The Expression And Regulatory Mechanisms Of HnRNP F And HnRNP E2 In Head And Neck Squamous Cell Carcinoma

Background:Head and neck squamous cell carcinoma is the most common malignancy in head and neck.Although advanced in the option of treatment methods,due to its strong invasion and metastasis capabilities,the prognosis of patients has not been improved significantly company with high mortality.It is still posing a significant threat to the life and health of patients.Heterogeneous nuclear ribonucleoproteins are a superfamily of nuclear proteins,among which hnRNP F and hnRNP E2 are RNA-binding proteins with multiple functions.They play an important role in the biological processes of mRNA splicing,polyadenylation site selection,mRNA stability,mRNA transport and so on.There is increasing evidence that both hnRNP F and E2 are significantly associated to the occurrence and development of tumors,but the relationship between hnRNP F and E2 and head and neck squamous cell carcinoma is largely unknown.Objective:The aim of this study was to investigate the expression of hnRNP F and E2 and the prognostic significance in head and neck squamous cell carcinoma.Methods:1.The sequence data of head and neck squamous cell carcinoma samples were selected from TCGA database to analyze the expression of hnRNP F gene and its relationship with clinical grade in head and neck squamous cell carcinoma.And then the effect of hnRNP F gene on the survival of patients with head and neck squamous cell carcinoma was obtained by Kaplan-Meier method on the cbioportal platform.Data about human hnRNP F were retrieved from the Oncomine database and the role of hnRNP F in OSCC was analyzed.RNAi technique was used to reduce the expression of hnRNP F in CAL 27 cells,and cell counting was used to detect the proliferation ability of CAL 27 cells.2.The hnRNP E2 data was retrieved from TCGA database,and the expression of hnRNP E2 in HNSCC,OSCC and 10 other cancers was analyzed.The GraphPad Prism software and Log-rank(Mantel-Cox)test method was used to analyze the relationship between patients’ survival time and hnRNP E2 expression level.The Oncomine database was used to meta-analyze the expression of hnRNP E2 in head and neck squamous cell carcinoma and normal head and neck tissues.The alternative splicing of hnRNP E2 new exon was detected by RT-PCR.3.Transfection technology was used to increase the expression of hnRNP E2 in HEK293 cells.Reverse transcription PCR and western blotting were used to detect the autoregulation mechanism of hnRNP E2.Results:1.In the TCGA database,we found that the expression of hnRNP F mRNA in head and neck squamous cell carcinoma tissues was significantly higher than that of normal mucosa tissues,and the difference was statistically significant(P <0.05);but the expression of hnRNP F was not significantly related to the clinical grade of tumors;Survival analysis found that the expression level of hnRNP F in patients with head and neck squamous cell carcinoma was related to the disease-free survival rate(P = 0.03).A total of 9 studies in the Oncomine database involved the expression of hnRNP F in oral cancer and normal tissues.Through meta-analysis,we found that the expression of hnRNP F in oral cancer was significantly higher than that in normal tissues(P =0.003).In the CAL 27 cell line,the downregulation of hnRNP F inhibited cell growth.2.By mining the TCGA database and the Oncomine database,it was found that the expression level of hnRNP E2 in head and neck squamous cell carcinoma and oral squamous cell carcinoma was not significantly different from that of normal mucosa tissues.In addition,we found that the expression level of hnRNP E2 in liver cancer and breast cancer was significantly lower than that in normal controls,but the expression level of hnRNP E2 in some cancers was significantly higher than that in normal controls.The median survival time of patients with high expression of hnRNP E2 in head and neck cancer and oral cancer was significantly lower than that in patients with low expression of hnRNP E2.In this study,we found for the first time that there is a new exon between exon 6 and exon 7 of hnRNP E2.This new exon can generate three kinds of isomers by alternative splicing,two of which contain early termination codon and are degraded by NMD pathway.The expression level of this new exon is positively correlated with the survival of patients with head and neck cancer and oral cancer.3.In HEK293 cell line,overexpression of hnRNP E2 did not affect the expression of endogenous hnRNP E2,suggesting that hnRNP E2 may not have a self-regulating mechanism.Conclusion:1.The results of bioinformatics analysis show that hnRNP F is highly expressed in head and neck squamous cell carcinoma,and it is related to the poor prognosis of head and neck squamous cell carcinoma.Additionallly,hnRNP F is very important for the proliferation of OSCC cells.2.The expression level of hnRNP E2 in head and neck squamous cell carcinoma and oral squamous cell carcinoma is not significantly different from that in normal mucosa,but is related to the prognosis of head and neck squamous cell carcinoma and oral squamous cell carcinoma.3.There is a new exon in exon 6 and 7 of hnRNP E2,and its expression level is positively correlated with the survival of patients with head and neck cancer and oral cancer.4.There may be no self-regulation mechanism in hnRNP E2.