The Mechanism And Application Of ZNF582 Meth-Ylation In Cervical Carcinogenesis

Cervical intraepithelial neoplasia grade 3（CIN3）is a high-grade squamous intraepithelial lesion（HSIL）of the cervix.It is a precancerous lesion of cervical cancer and can develop into cervical cancer or regress to normal or low-grade squamous intraepithelial lesions（LSIL）.Therefore,there is an urgent necessity for precise triage of cervical precancerous lesions.ZNF582（Zinc finger protein 582）methylation has good accuracy in cervical cancer screening,but its role and related mechanism in CIN3 triage are still unclear.Purpose:To evaluate the accuracy of ZNF582 methylation and comparing with common screening strategies in predicting the pathology of surgical specimens from patients with CIN3.To explore the mechanism of ZNF582 in the malignant transformation of cervical lesions.Methods:1.A total of 277 patients underwent cervical cold knife conization（CKC）due to CIN3 in 5 hospitals including Central South University Xiangya Hospital from January 2019 to December 2020 were included.Preoperative cervical exfoliated cell samples were collected for specificity by methylation.The methylation level of ZNF582 was detected by methylation specific PCR（MSPCR）and represented by ΔCpzNF582.The methylation level of ZNF582 was negative corelated to ΔCpzNF582.According to the pathology of CIN3 conization specimens,the subjects were divided into 3 groups:regressed to normal/LSIL group,HSIL group and developed to cervical squamous cell cancer group.According to the pathological outcomes and ZNF582 methylation levels,ROC curves were drawn to clarify the accuracy of ZNF582 methylation for predicting the pathology of CIN3 conization specimens,and to determine the cut-off value.2.The expression of ZNF582 in HSIL and cervical cancer was detected by immunohistochemical experiments,and the relationship between ZNF582 methylation and expression level were evaluated.The KM（Kaplan-Meier）curve was drawn using TCGA（The Cancer Genome Atlas）database information to clarify the effect of ZNF582 expression on the prognosis of cervical cancer.Culturing a ZNF582 overexpressing stable cell line,the proliferation,migration,and invasion capacity of cervical cancer cells were detected by CCK-8（Cell Counting Kit-8）,clone formation,wound test and Transwell.The flow cytometry was used to evaluate the cell cycle and apoptosis rate.3.RNA sequencing analysis clarified the differential genes and enrichment pathways of ZNF582 overexpressing cell lines and the cells of control group,and the genes were verified by RT-PCR,Western Blot and immunohistochemistry.Results:1.Compared with HSIL,the methylation level of ZNF582 in squamous cervical cancer was increased;the accuracy rate of ZNF582 methylation in identifying squamous cervical cancer was about 77.3%,which was significantly higher than that of common screening methods.WhenΔCp_ZNF582<7.09,the risk of squamous cervical cancer increased 6.53 folds.When ΔCp_ZNF582≥19.18,the possibility of normal/LSIL increased 4.33 folds.2.Compared with HSIL,the expression level of ZNF582 in cervical cancer cells was decreased;The expression level of ZNF582 was negatively correlated with its methylation.Low expression of ZNF582 was associated with poor prognosis of cervical cancer.After the overexpression of ZNF582,the proliferation and migration were decreases,and the apoptosis rate was increased.ZNF582 overexpression did not affect cell cycle and invasion.3.After the overexpression of ZNF582,the expression levels of 11 genes related to tumor cell proliferation and apoptosis,such as KIF7,were changed.It was verified that the expression level of KIF7 increased after the overexpression of ZNF582,and the expression levels of ZNF582 and KIF7 were positively correlated.Knocking down of KIF7 could reduce the cell proliferation inhibition caused by ZNF582 overexpression.Conclusion:ZNF582 methylation can be used for CIN3 triage with better accuracy than common screening strategies.ZNF582 methylation decreased its expression level and function.ZNF582 inhibit the proliferation and promote apoptosis of cervical cancer cells by up-regulating of KIF7 expression.