Exploring transcriptomic databases: unraveling circadian gene disruptions in lower grade glioma

The study explored the role of circadian rhythm genes (CRGs) in lower grade glioma (LGG) development and found that certain genes, such as CRY1, NPAS2, and RORB, were associated with increased or decreased risk of LGG. The study also investigated the correlation between CRGs and immune cell infiltration, revealing a negative association with macrophage infiltration and a positive correlation with B cell and CD8 + T cell infiltration. Additionally, the study identified major mutated CRGs, including PER2, BMAL1, CLOCK, and BMAL2, and their potential interaction with other CNS-associated genes. The study suggests that CRGs play a crucial role in immune response and tumorigenesis in LGG patients and warrants further investigation.

the tumor microenvironment.Additionally, the study explored the relationship between CRGs methylation levels and transcriptional regulation.By analyzing DNA methylation and gene expression data from brain tumor samples, we identified potential associations and correlations.To further investigate causal relationships, Mendelian randomization was employed.This allowed us to explore the potential causal effects of multiple genes on brain tumor development and immune cell infiltration.
Overall, our study utilized a multidimensional approach, integrating various datasets and conducting extensive statistical and bioinformatics analyses.By investigating the expression, methylation, and causal effects of CRGs, as well as their correlations with immune cell infiltration, we aimed to provide insights into the complex biology of brain tumors.

Immune cell infiltration levels
It has been discussed that EPIC and QUANTISEQ results should be recommended as a routine computational method used to assess immune cell infiltration in tumors 16 .We selected EPIC results about B cell, CD4 + T cell, CD8 + T cell and Macrophage, QUANTISEQ result from Myeloid dendritic cell and CIBERSORT result from Neutrophil.As shown in Table 1, the expression of most CRGs showed a negative correlation with macrophage infiltration.PER1 showed an opposite trend compared to other CRGs.The expression of most CRGs had a strong positive correlation with B cell and CD8 + T cell infiltration, followed by CD4 + T cells and myeloid dendritic cells.The correlation between the expression of most CRGs and neutrophil infiltration was not significant, except for CLOCK, CRY2, and NR1D1.

CRGs in LGG with gene mutation and correlation
LGG mainly mutated CRGs are PER2, BMAL1, CLOCK, and BMAL2 (Fig. 3).According to the recent study, the significantly mutated genes in LGG are ARIDIA, CIC, EGFR, IDH1, KAT6B, PIK3R1, TP53 and FLG 17 .In differential analysis of CRGs expression between mutated and non-mutated groups of the mutated genes above in LGG (Figure S3), we found that NPAS2 showed significant differential expression between the ARID1A mutated group and the non-mutated group.expression between the TP53 mutated group and the non-mutated group.Also, we discovered EGFR, IDH1 and TP53 mutated groups have more altered CRGs via Venn network analysis (Fig. 4A, B).
Then we did the differential gene analysis in immune related genes among CRGs mutation groups.Differential gene expression analysis revealed significant differences between mutated and non-mutated groups for several immune-related genes (Figure S4).Specifically, AADAT showed significant differential expression in ARNTL2 mutated groups (Wilcoxon, p = 0.026), while AAAS showed significant differential expression in the same group (Wilcoxon, p = 0.044).In CREBBP mutated groups, differential expression was found for APITD1, APOBEC3B, LAMP3, CXCL9, TIGIT, TNFRSF9, GPNMB, MMP9, PHEX, and TAP1.Furthermore, the PER3 mutated group exhibited significant differential expression of NR1D1 (Wilcoxon, p = 0.046), while CREBBP mutated groups showed significant differential expression of ARNTL2 and CRY1.
In the correlation analysis of CRGs, significant associations were identified between different genes (Fig. 5A,  B).Particularly, a strong positive correlation was observed between NPAS2 and ARNTL2.Additionally, a strong positive correlation was also found between CRY2 and NR1D2.On the other hand, a strong negative correlation was observed between NPAS2 and PER3.Similarly, a strong negative correlation was identified between CRY2 and ARNTL2, further highlighting a potential regulatory interaction between these CRGs.

Mendelian randomization (MR) studies
In two-sample MR study (Table 2), CREB-binding protein (CREBBP) has a positive causality with benign CNS tumors (Inverse variance weighted beta = 0.2329, p = 0.01911).Besides, TNF receptor superfamily member 11b (TNFRSF11B) shows weak positive causality with benign CNS tumors as well (Weighted median beta = 0.1243, p = 0.03215).Apolipoprotein B has a negative causality with neutrophil cell count and sleeping disorders including trouble falling or staying asleep, or sleeping too much.GPNMB shows positive causality with CD8 + T cell Absolute Count (Wald ratio beta = 0.2068, p = 0.004226).CXCL9 has weak negative causality with daytime nap meanwhile its reduction can be explained by brain tumor.All single SNP analysis, method comparison plots, leave-one-out analysis and funnel plots are shown in Figure S6.
What's more, we have discovered daytime nap reasons apolipoprotein B and apolipoprotein causes the brain tumor (Table 2).However, there is no causality between daytime nap and brain tumor.Thus, this is the complete mediation case.www.nature.com/scientificreports/ In multivariate MR study (Table 3), we tried to discover caualities between multiple kinds of instruments affected by circadian rhythm and CREBBP.These instruments include: variation in diet, leisure/social activities (Sports club or gym), daytime dozing / sleeping (narcolepsy) and mixture of day and night shifts.No significant results were gained (significance level: 0.05).

Discussion
We mainly explored core CRGs in mRNA expression level, survival analysis, and immune cell infiltration levels.We found out CRY1 is significantly associated with increased risk of LGG.Survival analysis also showed CRY1 caused lower OS and DFS.CRY1 has been found to be pro-tumorigenic by promoting DNA repair and cell survival through temporal transcriptional regulation 18 .CRY1 is hormone-induced in tumors, indicating that circadian hormone might be able to affect LGG prognosis.Also, NPAS2 and RORB were both found to decrease LGG risk.It is suspected that NPAS2 modulates tumorigenesis through the following two aspects: directly carcinogenic mechanism and interaction with other clock proteins 19,20 .Both NPAS2 and RORB also showed lower OS and DFS in survival analysis.However, NPAS2 might have bidirectional effects in different tumors and deserve further studies.Likewise, although NR1D2 and PER3 do not show significant differece between tumor and www.nature.com/scientificreports/normal tissues, they do show significant correlation with survival conditions.It indicates that the mechanism behind how genes influence tumors is complicated and equally-expressed genes deserve more attention in the future studies.
In survival analysis, in ARNTL2 group two KM curves got intersected.From 0 to 8 years, the survival rate was higher when the ARNTL2 expression is higher.It demonstrated that ARNTL2 is anti-tumor during this time.However, after 9 years, it showed the opposite outcome which has been proved to be a potential oncogene, contributing to immunosuppressive tumor microenvironment 21 .Thus, contradictory results shows the value of continuing to dig up how ARNTL2 influences LGG.
In immune cell infiltration analysis, we detected the negative association between macrophage infiltration and most CRGs.As we have known, tumor-associated macrophages facilitate tumor proliferation, survival and migration 22 .However, macrophages in tumor micro-environment are categorized into pro-tumorigenic and antitumorigenic 23 , thus making the mechanism of different CRGs on LGG through macrophage infiltration more complicated.Particularly, the correlation between the PER1 gene and immune cell infiltration often exhibits an opposite direction compared to other genes.The downregulation of PER1 can lead to decreased sensitivity of U343 glioma cells to X-ray irradiation by altering the expression of genes associated with cell cycle arrest and apoptosis, such as c-myc, P53, p21, cdc2 and cyclineB1.This is achieved through the activation of the CHK2-P53 pathway, ultimately resulting in reduced apoptosis 24 .Further studies are needed to figure out how PER1 inhibits tumor growth via affecting different immune cell infiltration.
Most CRGs correlated strongly with B cell and CD8 + T cell infiltration in LGG.This indicates that most CRGs promote or suppress glioma mediated by both immune cells.No significant correlation with neutrophil infiltration except CRY2 and NR1D1 whose high expression has been estimated to high OS and DFS.Altered expression of CRGs including CRY2, PER2 and BMAL1 has been explored to be explained by reduced neutrophil infiltration in liver 25 .We still has no clue how these genes get modulated by neutrophil infiltration in brain which might uncover the specific function of CRY2 and NR1D1 on glioma development.
Major mutated CRGs in LGG are PER2, BMAL1, CLOCK and BMAL2, which might indicate these genes are most influencial CRGs to glioma development or metastasis.Besides, significant expression difference of most CRGs existed in TP53, EGFR and IDH1 mutated groups.This might imply that CRGs greatly influence LGG patients under those mutation background.In addition, within CRGs mutated groups, some CNS-associated genes like AADAT and AAAS showed significant differencial results.AADAT has been firstly investigated in immune response in meningitis patients 26 .AAAS may be involved in normal development of the peripheral and central nervous system [27][28][29] .Also we have discovered AAAS has a significant correlation with many CRGs.There is no clear evidence that both genes are related to LGG but it might hint the interaction between CRGs and these genes thus modulating CNS system via circadian rhythm.
Besides, in mutation differential analysis we have found out many significant results from CREBBP within extended core clock network genes while core CRGs got nothing.CREBBP is a tumor suppressor, whose decreased expression cooperates with the oncogene MYCN to induce malignant brain tumors in mice 30 .In our two-sample MR study, CREBBP affected benign brain tumors.We found many immune-related genes (APITD1 www.nature.com/scientificreports/、APOBEC3B、LAMP3、CXCL9、TIGIT、TNFRSF9、GPNMB、MMP9、PHEX、TAP1) expressed differently between CREBBP-mutated group and non-mutated group.Notably, MMP might be important to cancer cell morphology and angiogenesis with the extracellular matrix 31 .Some of them are even correlated to each other.
In our MR studies, we have found some caualities associated with those immune-related genes.Among them, another TNF receptor superfamily has been proved to cause benign CNS tumors.When TNFRSF is present in a gene collection, it can be a valuable molecular marker, a reliable predictor of survival for GBM patients, and a potential target for cancer immunotherapy 32 .Studies have also shown that immune system matters a lot to tumor development 33,34 .And CXCL9 modulating daytime nap also builds the bridge between immune response and circadian rhythm.Apolipoprotein B can control sleeping disorders and also neutrophil cell count.In mediation effect analysis, we proved apolipoprotein B completely mediates between daytime nap and brain tumor.This signals that this molecule might be necessary target between circadian rhythm disruption and brain tumor.Also GPNMB affects CD8 + T cell.In this way, continued research about APOBEC3B and GPNMB in immune cell infiltration and circadian rhythm should be carried out.These genes might become potential immune targets to LGG.Some CRGs have shown strong positive or negative correlation with other CRGs.Moreover, some CRGs mutations have differential expression of some other CRGs.Especially, the negative correlation between NPAS2 methylation and its transcription levels indicates that the regulation of this gene may be inhibited in LGG.Methylation of NPAS2 may disrupt the normal functioning of circadian rhythm, leading to aberrant timing of  LGG is undoubted and awaits more studies.
Our study does have several limitations.One potential limitation is our research lacks in vitro experiments to support the theoretical research points.We look forward to future studies that can complement and refine our findings.Besides there are studies showing that miRNAs associated with cancer immunity can be a useful prognostic marker, so more non-coding RNA researches are required to improve our results 35,36 .
In summary, CRGs play a crucial role in immune response and tumorigenesis in LGG patients.The bridge between circadian rhythm and glioma is expected to strengthened in the near future.

Figure 1 .
Figure 1.Circadian gene expression in LGG (red boxes) compared to normal brain samples (grey boxes); TPM Transcripts Per Million; Significance level is 0.01.

Figure 3 .
Figure 3. Frequency of mutations in LGG based on CBioPortal.

Figure 4 .
Figure 4. (A) Venn network analysis of circadian genes expression in major mutations from LGG. (B) Upset plot for different amount of valuable circadian genes among different gene mutations (FLG, KAT6B, CIC, IDH1, EGFR and TP53).

Figure 5 .
Figure 5. Correlation and Co-Expression Analysis of Circadian Genes in Relation to LGG. (A) Correlation analysis between circadian genes.Corr: correlation effect.(B) Co-expression analysis of circadian genes by both Spearman and Pearson statistics.

Figure 6 .
Figure 6.Correlation between mRNA expression of circadian genes and their methylation by both Spearman and Pearson statistics.

Table 2 .
Two-sample mendelian randomization study.MR Mendelian randomization, nsnp number of single nucleotide polymorphisms, b effect sizes for each SNP, se standard error, pval p value *p-value threshold for exposure selection is adjusted to 5 × 10 6 .