Influence And Mechanism Of Circadian Clock Gene CRY1 On The Biological Behavior Of Glioma

Objective:The human body follows a certain circadian rhythm for life,work,learning and other normal physiological activities,this rhythm is called the body’s biological clock.New evidence suggests that changes in circadian rhythms are closely related to tumorigenesis and development,and that chronospecificity of gene phenotypes may be used as a strategy to influence tumor growth and improve therapeutic efficacy.Based on previous studies,we found that CRY1(Cryptochrome Circadian Regulator 1)is a key circadian clock gene,and its dysregulation is beneficial to tumor development.Therefore,it is urgent to investigate the effect and mechanism of CRY1 gene on the biological behavior of glioma,which plays an important role in guiding clinical treatment.Materials and Methods:Gene Expression data were downloaded from GEO(Gene Expression Omnibus data base)and TCGA(The Cancer Genome Atlas)databases to analyze CRY1 Gene Expression in glioma.Survival was analyzed by Kapian-Meier analysis and Cox proportional hazards regression model.c Bio Portal cancer genome website(https://www.c Bio Portal.org/),for CRY1 mutation frequency,mutation type and location information,kaplan-meier Kaplan-Elana Meyer curve and log-rank log-rank test were used to explore the overall,disease-specific,disease-free and progression-free survival prognostic curves of CRY1 mutations.Minimal absolute contraction and selection operator(LASSO)regression was used to screen the most survival-related immune cells.Wilcoxon rank sum test was used to analyze the levels of CRY1 gene mutation and immune cell infiltration without CRY1 mutation.GSEA gene probe enrichment analysis of glioma-related biological function of co-expression of signaling pathways.RNA-Seq analysis confirmed the previous results.Two cell lines,U118 and U251,were stably transfected with the lentivirus,and the CRY1 overexpression and interference model was constructed.The transfection effect was observed by inverted Fluorescence microscope,the m RNA and protein expression levels were detected by real-time fluorescence quantitative PCR(q RT-PCR)and Western Blot.The effect of CRY1 expression on the proliferation of U118 and U251 glioma cells was examined by plate cloning and CCK-8.The effect of CRY1 expression on the migration of U118 and U251 glioma cells was examined by scratch test.The effect of CRY1 expression on the invasion of U118 and U251 glioma cells was examined by Transwell assay.The effect of CRY1 on the cell cycle of U118 and U251 glioma cells was examined by flow cytometry.Annexin V-FITC method was used to examine the effect of CRY1 expression on the apoptosis of U118 and U251 glioma cells.The transfected cells were inoculated subcutaneously into nude mice,the size and weight of the tumor were observed and measured regularly,and the tumor was removed and paraffin-embedded and made into sections,the expression of proliferation index Ki-67 was detected by immunohistochemistry.Results:The circadian clock gene CRY1 is abundant in gliomas,and the high expression of CRY1 is associated with poor prognosis and poor pathological findings.GSEA(Gene Set Enrichment Analysis)software was used to screen out seven signaling pathways related to glioma,which were involved in up-regulation of tumorigenesis,by studying the mutation frequency,mutation type and locus information of CRY1 gene in TCGA database,we found that the type of CRY1 gene mutation was associated with good survival prognosis,the bioinformatics method examined the association between CRY1 and several immune-related pathways.Transcriptome RNA-seq sequencing showed that the expression of 131 genes was significantly affected,including 94 up-regulated and 37down-regulated highly differentially expressed genes.KEGG enrichment analysis and GO classification analysis showed that differentially expressed genes were enriched in biological processes,CRY1 is involved in apoptosis,regulation of cellular immunity,anti-virus,enhancement of cytotoxicity,leukocyte chemotaxis and anti-cell proliferation.The periodic expression pattern,m RNA and protein expression levels of CRY1 were affected by the establishment of stable lentivirus model with overexpression and interference.A lot of in vitro experiments have proved that overexpression of CRY1 can inhibit the proliferation,migration,invasion,interfere with the cycle and promote the apoptosis of tumor cells,overexpression of CRY1 can inhibit the growth of glioma.Conclusions:High expression levels of CRY1 are strongly associated with poor prognosis in gliomas,and our results confirm that CRY1 is a novel tumor suppressor gene that may serve as a potential therapeutic target for the study of gliomas,it has potential significance for biomarkers of timing therapy of glioma.