| Objective: Head and neck squamous cell carcinoma(HNSCC)is the most common malignancy in the head and neck region.It has a high mortality,and the 5-year overall survival rate(OS)of HNSCC patients is only about 50%,mainly due to the high rate of lymphatic metastasis and postoperative recurrence.Studying the molecular mechanisms that affect the invasion and metastasis of HNSCC,will contribute to determining molecular targets and finding biomarkers for the prognosis of patients.Mena protein belongs to the Ena/VASP(Enabled/VASP)family,encoded by the ENAH gene located on chromosome1.Mena protein implicates in connecting the signal pathways to the remodeling of the actin cytoskeleton.Among them,TGFβ signal is essential to promote EMT which is positively related to tumor progression.Studies in breast cancer and lung carcinoma cells have found that Mena and its isoforms are closely associated with the TGFβ-induced EMT process.TGFβ is involved in the regulation of alternative splicing of ENAH.It has been shown that Mena protein is highly expressed in many types of tumors and plays a role in promoting tumor invasion and metastasis.However,the potential role and mechanism of Mena in the progression of HNSCC remain unclear.Therefore,this study will explore the expression of Mena in the human tissue of HNSCC,the relationship with clinicopathological features,and the regulation of TGFβ signals on Mena protein and its isoforms.Research methods: 1.Download the HNSCC gene data from the TCGA database to analyze the expression of ENAH in HNSCC and the normal epithelium,and analyze the survival rate;2.Acquire 91 HNSCC patients’ tissues from the Stomatological Hospital of China Medical University from 2020 to 2022,and construct HNSCC tissue microarray(TMA);carry out the immunohistochemical staining of Mena in TMA tissue sections;perform the semi-quantitative analysis to analyze the relationship between Mena and clinicopathological characteristics using Chi-square test;3.The expression of Mena in the tumor tissues and the adjacent normal tissues of HNSCC was determined by RT-q PCR and Western blot;4.Detect the invasive capacity of SCC cells induced by TGFβ signals by the transwell invasion assay;Detect the expression of Mena and its isoforms in SCC cells upon TGFβ stimulation by RT-q PCR,Western Blot,and immunofluorescence staining combined with the three-dimensional clone model;5.Determine the network of ENAH by the bioinformatic analysis of protein-protein interaction(PPI),and find the potential partner genes for ENAH.Results: 1.Bioinformatic analysis showed that the expression of ENAH in HNSCC was significantly higher than the normal epithelium(P<0.05),but the expression of ENAH was not correlated with the overall survival rate(P=0.357);2.Immunohistochemical staining of Mena showed that the expression of Mena in HNSCC was significantly higher than the normal epithelium(P <0.001);The expression of Mena was not related to sex(P=0.2807),age(P=0.522),smoking history(P=0.6355),drinking history(P=0.5292),primary focus(P=0.3657),recurrence history(P=0.7571),personal cancer history(P=0.2951),family cancer history(P=0.8077),preoperative radiotherapy and chemotherapy history(P=0.0569),tumor pathological grade(P=0.182),tumor size(P=0.1398),but was related to lymph node metastasis(P<0.001),TNM stage(P<0.001).3.The results of RT-q PCR and Western blot showed that the expression of Mena in the tumor tissue was higher than in the matched adjacent normal tissues;4.TGFβ promotes the invasion of squamous cell carcinoma cells by the transwell invasion assay,and TGFβ induces the expression of Mena and its invasive isoforms;5.Through the network of ENAH by the bioinformatic analysis,10 potential partner genes for ENAH were found.Conclusion: 1.The expression of cytoskeleton regulatory protein Mena in HNSCC was higher than that in the normal epithelium.The expression of Mena was correlated with the lymph node metastasis and TNM stage;2.TGFβ signal promotes the expression of Mena and its invasive isoforms in the squamous cell carcinoma cells,and enhance the invasive capacity of the squamous cell carcinoma cells. |
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