| Chapter 1 Screening of epigenetic regulators that potentially regulate ferroptosis based on ferroptosis core index in HNSCCObjective:Head and neck squamous cell carcinoma are among the most common malignancies of the upper respiratory tract.The long-term clinical efficacy of various treatments has stagnated after decades of research.As a new mode of programmed cell death,ferroptosis has gradually become one of the most promising treatment strategies for overcoming tumors throughout its development in nearly a decade.Epigenetics,a sophisticated regulatory mechanism that does not alter DNA sequences,is still underexplored in ferroptosis.To select epigenetic regulators who potentially regulate ferroptosis by bioinformatics analysis(based on mRNA expression database and clinical survival data of patients with head and neck squamous cell carcinoma in the TCGA database)combined with experimental validation.Methods:41 core genes of ferroptosis were obtained from the PathCards(https://pathcards.genecards.org/)website,and gene expressions in GDC TCGA Head and Neck Cancer(HNSC)were downloaded from UCSC XENA(https://xenabrowser.net/datapages/).RNAseq counts data(n=546)and patient OS survival data(n=611).The R package "ssgsea" was used for scoring FCI.After further clustering,the differential genes between high and low groupings were analyzed to extract the overlapping gene sets in the epigenetic database and the Kaplan-Meier survival analysis of FCI high and low scores.Low expression epigenetic regulators with poor prognosis in the tumor were selected for further in vitro model verification of ferroptosis.In vitro ferroptosis models for head and neck squamous cell carcinoma were established by Fadu and HN8 cell lines and classical ferroptosis-inducing agents(FINs),further screening for epigenetic regulators that potentially regulate ferroptosis.Results:We obtained 495 valid sample information by rejecting nontumor patients and paraffin specimen data by R-studio.GSVA scoring("ssgsea")scored by 41 ferroptosis genes(input gene sets),and we obtained FCI.FCI divided into 3 clusters by unsupervised clustering.There were 19 differentially expressed epigenetic factors between the high(n=85)and low group(n=80),of which 10 were expressed differently between tumor and paratumor.At last,four epigentic regulators(ZBTB7C,SATB1,SCML4,CRB2)in the tumor were further screened by the prognosis screening of these 10 differently expressed genes.In in vitro iron death model experiments,we observed that the mRNA and protein levels of CRB2 were significantly improved after inducing iron death in the head and neck squamous cell lines(Fadu and HN8).Conclusion:Based on bioinformatics analysis and in vitro model verification of ferroptosis in head and neck squamous cell carcinoma cells,we preliminarily obtained CRB2,an epigenetic regulatory factor,that potentially regulates ferroptosis.Chapter 2 Epigenetic Regulator CRB2 Promoted FINsinduced ferroptosis in HNSCCObjective:To further experimentally verify the epigenetic factor CRB2,which is potentially regulating ferroptosis and clarify the role of CRB2 in ferroptosis in HNSC.Methods:The cell lines of HNSC commonly used in the researcher’s laboratory was selected to clarify the basal expression of CRB2 protein.Cell lines with low CRB2 expression were selected for gene overexpression regulation,and high-expression cell lines were selected for siRNA knockdown regulation.Ferroptosis was induced in HNSC in the experimental and control groups with classical FINs(Erastin and RSL3),respectively,and the ferroptosis effect of FINs was inhibited with the classic ferroptosis inhibitor Ferrostatin-1.We detected cell viability in different groups using the CCK8 method,cellular ROS levels with C11BODIPY probes,and glutathione metabolism levels with GSH-GSSG kits.Nude mouse transplant tumor model was established,inoculated with CRB2 stable transfection Fadu cell line and control,and then divided into solvent group and FINs group,and the effect of CRB2 on inhibiting HNSC growth was evaluated by measuring tumor size and anatomical weighing of tumors.Results:CRB2 was lowly expressed in Fadu cells and Tca8113 cell lines and highly expressed in Tu686 and HN8 cell lines.Fadu and Tca8113 cell lines with overexpressing CRB2 were significantly reduced in cell viability,intracellular ROS levels,GSH levels,but GSSG levels were significantly increased under the induction of FINs.The nude mouse transplant tumor model confirmed that CRB2 inhibits the growth of head and neck tumors in vivo,and overexpressing CRB2 tumor has significant growth inhibition compared with other groups under the action of FINs,and the dry weight of tumors is the lightest.After knocking down CRB2 expression in Tu686 and HN8 cell lines with the siRNA method,the cell viability of experimental group was significantly improved after FINs induction,the intracellular ROS level was significantly reduced,the GSH level was significantly increased,and the GSSG level was significantly reduced.Conclusion:By in vitro cell viability detection,intracellular ROS level detection,GSH/GSSG level detection,and nude mouse transplant tumor model verification,it is suggesting that CRB2 has the effect of promoting FINs-induced ferroptosis in HNSC.Chapter 3 CRB2 Promotes ferroptosis by inhibiting expression of SLC7A11 and GPX4 in HNSCCObjective:To further explore the mechanism of CRB2 promoting ferroptosis in HNSC based on the function of CRB2 in Chapter 2.Methods:Transcriptome sequencing was performed between CRB2 overexpressed Fadu cell line(n=3)and control cell line(n=3),KEGG signaling pathway and GO molecular function enrichment analysis were performed on the differential genes between the two groups,and the differential TOP100 gene and the key ferroptosis defense gene were further verified.Finally,the effect of CRB2 on downstream ferroptosis-related proteins was further confirmed by immunohistochemistry of nude mouse transplanted tumor.Results:Transcriptome sequencing of CRB2 overexpressed Fadu cell line vs.control group yielded 642 differential expression genes(p<0.05,logFC>1),of which 5 ferroptosis-related genes(DRD5,SLC7A11,SESN2,CHAC1,and TSC22D3)were included in the TOP100 differential expression genes.KEGG and GO analysis of all differential expression genes initially explored the functions and pathways of CRB2 downstream genes enriched in the cell cycle,oxidative phosphorylation,cell adhesion,and chromatin,etc.qPCR validation of the sequenced 5 genes and 3 key ferroptosis defence proteins revealed mRNA expression of SLC7A11 were decreased in the CRB2 overexpressed Fadu and Tca8113 cell lines.WB experiment confirmed a significant decrease in SLC7A11 and GPX4 protein levels in CRB2 overexpressed Fadu and Tca8113 cell lines.Immunohistochemistry confirmed a significant reduction in SLC7A11 and GPX4 protein expression in CRB2 overexpressed nude mouse transplanted tumor tissues.In contrast,no significant changes were observed in other ferroptosis defense proteins(FSP1,DHODH).Conclusion:Through CRB2 overexpression and Vector control Fadu cell line sequencing binding experimental verification,it is preliminarily shown that CRB2 promoted ferroptosis in HNSC by acting on the SLC7A11-GPX4 signaling pathway.Chapter 4 CRB2-Mediated H4K20me2 in epigenetic regulation of SLC7A11 promotes ferroptosis in HNSCCObjective:To further investigate whether CRB2 can regulate downstream ferroptosis related protein through epigenetic mechanisms.Methods:WB verified the histone modification levels of H4K20me1,H4K20me2 and H4K20me3 in overexpressed CRB2 and vector controls in Fadu and Tca8113 cell lines,respectively.The DNA bound to CRB2 and vector control Fadu cells was extracted with the chip-grade antibody with altered histone levels,and the CHIP-qPCR confirmed that CRB2 was directly regulated by SLC7A11 and/or GPX4.Finally,the transcriptional regulation effect of CRB2 on SLC7A11 was confirmed by dual luciferase reporter assay.Results:WB results of Fadu and Tca8113 cell lines showed that the H4K20me2 level was increased in the overexpressed CRB2 group,and the expression of H4K20me1 and H4K20me3 was unchanged.Continuing to extract the DNA bound to CRB2 and vector control Fadu cells with a CHIP-grade antibody of H4K20me2,CHIP-qPCR verification of the promoter sequence primer of the designed SLC7A11 and GPX4 was performed.It was found that H4K20me2 could bind to the promoter sequence of SLC7A11 and silence the transcription,while the promoter binding to GPX4 did not change.Finally,we confirmed the inhibition of CRB2 on the transcriptional activity of SLC7A11 by dual luciferase reporter assay.Conclusion:CRB2 may inhibit transcription of SLC7A11 by epigenetic silencing mechanisms with H4K20me2 and thus promote ferroptosis in HNSC.Chapter 5 Relationship of CRB2 and clinicopathological features of HNSCC and prospectObjective:The first four chapters clarify the role of CRB2 in promoting ferroptosis in HNSC and its mechanism of action,and this chapter will continue to study the potential clinical value and the prospect of CRB2.Methods:The differences in mRNA expression of CRB2 in HNSC and para cancer in TCGA-HNSC were investigated,and the protein expression level of CRB2 in HNSC was assessed using the human proteomics database.The correlation between CRB2 and clinicopathological parameters and its value as an independent prognostic risk factor were analyzed and evaluated by CRB2 alone and with SLC7A11 expression.Finally,we conducted a preliminary study of immune infiltration of high and low CRB2-expressed patients by CIBERSORT and TIMER.Results:In the TCGA-HNSC library,CRB2 expression in tumors was significantly lower than that in pericarcinomatous tissue(p<0.05),and IHC in the human proteomics database showed low CRB2 expression in HNSC tissues.Patients with high CRB2 expression of head and neck squamous cell carcinoma had better overall survival(p=8.747e-04)and longer disease-free survival(p=1.382e-02).At the same time,prognosis stratification was better in combination with SLC7A11 expression(overall survival(5.112e-03),disease-free survival(p=7.043e-03)).It was found that CRB2 expression was positively correlated with infiltration of B cells,CD8+T cells,T follicular helper cells and NK cells but negatively associated with infiltration of myeloid suppressor cells,M0 macrophages,neutrophils and eosinophils.Conclusion:CRB2 is significantly lower expressed in HNSC tissues,the prognosis of patients with high expression is better,and the prognostic stratification effect combined with SLC7A11 is better.At the same time,CRB2 is statistically correlated with the survival status of patients and can be used as an independent prognostic factor for clinical low score insurance.Increased anti-tumor immune cell infiltration and decreased immunosuppressive cells in patients with high CRB2 expression may indicate that it can improve anti-tumor immunity in patients.Fig 25,Table 5,references 83... |
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