-associated prognostic model,and validate its predictive ability for recurrence-free survival and the benefit of receiving immunotherapy in colon cancer patients.In addition,we also investigated the effect of melatonin,which can maintain circadian rhythms,on the growth and metastasis of colon cancer cells.Methods:In this study,we first performed the consensus clustering analysis of colon cancer patients downloaded from public databases based on 23 circadian rhythm-related genes to determine the expression of circadian rhythm-associated genes and different circadian rhythm-associated subtypes in colon cancer.The ss GSEA,CIBERSORT,and MCPcounter algorithms were used to quantify the relative infiltration levels of cells in the tumor microenvironment.The prognostic model CRscore was constructed by identifying differential genes between different subtypes to assess the circadian rhythm-related modification patterns of individual tumors.Differential expression of the circadian rhythm core gene BMAL1 in colon cancer and paracancerous tissues was detected by RT-q PCR.Then CCK8,Ed U assay,cell cycle and apoptosis,and scratch healing assay were applied to detect the effects of melatonin on proliferation,apoptosis,and migration of colon cancer cells.Finally,the changes in related protein expression after melatonin treatment of colon cancer cells were analyzed by flow cytometry.Results:1.To identify circadian rhythm-associated subtypes of colon cancer,23 circadian rhythm-related genes were first retrieved from the review.Gene expression data from TCGA and GEO database sources were then combined by the Combat algorithm to cancel batch effects and integrate clinical information as well.Circadian rhythm-related genes have somatic mutations and copy number variation in colon cancer and are differentially expressed in colon cancer and paracancerous tissues.Reduced expression of BMAL1,a core circadian gene,in colon cancer was demonstrated by RT-q PCR experiments.2.All colon cancer samples were clustered into two subtypes,CRcluster A and CRcluster B,using consensus clustering analysis.The prognosis of the two subtypes was significantly different by survival analysis,with the CRcluster A subtype having a longer recurrence-free survival.The results of GSVA enrichment analysis indicated that CRcluster B was significantly enriched in tumorigenesis development-related signaling pathways and stromal-related pathways were also significantly enriched in this cluster.The next immune infiltration analysis suggested differences in immune cell infiltration between subtypes.The CRcluster B subtype intermediated stromal score was higher by ESTIMATE analysis.The above results suggested that the poor prognosis of the CRcluster B subtype may be related to the immunosuppressive microenvironment.Next,with the LIMMA package we identified 45 genes that were differentially expressed between the two clusters and performed functional enrichment analysis,showing that these differentially expressed genes were significantly enriched in extracellular matrix-associated biological processes and tumor-associated pathways.Using univariate Cox regression analysis we identified 37 prognosis-related genes.Based on the differentially expressed genes associated with prognosis,we constructed the prognostic model CRscore.Survival analysis indicated that patients in the CRscore-Low subgroup had longer recurrence-free survival.By combining the characteristics of CRscore molecules and immunity,we suggested that patients in the CRscore-Low subgroup may gain more benefits by receiving immunotherapy.3.The results of the CCK8 cell proliferation assay and Ed U assay demonstrated that melatonin could inhibit the proliferation of colon cancer cells HCT116 and SW480;flow cytometry detected that melatonin promoted the apoptosis of colon cancer cells and kept more cells in the G1 phase thus inhibiting cell proliferation and promoting apoptosis.4.The results of the scratch assay and EMT-related protein assay indicated that melatonin could inhibit the migration of colon cancer cells.Conclusion:1.Circadian rhythms play a non-negligible role in the formation of the diversity and complexity of the immune microenvironment in colon cancer.2.Assessing the circadian modification patterns of individual tumors will help improve our understanding of tumor microenvironment characteristics and facilitate more effective immunotherapeutic strategies.3.The circadian rhythm-related prognostic model CRscore has good predictive value for colon cancer patients in terms of prognosis and immunotherapy outcome.4.Melatonin inhibits the growth and metastasis of colon cancer cells HCT116 and SW480.