Abnormal Transcription And Expression Of Transferrin Receptor 1 And SLC40A1 In Head And Neck Squamous Cell Carcinoma And Nasopharyngeal Carcinoma

Background:Head and Neck Squamous Cell Carcinoma(HNSCC)is the sixth most common malignancy in the world.HNSCC is related to smoking,drinking,and human papillomavirus(HPV)infection.HNSCC is concealed that most patients are in advanced stage at the time of the first visit.Although the treatments based on surgery,radiation therapy and simple systemic therapy are developing,the survival rate of patients is still low.Nasopharyngeal carcinoma(NPC)is a malignant tumor derived from nasopharyngeal epithelial tissue with unique geographical distribution and complex etiology.Epstein-Barr virus infection,genetic susceptibility,and environmental factors are reported to be related to NPC.The anatomical location of nasopharyngeal carcinoma is concealed,so most of the first diagnosis patients have already metastasized.It affects the therapeutic effect of radiotherapy-based treatment.At present,more and more studies have shown that iron metabolism abnormalities are closely related to tumors,the expression of iron transport key genes TFR1 and SLC40A1 and their molecular mechanisms and translational medical significance in HNSCC and NPC have not yet been elucidated.Objective:Through the integration of bioinformatics analysis and the validation of clinical samples,to explore the abnormal expression of TFR1 and SLC40A1 in HNSCC and NPC,and elucidate the relationship with the clinicopathological features of patients.To explore the effects of TFR1 and SLC40A1 on the malignant biological behavior of tumor cells.To explore new mechanisms of HNSCC and NPC pathogenesis from a metabolic perspective and find new molecular markers or therapeutic targets for tumor diagnosis and prognosis.Explore new strategies for finding new tumor markers and therapeutic targets by combining integrated bioinformatics analysis with multi-level sample validation and tumor biology experiments.Methods: 1.We analyzed the transcription of TFR1 and SLC40A1 in HNSCC from the GEO,TCGA,Oncomine database.2.We analyzed the association between abnormal transcription of TFR1 and SLC40A1 and clinicopathological parameters in HNSCC patients from the TCGA database.3.We analyzed the association between abnormal transcription of TFR1 and SLC40A1 and survival prognosis in HNSCC patients by K-M plotter database.4.We analyzed co-expressed genes with TFR1 and SLC40A1 by cBioportol,then perform GO analysis and KEGG-Pathway analysis to find possible functions and pathways.5.Target gene prediction and regulatory network mapping of TFR1 and SLC40A1 by GCBI database.6.The transcription of TFR1 and SLC40A1 in laryngeal carcinoma cell lines,nasopharyngeal carcinoma cell lines and control cell lines,as well as nasopharyngeal carcinoma tissues and control tissues were validated by Real-time quantitative PCR.7.The protein expression of TFR1 and Ferroportin in head and neck squamous cell carcinoma,nasopharyngeal carcinoma and normal control tissues were detected by immunohistochemical staining.8.Knocking down TFR1 gene in nasopharyngeal carcinoma cell line was determined by real-time quantitative PCR,and then we detected the malignant biological behavior of TFR1 by performing CCK8 cell proliferation assay.Result: 1.TFR1 was up-regulated in HNSCC and NPC.In the Oncomine database,two sets of GEO datasets and TCGA datasets,the transcription of TFR1 was up-regulation,and then confirmed by Real-time quantitative PCR in tumor cell lines and tumor tissues.The expression of TFR1 was up-regulated in the HNSCC tissue chip and the NPC tissue chip by immunohistochemical staining.2.SLC40A1 was down-regulated in HNSCC and NPC.Through the integration of bioinformatics analysis,it was found that the transcription of SLC40A1 was down-regulated in HNSCC and NPC,and then confirmed by Real-time quantitative PCR.The expression of SLC40A1 was downregulated in the HNSCC tissue chip and the NPC tissue chip by immunohistochemical staining.3.The transcription of TFR1 gene was related to the clinicopathological features of HNSCC patientsBy analyzing the relationship between TFR1 transcription and clinicopathological features in TCGA-HNSCC patients,we found that the high transcription of TFR1 gene was related to tumor lymphatic invasion,clinical stage,lymph node metastasis,and patient drinking history.Further verification of HNSCC tissue microarrays suggested that high expression of TFR1 gene was related to the distant metastasis and higher T stage in HNSCC patients.4.The transcription of SLC40A1 gene was related to clinicopathological features of HNSCC patients,but the relation was not found in protein levelBy analyzing the relationship between SLC40A1 transcription and clinicopathological features in TCGA-HNSCC patients,it suggested that the low transcription of SLC40A1 gene was associated with high clinical stage and high T stage.Head and neck tumor tissue microarray results were not found to be associated with clinicopathological features of HNSCC patients.5.High transcription of TFR1 gene was related to poor survival prognosis in patients with HNSCCThe KM plotter database suggested that abnormally high transcription of the TFR1 gene was related to low OS in HNSCC patients.Further analysis revealed that abnormally high TFR1 transcription in Grade1 patients,white patients,female patients,and patients with high-mutation-loaded patients was related to worse OS.6.Low transcription of SLC40A1 gene was related to poor survival prognosis in patients with HNSCCThe KM plotter database suggests that abnormally low transcription of the SLC40A1 gene was related to low OS in HNSCC patients.The low transcription of SLC40A1 in Grade1 patients,Grade2 patients,Stage4 patients,male patients,black American patients,and high-mutations-loaded patients was related to worse OS.7.Knockdown of TFR1 gene inhibited the proliferation of NPC cellsAfter the construction of the knockdown TFR1-TWO3 cell line and control cell line,we found that knockdown of TFR1 inhibits proliferation of nasopharyngeal carcinoma cells by CCK8 proliferation experiment.Conclusion: 1.TFR1 was up-regulated in HNSCC and NPC cell lines and tissues;2.SLC40A1 was down-regulated in HNSCC and NPC cell lines and tissues;3.High transcription of TFR1 suggested poor lymphatic invasion,clinical stage,and poor survival prognosis in patients with HNSCC;4.Low transcription of SLC40A1 suggested poor clinical stage and survival prognosis in patients with HNSCC;5.Low expression of TFR1 can inhibit the proliferation of NPC cells.In summary,the iron transport key genes TFR1 and SLC40A1 were abnormally expressed in HNSCC and NPC,and affected the clinical stage and prognosis of the patients.In addition,we found low expression of TFR1 can inhibit the proliferation of NPC cells.These results suggested that iron metabolism abnormalities may play an important role in the pathogenesis of HNSCC and NPC.TFR1 and SLC40A1 may be potential tumor markers for HNSCC and NPC diagnosis,prognosis and intervention measures.