The Mechanism Of Foxd1 Regulating The Proliferation And Invasion Of Head And Neck Squamous Cell Carcinoma

Background and Purpose:Head and neck squamous cell carcinomas(HNSCC)accounts for 90% of head and neck malignant tumors.The onset is insidious and there are no obvious symptoms in the early stage.Therefore,the incidence is mostly in the middle and late stages,and the mortality rate is high.The median survival time is only about 10 months.Due to the special location of the disease,the appearance and function of the head,face and other organs are seriously damaged,resulting in a serious impairment of the patient’s quality of life.The lack of clear biological markers and effective treatment is the main reason for the high mortality rate of HNSCC.Therefore,finding new markers that may lead to the onset and progression of HNSCC is of great significance for early diagnosis and determination of new therapeutic targets.FOXD1 is a member of the transcription factor FOX(forkhead box)family,which locates on chromosome 5q13 and has the function of enhancing and / or inhibiting transcription.It was widely involved in embryonic development,cell cycle regulation,metabolic regulation,stem cell niche maintenance,signaling transduction and other physiological processes.Recently,many studies have shown that FOXD1 was up-regulated in cancer,such as non-small cell lung cancer,cervical cancer,pancreatic cancer,colon cancer,and breast cancer.FOXD1 was the key factor for the poor prognosis of patients.This showed that FOXD1 was an oncogene,and its expression level was closely related to the occurrence and development of tumors.In terms of cell function,FOXD1 might affect the regulation of the cell cycle and promote the proliferation,invasion and distant metastasis of tumor cells directly.It prompted us: FOXD1 might become a new tumor marker and therapeutic target for malignant tumors.Although the biological roles of FOXD1 have been reported in other tumors,the specific way of FOXD1 in head and neck squamous cell carcinoma is still not yet clear.This topic aims to clarify the role of FOXD1 in head and neck squamous cell carcinoma,and to explore its possible molecular mechanism,for providing new ideas and perspectives for the diagnosis and treatment of head and neck squamous cell carcinoma.Methods:1.Use the online software GEPIA:Gene Expression Profiling Interactive Analysis(http://gepia.cancer-pku.cn/)to analyze the FOXD1 expression in tumors.RT-PCR and IHC were used to verity the expression of FOXD1 in HNSCC and non-cancerous epithelial tissues of the head and neck.2.The small molecule interfering RNA(si-RNA)was used to silence the expression of FOXD1 in FaDu cells of HNSCC.Western blot was used to detect the silencing effect and the changes in the expression level of downstream genes.MTT assay was used to detect the growth ability of cells,clone formation assay were used to detect the ability of cells to form clones;Flow cytometry was used to detect changes in cell cycle.Transwell test detected the invasive ability of cells.3.RNA sequencing was used to sequence Fadu cells before and after FOXD1 silencing.GO enrichment analysis was performed on genes with significant different in expression(log2 > 4 times).To Analyse the targets and signaling pathways may regulated by FOXD1.4.Use the online database GEPIA to analyze FOXD1 and candidate signal paths and their key factors.5.Use online databases GEPIA and TIMER: Tumor IMmune Estimation Resource(https://cistrome.shinyapps.io/timer/)to predict the correlation between FOXD1 and the expression of key factors of EMT and TIL.Results:1.According to GEPIA analysis,the expression level of FOXD1 in head and neck squamous cell carcinoma was significantly increased(p<0.05).Compared with patients with low expression of FOXD1,DFS and OS were significantly shortened(p <0.05).RT-PCR and immunohistochemical staining showed that the expression level of FOXD1 in head and neck squamous cell carcinoma was significantly increased.2.Using small RNA interference to silence the expression of FOXD1.RT-PCR showed the silencing efficiency of si-FOXD1 was statistically significant(p<0.05).Western blot showed that the expression level of FOXD1 protein in si-FOXD1 decreased significantly(p<0.05).MTT assay found that the growth rate of tumor cells after si-FOXD1 decreased significantly(p<0.05).Clone formation assay found that after si-FOXD1,the cloning ability of cells was significantly reduced(p<0.05).Flow cytometry found that the cell cycle was blocked in the G1 phase after silencing FOXD1.3.The Transwell assay found that the invasion ability of si-FOXD1 cells decreased significantly(p<0.05).Western blot found that the expression of cyclinD1 and metal matrix proteinase MMP-2/MMP-9 in si-FOXD1 group decreased significantly(p<0.05).4.The RNA sequence sequencing was used to found out significantly different genes between si-FOXD1 and si-ctrl cells.GO enrichment analysis found that the WNT / β-catenin signaling pathway was one of the most obvious signaling pathways for differential gene enrichment(p<0.05).5.The online database GEPIA and TIMER predictive analysis showed that in head and neck squamous cell carcinoma,FOXD1 was positively correlated with the expression of EMT markers: CDH2(N-cadherin),Vimentin,SNAIL1,SNAIL2,TGFβ1 and TWIST1(p<0.05).FOXD1 was negatively correlated with the expression of TIL key factor: B cells,CD8 + T cells,and CD4 + T cells(p<0.05).Conclusions:1.The expression of FOXD1 is increased in head and neck squamous cell carcinoma,and the high expression of FOXD1 is associated with poor prognosis.2.FOXD1 may promote the proliferation and invasion of head and neck squamous cell carcinoma cells by regulating the WNT / β-catenin signaling pathway.3.FOXD1 may promote the cell invasion by inducing EMT in head and neck squamous cell carcinoma.The high expression of FOXD1 may be related to tumor immune escape.4.FOXD1 may become a diagnostic marker and a new target for the treatment of head and neck squamous cell carcinoma.