A Study On The Association Of TEAD4 With The Proliferation And Invasion Of Cervical Squamous Carcinoma

Objectives:As a member of TEAD transcription factor family,the transcriptional activation domain of TEAD4（transcriptional enhancer associate Domain family member 4）molecule can bind with YAP（the main coactivator of Hippo signaling pathway）to form a transcriptional regulatory complex which regulates the transcription of downstream target genes.TEAD4 has recently been found to be associated with adverse biological behaviors of gastric cancer,lung adenocarcinoma,oral squamous cell carcinoma and other malignant tumors,and it has been shown by studies that there is a high expression level of TEAD4 in keratinocytes induced by human papilloma virus type 16（HPV16）infection.However,the relationship between TEAD4 and Cervical squamous cell carcinoma（CSCC）has not been reported.The study aims to investigate the association of TEAD4 with the proliferation and invasion capacity of CSCC,and further to preliminarily explore the regulatory pathway,so as to provide scientific basis for clinical diagnosis and novel therapeutic avenue of targeting TEAD4 in CSCC.Methods:1.Bioinformatics technical analysis:TCGA database was used to analyze the expression differences of TEAD4 and MMP9 in cervical cancer tissues and normal tissues,respectively.Then,the online database GEPIA was used to analyze the correlation between TEAD4 and MMP9 gene on the basis of pearson correlation analysis,and the online database OncoLnc was also used in combination with Kaplan-meier analysis assess the effect of TEAD4 expression on survival rate of cervical cancer patients.2.Clinicopathological specimen analysis:50 CSCC tissue specimens diagnosed by The Affiliated Hospital of Fuzhou Medical College of Nanchang University from2011 to 2019 were collected as the research objects,and 20 cervical epithelial specim ens from chronic cervicitis cases were collected as the control.group.Immunohist ochemistry Max Vision^TM was performed to detect the expressi ons level of TEAD4and MMP9 in the paraffin-embedded blocks of tissue specimens of CSCC and chronic cervicitis cases.The expression differences of TEAD4 in CSCC and chronic cervicitis tissues were analyzed.The relationship between the expressions of TEAD4and MMP9 in CSCC tissues and clinicpathological parameters was analyzed.And the correlation between TEAD4 and MMP9 was also evaluated.3.Cell experiments:SiHa cells were into control group,vector group and pCMV3-TEAD4 transfection group.The control plasmid and pCMV3-TEAD4plasmid were transfected with liposome Sinofection.The expression levels of TEAD4,MMP9 and phosphorylated ERK proteins were measured by Western blotting.The capacity of Cell proliferation and invasion were capacity was assessed by CCK8and transwell chamber assay,respectively.4.Mechanism study:With pre-treatment using ERK inhibitor U0126 in SiHa cells,TEAD4 gene was transiently transfected into the cells,and the expression levels of ERK,phosphorylated ERK and MMP9 were measured by Western blotting.Results:1.TCGA database analysis showed that both TEAD4 and MMP9 were highly expressed in cervical cancer tissues（P<0.05）,and TEAD4 was positively correlated with MMP9 gene by Pearson correlation analysis using GEPIA（R=0.17,P<0.01）;With online database OncoLnc,Kaplan-Meier curve analysis indicates the cervical cancer patients with low TEAD4 expression level showed a higher overall survival rate than those with high TEAD4 expressions level,however,the difference was not significant（P>0.05）.2.Immunohistochemical method was used to detect the tissue protein expression level,and image acquisition software was used to select 3 representative fields and the photos were input into the oftware Image Pro-Plus 6.0 for optical density analysis.The results showed that,The average expression level of TEAD4 in cervical squamous cell carcinoma（0.186±0.0355）was significantly higher than that in the control group（0.0494±0.0105,P<0.001）.The mean expression level of MMP9 in cervical squamous cell carcinoma（0.180±0.0310）was significantly higher than that in the control group（0.0608±0.0211,P<0.001）.Moreover,Correlation analysis showed that there was a positive correlation between TEAD4 expression and MMP9expression in cervical squamous cell carcinoma（R=0.394,P<0.01）.Further analysi s of clinicopathological parameters showed that TEAD4 expression was related with lymph node metastasis（P<0.05）,vascular invasion（P<0.05）,parametrial involvem ent（P<0.05）and staging（P<0.05）,but not related to the patient age（P>0.05）,tu mor diameter（P>0.05）and degree of differentiation（P>0.05）in CSCC.MMP9 expre ssion was related with lymph node metastasis（P<0.05）,parametrial involvement（P<0.05）,but not related to the patient age（P>0.05）,tumor diameter（P>0.05）,vasc ular invasion（P>0.05）,degree of differentiation（P>0.05）and staging（P>0.05）in CSCC.3.As a result of western blotting measurement,the SiHa cells with TEAD4 gene transfection showed a notably higher TEAD4 expression level than the cancer cells of control and vector group（P<0.01）.The proliferation and invasion capacity of SiHa cell with TEAD4 gene transfection was clearly increased in comparison with those cells of control and vector group（P<0.05）.4.The expression levels of MMP9 protein and phosphorylated ERK in all the groups of SiHa cells were detected by Western blotting,and the results showed that the expression levels of MMP9 and phosphorylated ERK in cancer cells of TEAD4gene transfection group were significantly higher than those in the cancer cells of control group（P<0.05）.5.After SiHa cells were pretreated with ERK inhibitor U0126 and transfected with pCMV3-TEAD4 plasmid,the expression levels of phosphorylated ERK and MMP9 in SiHa cells of the combined treatment group were significantly decreased in comparison with the SiHa cells with normal TEAD4 gene transfection（P<0.01）.Conclusion:1.TEAD4 was highly expressed in CSCC,and was related to the cancer lymph node metastasis,vascular invasion,parametrial involvement and staging.2.High expression of TEAD4 can promote the proliferation and invasion of CSCC,and the molecular mechanism involves the TEAD4-mediated ERK activation which upregulates the downstream expression of MMP9.