Hypoxia-induced MiR-421 Of Exosomes For Head And Neck Squamous Cell Carcinoma And Its Mechanism Of Action

Objective: Head and Neck Squamous Cell Carcinoma(HNSCC)is a group of malignant tumours of the head and neck that is the sixth most prevalent in the world.Radiotherapy is the main treatment modality for patients with HNSCC other than surgery,but approximately 15%to 40% of patients with advanced disease experience local recurrence after radiotherapy,and the development of radioresistance during radiotherapy is the main reason for poor treatment outcomes.The interaction between radiation resistance and the hypoxic microenvironment of tumour cells affects the development of HNSCC,and a clearer understanding of the HNSCC tumour microenvironment will help to find more effective targeted therapies for this disease.Cancer-associated fibroblasts(CAFs)are the largest stromal cell component of the HNSCC tumour microenvironment,while the hypoxic microenvironment of tumour cells is rich in exosomes,which are key messengers of cell-cell communication.In this study,we aimed to investigate whether hypoxia-induced exosomes from CAFs(CAFs-exosome)are involved in mediating resistance to radiotherapy in head and neck squamous cell carcinoma(HNSCC)and to explore the underlying mechanisms.Methods: In this project,the HNSCC cell line Cal27 was cultured under normoxic and hypoxic conditions and irradiated by X-rays.Cell viability was assayed by the CCK8 assay,proliferation capacity was assayed by clone formation assay,apoptosis was assayed by flow cytometry and autophagic flux was assayed by Cyto-ID(?) Autophagy Assay Kit.Gene and protein expression was detected by Western blot,q RT-PCR and immunohistochemistry.A luciferase reporter assay was used to verify the interaction of miR-421 with m TOR.CAFsexosomes were isolated by ultrafiltration and validated by electron microscopy and nanoparticle tracking techniques.Cal27 cells which were treated were injected subcutaneously into the left abdomen of nude mice to assess the effect of CAFs-exosomes on the radioresistance of HNSCC tumours.Results: The irradiation-treated Cal27 cell lines were co-cultured with cancer-associated fibroblasts(CAFs)and normal fibroblasts(NFs)under normoxia and hypoxia,respectively.The results of the CCK8 assay showed that Cal27 cells in the CAFs-hypoxia group were the most viable,the clonogenesis assay indicated that Cal27 cells in the CAFs-hypoxia group were the most proliferative,and flow cytometry showed that Cal27 cells in the CAFs-The apoptosis of Cal27 cells was most significantly inhibited in the hypoxic group.The autophagic flux assay showed an accumulation of the autophagy marker LC3 II under hypoxic conditions,indicating that hypoxia promotes autophagy in Cal27 cells.Western blot assays indicated that hypoxic conditions induced high expression of HIF-1α,and also showed that Cal27 cells co-cultured with CAFs had increased expression of the autophagy-related protein Beclin1,and the autophagy marker.These results suggested that CAFs could promote proliferation and autophagy and inhibited apoptosis in irradiated Cal27 cells under hypoxia,resulting in radioresistance.We next explored the mechanisms by which CAFs affect the phenotype and radiosensitivity of the HNSCC cell line Cal27,where CAFs secreted exosomes that induced proliferation and autophagy in irradiated Cal27 cells under hypoxia and inhibited apoptosis,resulting in radioresistance.In clinical samples,the development of radioresistance was verified to be due to hypoxia and accompanied by autophagy.We found that exosomes secreted by CAFs induced tumour size and volume in mice,particularly under hypoxic conditions,through experiments in a mouse tumour model.H&E staining confirmed that exosomes secreted by CAFs enhanced the radioresistance of Cal27 cells under hypoxic conditions.Immunohistochemical results showed that m TOR expression was down-regulated and Belicn1 expression was increased in Cal27 cells treated with irradiation and exosomes secreted by CAFs under hypoxic conditions,suggesting that the process was mediated by accelerated autophagy.After clarifying the m TOR changes,we continued to investigate the regulatory mechanisms underlying the changes in m TOR expression.A database was used to predict upstream regulators of m TOR.q RT-PCR verified miR-421 as an upstream regulator of m TOR,and a dual luciferase reporter assay verified direct contact of miR-421 with m TOR m RNA.The ability of miR-421 to promote proliferation and inhibit apoptosis in irradiated Cal27 cells was verified by CCK8 assay,single cell cloning assay and flow cytometry.Autophagy flux results showed that miR-421 mimics promoted autophagy and miR-421 inhibitors reduced autophagy in Cal27 cells.The expression of autophagy-associated proteins m TOR,Beclin1 and LC3II/I further validated this result,indicating that miR-421 enhanced the radiation resistance of HNSCC by accelerating autophagy.Conclusion: Under hypoxic conditions,exosomes secreted by CAFs promoted proliferation and autophagy of irradiated Cal27 cells and also inhibited apoptosis.miR-421 expression levels in CAFs-exosomes were upregulated,accelerating Cal27 cell proliferation and autophagy and enhancing radioresistance of HNSCC by negatively regulating the expression of its target gene m TOR.