| Objectives:The aim of this study is to investigate the role and mechanism of m6A/KRT7-AS/KRT7 molecular axis in the resistance of head and neck squamous carcinoma to radiotherapy,and to provide new ideas for the clinical sensitization of head and neck squamous carcinoma to radiotherapy.The treatment of head and neck squamous carcinoma is mainly based on surgery and radiation therapy,and radiation therapy as the main treatment for head and neck squamous carcinoma can reduce the recurrence and metastasis of patients through various mechanisms in order to achieve survival benefits for patients.However,the widespread existence of resistance to radiotherapy has greatly limited its efficacy and become an important cause of treatment failure,recurrence and metastasis in head and neck cancer.In this study,we investigated the role and mechanism of m6A/KRT7-AS/KRT7 molecular axis in radiotherapy resistance in head and neck squamous carcinoma,and laid a solid foundation for further clinical studies.For example,we study the use of targeted drugs in head and neck squamous carcinoma patients to make the tumor more sensitive to radiotherapy.Methods:1.By downloading the sequencing data and clinical information of head and neck squamous carcinoma patients from TCGA database,we divided the patients into radiotherapy-resistant and radiotherapy-sensitive groups,and performed differential gene analysis on the genomic data of the two groups to derive the set of genes that may mediate radiotherapy resistance in head and neck squamous carcinoma.2.RNA-seq was performed on the radiotherapy-resistant cell line and the original cell line established by the group to further narrow down the set of genes that may mediate radiotherapy resistance in head and neck squamous carcinoma,and KRT7,the most likely gene that regulates radiotherapy resistance,was screened for biological mechanism study.3.m6 A site prediction site and analysis of sequencing data were used to clarify the regulatory role of m6A/KRT7-AS on KRT7 by q PCR,Western Blot and other experiments.4.Colony formation,flow cytometry and other experiments were used to further verify the influence of KRT7 and PI3K/AKT/m TOR signaling pathway on radiotherapy resistance of head and neck squamous cell carcinoma.Results:1.By analyzing the differential genes of patients in the radiotherapy-resistant and radiotherapy-sensitive groups and the differential m RNAs of the radiotherapy-resistant and primitive cell lines in the TCGA database,in which a total of 11 genes were expressed,the entries obtained from GO enrichment analysis were associated with biological functions such as epithelial development,epithelial cell variation,keratinization process and keratinocyte variation.Based on these results,we hypothesize that KRT7 may mediate resistance to radiotherapy in head and neck squamous carcinoma.2.By examining the RNA and protein expression of methylation genes in radiotherapyresistant cell lines and primary cell lines,the results showed that the methyltransferase METTL3 was lower expressed in the radiotherapy-resistant cell line.The m6 A site prediction database revealed no plausible m6 A modification sites on KRT7 m RNA,while multiple high confidence modification sites existed in its upstream KRT7-AS,and simultaneous knockdown of METTL3 and KRT7-AS could reduce the expression of KRT7 m RNA and protein.Subsequent m6A-RIP-q PCR experiments verified the presence of m6 A modification on KRT7-AS,thus clarifying the upstream or downstream relationship of the m6A/KRT7-AS/KRT7 gene axis.3.Under radiotherapy conditions,the clonogenic ability of the two cell lines was examined by clonogenic assay after modulation of the KRT7-AS/KRT7 axis,and the results showed that the clonogenic ability of the cells changed after the expression of KRT7-AS and KRT7 were regulated.Western Bolt results showed the expression of p-AKT and p-m TOR increased after the regulation of the KRT7-AS/KRT7 axis,suggesting that KRT7 may regulate radiotherapy resistance in head and neck squamous carcinoma through this pathway.4.After downregulating KRT7 and adding the mTOR inhibitor rapamycin,the expression of p-m TOR protein was changed and the apoptosis rate of cells after radiation irradiation wasaltered,suggesting that the PI3K/AKT/m TOR signaling pathway may regulate the radiation therapy resistance of head and neck squamous carcinoma.Conclusion:KRT7 was lower expressed in radiotherapy-resistant patient groups and radiotherapyresistant cell line,and its upstream m6A/KRT7-AS axis could regulate KRT7 expression.KRT7 could mediate radiotherapy resistance in head and neck squamous carcinoma through regulating PI3K/AKT/m TOR signaling pathway. |
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