Study On The Clinical Implications And Regulation Mechanism Of Tophagy-related Protein 12 In Cervical Lesions

Background andObjectivesCervical cancer is the most common gynecological malignant tumor in women.The annual incidence of new cases is about 130,000,accounting for 28%of the total number of cases in the world.About 20,000 women die of cervical cancer every year.The main etiology of cervical cancer is the infection of human papillomavirus(HPV).Only a small percentage of women infected with high-risk HPV will develop precancerous lesions,namely cervical intraepithelial neoplasia(CIN)and cervical cancer.The main cause of HPV is the integration of the E6 and E7 oncogenes into the host DNA,resulting in the inactivation of tumor suppressor genes p53 and pRB.However,in the development and progression of cervical cancer,only HPV infection is not enough to cause cervical cancer,and abnormal regulation of genes,abnormal metabolism of cells,and instability of genomes also play an important role.Autophagy is a type ? programmed cell death that is widespread in eukaryotic cells.During stress,such as hypoxia,changes in growth factor levels,exposure to cytotoxic conditions,autophagy is up-regulated.Autophagy is closely related to tumorigenesis and development.Its role in tumorigenesis and development has two sides.On the one hand,autophagy can act as a tumor suppressor to inhibit tumorigenesis.On the other hand,autophagy can promote tumorigenesis.The autophagy-associated protein ATG12 is a protein encoded by the ATG12 gene.In mammalian cells,ATG12 forms a complex with ATG5,which plays an important role in the formation of autophagy.Studies have shown that autophagy-related proteins,such as Beclinl,are involved in the occurrence and development of cervical cancer and play an important role in the development of cervical cancer.However,the role of ATG12 in cervical cancer and precancerous lesions is not clear.The purpose of this study:(1)to detect the expression and clinical significance of ATG12 in normal cervical epithelium,cervical intraepithelial neoplasia and cervical cancer tissue;(2)to explore the value of ATG12 in predicting recurrence of lesions after cervical conization;3)to explore the regulatory mechanism of ATG12 at the cellular level.Chapter?Expression and clinical significance of autophagyassociated protein ATG12 in cervical lesionsObjectivesThe purpose of this study was to detect the expression and clinical significance of ATG12 in normal cervical epithelium,cervical intraepithelial neoplasia and cervical cancer.MethodsImmunohistochemistry was used to detect the expression of ATG12 in cervical squamous cell carcinoma specimens,CIN1 specimens,CIN2-3 specimens,and normal cervical tissue specimens.ResultsA total of 120 cervical tissue specimens were collected and immunohistochemical staining was performed.The results showed that ATG12 was mainly expressed in cytoplasm.Among 30 normal cervical tissues,28 were positive,the positive rate was 93.3%,and 27 of 30 CIN1 tissues were positive,the positive rate was 90.0%,and 30 cases in CIN2-3 tissues were positive the positive rate was 53.3%,only 7 of 30(23.3%)cervical squamous cell carcinomas were positive.The positive rate of ATG12 in CIN2-3 and cervical squamous cell carcinoma was significantly lower than that in normal tissues and CIN1 group.There was no difference between normal tissue and CIN1 group.No difference between CIN2-3 and cervical squamous cell carcinoma.ConclusionThe expression of ATG12 in cervical precancerous lesions CIN2-3 and cervical cancer decreased,suggesting that ATG12 may be involved in the development of cervical cancer.Chapter?Correlation between ATG12 and recurrence of lesions after cervical conizationObjectivesThrough retrospective case-control study,we looked for factors related to the persistence or recurrence of cervical lesions,and explored the clinical significance of autophagy-related protein ATG12 in the persistence or recurrence of cervical conization.Methods and resultsThe clinical and pathological features and follow-up information of 352 patients who underwent cervical conization in Laiwu Maternal and Child Health Hospital from January 2009 to December 2015 were retrospectively collected.TCT and HPV were reviewed at 3 months,6 months,and 12 months after surgery.Thereafter,a combination of cytology and HPV was performed every 6-12 months.The indication for referral colposcopy is ASCUS and more severe lesions or HPV persistence positive for more than 1 year.The definition of persistent lesions is a high-grade CIN confirmed by biopsy within half a year after conization.The definition of recurrence of lesions is a high-grade CIN confirmed by biopsy for more than half a year.Patients with residual or recurrent lesions after cervical conization were used as the case group.Patients who underwent cervical conization in our hospital and were followed up for 2 years without recurrence were used as the control group,and matched according to the case group:control group =1:1 ratio.The paraffin-embedded specimens of the initial cervical conization and the primary cervical conization of the patients in the patients with persistent or recurrent disease were selected.The expression of ATG12 in the two groups was detected by immunohistochemistry.ResultsAfter screening according to inclusion criteria and exclusion criteria,the final 298 patients were included in the study.The median follow-up time was 25 months,with a minimum of 12 months and a maximum of 64 months.A total of 72 women were referred for colposcopy during the follow-up period.A total of 37 cases were satisfied with colposcopy,accounting for 51.39%;35 cases were unsatisfactory,accounting for 48.61%.Sixty-one patients underwent colposcopic cervical biopsy.Biopsy pathology confirmed that a total of 28 patients with pathology were CIN2-3,diagnosed with persistent or recurrent disease.Twenty-eight patients in the recurrence group were the case group,and 28 patients who were not relapsed were the control group.The age of the two groups,the HPV of the 3 months postoperatively,the margin of the cervical conization and the expression of ATG12 were compared.The results showed there was no difference in age between the two groups.The positive rate of HPV in the recurrent group was 89.3%(25/28),and the positive rate in the non-recurrent group was 35.7%.The positive rate of HPV in the recurrent group was significantly higher than that in the non-recurrent group(p<0.001).The positive rate of ATG expression in the recurrent group was 25%(7/28),and the positive rate of ATG in the non-recurrent group was 71.4%(20/28).The positive rate of ATG in the non-recurrent group was significantly higher than that in the recurrent group(p=0.001).The loss of ATG12 expression,positive HPV and positive margin were risk factors for persistent or recurrent lesions after cervical conization.HPV positive odds ratio(OR)=15,95%confidence interval(CI)=(3.7,61.1).The OR value of ATG12 expression deletion was 7.5,95%CI(2.3,24.5).The OR value of positive margin was 10.8,95%CI(1.2,93.5).ConclusionLoss of ATG12 expression,HPV positive conization and positive margin are risk factors for persistent or recurrent lesions after cervical conization.Chapter?MiR-378 promotes migration and invasion of cervical cancer cells by regulating ATG12ObjectivesThe purpose of this part of the study is to explore the apparent regulation mechanism of ATG12,to explore the interaction between ATG12 and miR-378 and its role in cervical cancer cells and its clinical significance.MethodsSixty cases of normal cervical tissue from hysterectomy for benign gynecological diseases,70 cases of CIN ? tissue and 55 specimens of cervical squamous cell carcinoma were collected.Conventional culture of human cervical cancer cell lines HeLa and C-33A cells is utilized.TargetScan software predicts miRNAs that regulate ATG12,initially predicting that ATG12 is a target gene for miR-378.Construction of small interfering RNA(si-ATG12)down-regulated ATG12 expression;miR-378 overexpressed lentivirus GIPZ-miR-378 and mi R-378 interfering with viral vector;real-time quantitative PCR for detection of ATG12 mRNA and miR-378 relative expression The expression of CCND1,phosphorylation-Rb and ATG12 protein was detected by Western blot;the dual luciferase reporter system was used to verify the regulation of miR-378 on ATG12.ResultsThe target miRNAs were predicted by Targetscan,and the results showed that the 3'UTR 449-455 of ATG12 is complementary to miR-378.The luciferase reporter assay showed that miR-378 can inhibit pmirGLO in HeLa and C33-A cells.The luciferase activity of the-ATG12-3'-UTR wild-type transfected cells had no effect on the pmirGLO-ATG12-3'-UTR mutant transfected cells,while the miR-378 negative control was pmirGLO-ATG12-3'-The luciferase activity of UTR wild-type transfected cells had no effect.It is indicated that ATG12 is a direct target gene of miR-378 and miR-378 regulates the expression of ATG12.Interfering RNA si-ATG12 significantly down-regulated the expression of ATG12 mRNA and protein;down-regulated ATG12 inhibited the invasion and migration of cervical cancer cell line C33-A.Real-time PCR showed that the expression of miR-378 was significantly increased in CIN3 and cervical squamous cell carcinoma compared with normal cervical tissues(p<0.001),but there was no significant difference between CIN3 tissue and cervical squamous cell carcinoma.Further analysis of cervical squamous cell carcinoma showed that the expression of miR-378 in cervical cancer tissues with lymph node metastasis was significantly higher than that in cervical cancer tissues with negative lymph node metastasis(p<0.05).Successfully constructed miR-378 overexpression and interference with lentiviral vector,miR-378 overexpression enhanced the migration and invasion ability of cervical cancer cell lines HeLa and C33-A cells,and down-regulation of miR-378 inhibited cervical cancer.HeLa and C33-A Cell migration and invasion ability.The expression of p-Rb,ATG12 and CCND1 protein was significantly decreased in miR-378 overexpressing C-33A cells.Similarly,in HeLa cells of miR-378 inhibitor group,the expression of p-Rb,ATG12 and CCND1 protein was significantly higher than that of the negative control group,suggesting that miR-378 may promote cervical cancer metastasisthrough p-Rb,ATG12 and CCND1 proteins.ConclusionATG12 is a functional target of miR-378,and miR-378 promotes the invasion and metastasis of cervical cancer cells.The results of this study suggest that ATG12 and miR-378 may be therapeutic targets for cervical cancer patients,providing a new theory for the molecular mechanism of cervical carcinogenesis and metastasis.Conclusions1.ATG12 expressedin most normal tissues and CIN1,the positive rate of ATG12 in CIN2-3 and cervical squamous cell carcinoma was significantly decreased.2.The loss of ATG12 expression,HPV positive after conization and positive margin are risk factors for persistent or recurrent lesions after cervical conization.3.ATG12 is a functional target of miR-378,miR-378 may promote the invasion and metastasis of cervical cancer cells by regulating ATG12.