The Clinical Significance And Mechanism Of AGR2 In Cervical Lesions

Objective:To evaluate the clinical significance and mechanism of anterior gradient 2?AGR2?in the progression of cervical lesions.Methods:Paraffin specimens and clinical data of 46 chronic cervicitis,31 Low-grade squamous intraepithelial lesion?LSIL?,70 High-grade squamous intraepithelial lesion?HSIL?and 37 squamous cell carcinoma of cervix?SCC?tissues were collected,and immunohistochemical staining?IHC?was used to detect the expression of AGR2 and P16^INK4in these specimens.The pathological diagnosis of cervical lesions were divided into three categories:CC group versus cervical lesions group?LSIL+HSIL+SCC,LHS group?,LSIL and below group?CC+LSIL,CL group?versus HSIL and above group?HSIL+SCC,HS group?,and non-malignant cervical lesions group?CC+LISL+HSIL,CLH group?versus SCC group,and P16^INK4NK4 was used as control.Logistic regression analysis was used to evaluate the correlation between AGR2 expression and cervical lesions.OR value was obtained by Logistic regression analysis to evaluate the diagnostic significance of AGR2 in cervical lesions.Besides,receiver operator characteristic curve?ROC curve?and area under the curve?AUC?were performed to assess the diagnostic accuracy of AGR2,P16^INK4,and combination analysis of AGR2 and P16^INK4NK4 in cervical lesions.Next,paraffin specimens and clinical data of cold knife conization?CKC?and loop eleotrosurgical excisional procedure?LEEP?tissues confirmed to be margin positive or negative by pathologists were collected,and IHC was used to measure the AGR2 expression.The follow-up data was retrospectively analyzed to investigate the correlation between AGR2 expression and residual/recurrence after cervical conization.Then,the cervical squamous cell carcinoma?CSCC?cell lines Ca ski and SiHa were cultured,and the si RNA-AGR2 were transfected into CSCC cells,while siRNA-control was used as negative control.qRT-PCR and western blot were used to compare the expression of AGR2 in each group and verify the transfection efficiency.Based on the constructed cell models,the CCK-8 assay,colony-formation assay and cell cycle analysis with flow cytometry?FCM?were performed to investigate the effects of AGR2 ablation on proliferation ability in CSCC cells in vitro.Results:As the results showed,AGR2 was gradually increased with the severity of cervical lesions,and the positive expression rate of AGR2 was 4.3%,38.7%,65.7%and 89.2%in CC,LSIL,HSIL and SCC groups,respectively,and the difference among the four groups was statistically significant??2=82.760,P<0.001?.Among the group comparison,the difference of AGR2 expression rate in CC group versus LSIL group,LSIL group versus HSIL group and HSIL group versus SCC group had statistically significance??2=14.195,P<0.001;?2=10.667,P<0.001;?2=22.823,P<0.001?.Meanwhile,P16^INK4NK4 was gradually increased with the severity of cervical lesions,and the positive expression rate of P16^INK4NK4 was 19.6%,51.6%,70.0%and91.9%in CC,LSIL,HSIL and SCC groups,respectively,and the difference among the four groups was statistically significant??2=73.340,P<0.001?.Among the group comparison,the difference of P16^INK4in CC group versus LSIL group and HSIL group versus SCC group had statistically significance??2=7.971,P<0.001;?2=28.808,P<0.001?,however these was no significant difference between LSIL group versus HSIL group??2=6.084,P=0.014?.In addition,Logistic analysis results showed that the OR value of AGR2 were 6.440,3.022,2.204 in CC group versus LHS group,CL group versus HS group and CLH group versus SCC group,respectively,while the OR value of P16^INK4were 1.927,1.795,2.913,which indicated that AGR2 and P16^INK4were dramatically associated with cervical lesions,and could be risk factors of CSCC.Next,the results of ROC curve and AUC exhibited that,the AUC of AGR2were 0.815,0.817,0.84 in CC group versus LHS group,CL group versus HS group and CLH group versus SCC group,respectively,the AUC of P16^INK4NK4 were 0.786,0.788,0.860,and the AUC of combination analysis of AGR2 and P16^INK4were 0.858,0.851,0.902 in corresponding groups.The difference was not statistically significant in comparison of AGR2and P16^INK4?P>0.05?,however,the combination analysis of AGR2 and P16^INK4NK4 was obviously higher than AGR2/P16^INK4alone?P<0.05?,which indicated that combination analysis could improve the diagnostic efficiency of cervical lesions.Then,the results of IHC showed that,the expression rate of AGR2 in positive surgical margins?88.2%?was remarkable higher than negative surgical margins?41.2%??P<0.05?,and the follow-up results showed that the expression of AGR2 was associated with the secondary surgery rate and the progression of cervical lesions in positive margin patients.Finally,the knockdown of AGR2 CSCC cell models were constructed successfully.As the results showed,knockdown of AGR2 could significantly attenuate Cacx cell proliferation and the number of colonies,compared with negative controls?P<0.05?.Conclusion:AGR2 plays a role in tumorigenesis in cervical lesions,and certain clinical value in the diagnosis and prognosis of cervical lesions with different degrees,which may become a new target for the treatment of cervical cancer.