| Objective:Oral Squamous Cell Carcinomas(OSCC)is the most common types of head and neck malignant tumors,radical resection and regional lymph node cleaning is the most direct and most common method of treatment.Although with the discovery and application of a large number of adjuvant therapies,the prognosis of oral squamous cell carcinoma has been improved to some extent in recent decades,the five-year survival rate is still not high,maintaining about 40-50%.In view of the complexity and unknown of tumor occurrence and development,we intend to explore the pathogenesis and development mechanism of oral squamous cell carcinoma at the protein and molecular levels,with a view to providing a theoretical basis for immunotherapy or more targeted personalized therapy of oral squamous cell carcinoma in the near future.KIF20A is a gene associated with tumor occurrence and development,and its protein shows increased in a variety of tumors and is associated with poor prognosis of patients.The peptide vaccine derived from KIF20A is also one of the hottest topics in tumor immunotherapy research.Currently,there is no study on the mechanism of action related to KIF20A and oral squamous cell carcinoma.In this study,the expression of KIF20A in oral squamous cell carcinoma tissues and adjacent normal squamous cell epithelium was detected by immunohistochemistry,and the relationship between KIF20A and clinicopathologic parameters were analyzed.Furthermore,the effect of the biological characteristics and biological behavior of oral squamous cell carcinoma line HSC3 was studied through in vitro cell experiments after silencing KIF20A gene,so as to explore the role and possible mechanism of KIF20A in oral squamous cell carcinoma.Then provide a new idea for the occurrence and development mechanism of oral squamous cell carcinoma and for clinical personalized and immunotherapy for oral squamous cell carcinoma patients.Methods:1 A total of 87 oral squamous cell carcinoma specimens were collected from the Department of Pathology,Affiliated Hospital of Southwest Medical University.Normal squamous cell epithelium 1cm away from the tumor was used as normal control.Immunohistochemical techniques were used to detect the protein expression of KIF20A in oral squamous cell carcinoma tissues and adjacent normal tissues,and correlation analysis was conducted based on clinical and pathological parameters.Patients were followed up and survival data were collected for survival analysis.2.The effects of KIF20A on the biological behaviors related to oral squamous cell line HSC3 were investigated through in vitro cell experiments.The siRNA-KIF20A small interference fragment was transfected into HSC3 cells with Lip2000 to silence the expression of KIF20A.then the silencing efficiency was verified by Real-Time PCR and Western Blot.The changes of proliferation,migration and invasion ability of oral squamous cell line HSC3 were detected by CCK8,plate cloning,scratch and Transwell invasion assay.Western Blot was used to detect the changes of apoptosis-related proteins and ERK/MAPK signaling pathway proteins after silencing KIF20A.Results:1.Expression of KIF20A in oral squamous cell carcinoma(1)The expression of KIF20A was increased in oral squamous cell carcinoma(OSCC).The high expression of KIF20A in oral squamous cell carcinoma accounted for 72.4%(63/87),but only 18.4%(16/87)in adjacent normal tissue.The expression of KIF20A was statistically significant(p<0.001).(2)The expression of KIF20A was related to the gender of patients,tumor size,depth of invasion and ulcer(p<0.05).There was no correlation with age,tumor differentiation,nerve invasion,vascular invasion,local invasion,lymph node metastasis,clinical stage or recurrence(p>0.05).(3)Survival analysis Kaplan-Meier survival curve was drawn,and Log-Rank analysis showed that the expression of KIF20A was not correlated with the prognosis of oral squamous cell carcinoma(p=0.304).(4)By COX univariate and multivariate analysis,the larger the tumor was(p=0.001;Risk rate(HR)=1.688;95%confidence interval(CI)=1.249-2.281)and recurrence(p<0.001;Risk rate(HR)=4.292;95%confidence interval(CI)=1.965-9.377)will shorter the patient’s overall survival.2.Effects of KIF20A silencing on biological behavior of oral squamous cancer cells:(1)Silencing efficiency of Real-Time Time PCR and Western Blot detection:SiRNAKIF20A-3,SiRNAKIF20A-4 and SiRNAKIF20A-5 were transfected into oral squamous cell carcinoma HSC3 cells,and KIF20AmRNA relative expression and protein expression levels were detected by real-time PCR and Western Blot.The results showed that SiRNAKIF20A-4 and SiRNAKIF20A-5 transfected groups had the most obvious silencing effect(p<0.001),and were used as experimental groups for follow-up experiments.(2)The results of CCK8 and plate cloning showed that:By measuring the OD values of SiRNAKIF20A-4,SiRNAKIF20A-5 and SiRNANC at 24h,48h,72h and 96h respectively,CCK8 showed that cell proliferation was inhibited in the two groups after transfection(p<0.05),and the inhibition effect was the most obvious on the fourth day.The results of plate cloning experiment also showed that the number of cell colony formation was significantly decreased after two silences of KIF20A compared with the control group(p<0.05).(3)Scratch test showed that the cell migration ability was significantly lower than that of the control group at 24h and 48h after KIF20A silencing by area method,and the difference was statistically significant(p<0.05),indicating that KIF20A silencing could inhibit the migration ability of cells.(4)The results of Transwell assay showed that the number of cells passing through the compartment of the silenced KIF20A decreased compared with the control group(p<0.05),indicating that the cell invasion ability decreased after silenced KIF20A.(5)Western Blot detected the expression of apoptosis-related protein and ERK/MAPK pathway protein.The results showed that after KIF20A was silted,the expression of apoptosis-related protein Bcl-2 in HSC3 cells decreased and the expression of Bax increased compared with the control group.The expression levels of P-MEK and P-ERK1/2 proteins related to the ERK/MAPK pathway were decreased.Conclusions1.KIF20A was overexpressed in oral squamous cell carcinoma,and correlated with patient gender,tumor size,depth of invasion,and presence of ulcers.2.After silencing KIF20A,the ability of proliferation,migration and invasion of oral squamous cell cells decreased in vitro.3.After silencing KIF20A,the apoptosis of oral squamous cell cells increased.Meanwhile,KIF20A may promote cell apoptosis by promoting the phosphorylation of ERK1/2 and MEK,which are related to the ERK/MAPK signaling pathway. |
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