Expression And Significance Of PLK4 In Ovarian Cancer

Background and objective:Ovarian cancer(OC)is one of the three most common malignant tumors in the female genitourinary system.Its mortality ranks second in gynecological tumors,second only to cervical tumors.Ovarian cancer can be divided into four pathological types: epithelial tumor,sex cord stromal cell tumor,germ cell tumor and other types of tumors.Among them,epithelial ovarian cancer(EOC)occupies an important part of the pathological types of ovarian cancer,accounting for about 80 ～ 90%.Ovarian cancer occurs in the deep structure of the pelvic cavity.There are no obvious signs of early lesions,and there is a lack of specific clinical diagnosis methods.As a result,most patients with ovarian cancer have metastasized at the first diagnosis,and the 5-year survival rate is very low.The invasion and metastasis of epithelial ovarian cancer are mainly realized by epithelial mesenchymal transition(EMT).EMT is a process in which epithelial cells lack their apical basal polarity and cell-cell adhesion to form spindle mesenchymal cells with invasive function.Most of the tumor cells in EMT have decreased epithelial marker gene expression,as e-cadherin,and increased mesenchymal marker gene expression,as n-cadherin.Late clinical diagnosis,high degree of malignancy,easy metastasis,easy drug resistance and recurrence are the difficult problems in the treatment of ovarian cancer.Therefore,it is urgent to find new molecular markers,which is conducive to the early diagnosis of ovarian cancer,find new pathogenic mechanisms,and further develop new targeted therapeutic drugs to improve the prognosis of patients.Polo like kinase 4(PLK4)is a member of the serine / threonine protein kinase polo family.It can regulate the cell cycle of tumor cells by regulating the replication of centrosomes.Studies have found that PLK4 is highly expressed in breast cancer,neuroblastoma,lung cancer,liver cancer and colorectal cancer,and is closely related to the tumor growth,proliferation,metastasis and poor prognosis.Studies have shown that PLK4 inhibitors can inhibit the proliferation and migration of ovarian cancer cell lines in vitro,induce the apoptosis of tumor cells,and lead to the increase of mitotic deficient tumor cells.After knockdown of PLK4 in colorectal cancer and neuroblastoma,the expression level of epithelial marker E-cad increased,while the expression level of mesenchymal marker N-cad reduced,and the invasion and metastasis ability of tumor cells decreased,which may be realized by inhibiting the effect of EMT.However,there are still few studies on PLK4 in ovarian cancer,especially the study on the relationship between PLK4 and EMT in ovarian cancer.Based on this,this study first explored the expression of PLK4 in ovarian cancer by bioinformatics method,then explored the relationship between PLK4 expression and clinicopathological features by immunohistochemical method,and further explored the correlation between PLK4 and EMT molecular marker expression,in order to provide a new theoretical foundation for early diagnosis and targeted therapies of ovarian cancer.Methods:1.Bioinformatics:(1)The expression difference of PLK4 in tumor tissues and corresponding normal tissues was analyzed through the GEPIA database,and the expression of PLK4 in ovarian cancer was obtained in the database.(2)The expression of PLK4 in ovarian cancer and normal tissues was compared by HPA database.(3)The relevance between the expression level of PLK4 and the prognosis of patients with ovarian cancer could be explored by KM plotter database.(4)Construction of co-expression regulatory network of PLK4 in ovarian cancer by coexpedia database.(5)David 6.8 database could meet our needs for conducting the functions and pathways enrichment analysis of PLK4 and the selected key genes.(6)The correlation between the expression of PLK4 and the infiltration of six immune cells in ovarian cancer was studied by TIMER database.2.Immunohistochemistry:(1)From September 2019 to January 2021,40 patients with primary epithelial ovarian cancer who underwent surgery in Henan University People’s Hospital were selected,and 40 cases of benign ovarian tumor tissues were collected.(2)Immunohistochemical method was used to detect the difference of PLK4 expression between EOC and benign ovarian tumor.The clinicopathological features of patients with ovarian cancer were counted,including age,pathological type,FIGO stage,tumor size,unilateral and bilateral,degree of differentiation and lymph node metastasis.The relationship between PLK4 expression and clinicopathological features was analyzed.(3)To analyze the difference of the expression of EMT related molecular markers E-cad and N-cad in EOC and benign ovarian tumor,and The relationship between their expression level and clinicopathological features was analyzed.(4)The correlation between the expression levels of PLK4,E-cad and N-cad was compared.(5)SPSS 23.0 statistical software was used to compare and analyze the relevant data of the included cases.The measurement data were showed in the form of mean ± standard deviation(x ± s).T-test was used to conduct comparative analysis.The counting data were showed in number of cases or percentage(%).Chi square test was used to analyze the relevance between the expression of molecular markers and the clinicopathological features of epithelial ovarian cancer,and the difference of molecular markers between epithelial ovarian cancer group and benign lesion group.Spearman rank correlation was used to analyze the correlation between the expression of molecular markers in ovarian cancer.P < 0.05,the difference was considered to be statistically significant.Result:1.Bioinformatics(1)Through the GEPIA database,it was found that the expression of PLK4 m RNA in a variety of human tumor tissues was different from that in normal tissues.The expression level of PLK4 m RNA in ovarian cancer was sharply higher than that in normal ovarian.(2)The immunohistochemical results of HPA database showed that the staining intensity and the proportion of positive cells of PLK4 protein in normal ovarian tissues were significantly lower than those in ovarian cancer.(3)KM plotter database analysis found that high PLK4 expression predicted shorter PFS and OS.(4)By comparing the highly expressed genes and PLK4 co-expressed genes in ovarian cancer,21 key genes were obtained.These genes may be involved in the malignant biological behavior of tumors by regulating the cell cycle(5)The expression of PLK4 was directly proportional to the content of neutrophils and DC cells in the immune microenvironment of ovarian cancer(P < 0.05).2.Immunohistochemistry:(1)Comparing with the benign ovarian tumors,the positive expression rate of PLK4 in EOC was significantly higher than the former.The expression level of PLK4 was significantly correlated with the FIGO stage,differentiation and lymph node metastasis of epithelial ovarian cancer(P < 0.05).(2)Compared with benign ovarian tumors,N-cad was significantly higher in EOC tissues and E-cad was significantly lower in EOC tissues.The positive expression of N-cad was also significantly correlated with EOC stage,differentiation and lymph node metastasis,while the positive expression of E-cad was significantly correlated with EOC stage and lymph node metastasis(P < 0.05).(3)There was a significant positive correlation between the expression of PLK4 and N-cad in epithelial ovarian cancer(r = 0.361,P = 0.022),and a significant negative correlation between the expression of N-cad and E-cad in epithelial ovarian cancer(r =-0.409,P =0.009).There was no significant correlation between the expression of PLK4 and E-cad(r = 0.105,P =0.520).Conclusion:First,bioinformatics analysis showed that PLK4 was highly expressed in ovarian cancer.The positive expression of PLK4 suggested poor prognosis.PLK4 may participate in the occurrence and development of ovarian cancer by regulating cell cycle and immune cell infiltration.Next,The expression of PLK4 was significantly correlated with the stage,differentiation and lymph node metastasis of epithelial ovarian cancer by immunohistochemistry.Moreover,the results also showed that in the epithelial ovarian cancer,the expression of PLK4 and N-cad was positively correlated and the expression of N-cad and E-cad was negatively correlated.