Effects Of YAP On Proliferation And Apoptosis Of CAL27 Cell Line And The Possible Mechanism

Backgrounds and Objectives:Oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors in the maxillofacial region,and about 250000 new cases are added every year in the world.China is a high incidence area of OSCC,and its incidence has been rising in recent years.Although new chemotherapy methods and targeted therapies have been applied in clinic,the effect is not ideal.Therefore,we need to further understand the underlie mechanism of oral squamous cell carcinoma.YAP,a key downstream effector of the Hippo signaling pathway,has been reported to be abnormally expressed in tumor tissues such as liver cancer and lung cancer.Therefore,we want to explore the expression level of YAP in OSCC,and its effects on tumor progression and the possible mechanism in vivo and in vitro,so as to provide a theoretical basis for YAP gene as a new therapeutic target for oral squamous cell carcinoma.Methods:1.Immunohistochemical staining was used to detect the expression level of YAP protein in different differentiation level oral squamous cell carcinoma tissues and normal oral epithelium tissues.2.To detect the expression level of YAP in four kinds of oral squamous cell carcinoma cell lines,and finally select CAL27 for subsequent experiments.CAL27 cells were transfected with lentivirus vector,and the oral squamous cell carcinoma cell lines were constructed to knockdown and overexpression of YAP gene and control group respectively.QRT-PCR and Western blot were used to detect the efficiency of knock down and overexpression.3.We used CCK-8 cell viability assay,EdU staining,clonogenic assay to detect the effects of knockdown and overexpression of YAP on CAL27 cell proliferation,and to detect the effect of YAP on cell cycle and apoptosis by flow cytometry.The expression of factors related to cell cycle and apoptosis such as C-MYC and BCL-2 were detected by QRT-PCR and Western blot.4.CAL27 cells were treated with a different concentration of Hippo-YAP signaling pathway inhibitor,Verteporfin.Cell proliferation was detected by CCK-8 cell viability assay,and cell cycle and apoptosis were detected by flow cytometry.QRT-PCR and Western blot were used to detect the changes of C-MYC and BCL-2 along with inhibitor concentration.5.The experiment of tumor formation in nude mice was designed.The knockdown group,overexpression group and corresponding control group CAL27 cells were injected into the subcutaneous tissue of nude mice to observe the growth of tumor and analyze the effect of different expression levels of YAP on the occurrence and development of oral squamous cell carcinoma.Results:1.YAP protein is not expressed or weakly expressed in normal oral epithelium.In oral squamous cell carcinoma,the expression level of YAP protein is high,and it is correlated with the degree of tumor differentiation.2.In CAL27 cells,the expression level of YAP gene and protein is moderate.After knockdown of YAP gene,the expression level of YAP and its downstream target genes CTGF,ANKRD and CYR61 decreased significantly,meanwhile the expression of YAP and total YAP in cells decreased.After overexpression of YAP,the expression level of YAP and its downstream target genes increased significantly,and the expression level of YAP in total and in nucleus increased.3.Compared with the control group,knockdown of YAP in CAL27 cells resulted in inhibition of proliferation,cell cycle arrest,and the proportion of early and late apoptotic cells increased;meanwhile the expression level of C-MYC and BCL-2 mRNA and protein levels decreased;the expression level of cell cycle-related CyclinDl decreased,and pro-apoptosis protein Caspase7 and BAX expression were increased.Overexpression of YAP led to the accelerated procreation and cell cycle progression,the proportion of apoptotic cells decreased,the expression level of C-MYC and BCL-2 increased,the expression level of CyclinD1 increased,and the expression level of BAX and Caspase7 decreased.4.With the increase of inhibitor concentration,the proliferation activity of CAL27 cells was gradually decreased,cell cycle arrest was more obvious,and the proportion of early apoptosis and late apoptotic cells increased.The transcriptional level of C-MYC and BCL-2 is consistent with that of CTGF and CYR61,that is,the higher the inhibitor concentration is,the lower the expression level;the expression level of C-MYC and BCL-2 decreases with the increase of inhibitor concentration.5.After knockdown of YAP,CAL27 cells grew slower in tumor tissues,smaller in tumor volume and lighter in tumor weight.Overexpression of YAP resulted in faster tumor formation,larger tumor size and heavier tumor weight.Conclusion:YAP is highly expressed in oral squamous cell carcinoma and is related to tumor grade.YAP can affect cell proliferation and apoptosis by regulating C-MYC and BCL-2 expression,thereby regulating tumor progression.Our results suggest that YAP plays an important role in the occurrence and development of oral squamous cell carcinoma.This will provide new ideas for molecular targeted therapy of oral squamous cell carcinoma.