Relation Between HPV16/18 And E-cadherin, β-catenin In Cervical Intraepithelial Neoplasia

IntroductionCervical cancer(CC) is one of the most frequently founded malignant tumors in women world wide.Current evidences implicated high risk humn papillomavirus(HPV) especially HPV16/18 infection as a causative part of the natural history of cervical carcinogenesis.HPV can be found in 99.7%of cervical cancer patients.It's critical for precaution of cervical cancer to detect HPV.However only a part of the patients who contracted HPV may advance to cervical cancer.E-cadherin is mediated by a class of cell-cell adhesion,with the organizational structure to maintain the integrity and polarity of the calcium-dependent transmembrane protein.E-cad function is not only required the existence of calcium ion,but also the needs of their Ligand catenin (catenin,cat) can be combined into a complex role,β-cat and E-cad direct binding,α-cat-mediated E-cad /β-cat and actin cytoskeleton-linked protein.Therefore,the maintenance of the polarity of epithelial cells and play an important role in differentiation,E-cadherin function may result in decrease or loss of cells-the role of intercellular adhesion reduced,resulting in tumor occurrence and development.In cervical intraepithelial lesions and cervical cancer in expression was low in the high expression of the normal control group,so if the factors on the etiology of cervical cancer and HPV biomarkers factor E-cadherin,β-catenin co-testing,to high-risk groups for cervical cancer screening,may have important significance.In our study,we detect HPV16/18 by the hybridization in situ and E-cadherin,β-catenin by immuohistochemical analysis,to research the value of them in cutting down the mortality of CC,and to supply rationale to exploitation of convenient samples. Materials and Methods1.Materials(1) specimen600 cervical liquid-based cells remained samples were obtained from the Department of gynecology in the First Affiliated Hospital of China Medical University, during Nov 20067to Nov 2008.All of them were treated as CC or CIN,Among them 13 samples as control gropes.All of them possess corresponding histological samples as controls.The patients with CC were 40,with CINI were 21,with CINⅡ\Ⅲwere 19. The average age is 48.4(range,26～77 years).The standard of cytology grouping based on histodiagnosis.All patients had not the history of the operation on cervix,pregnancy and radiation therapy in the cavity of pelvic.(2) Main agents,equipment and sourceKit of HPV16/18 in situ hybridization was purchased from MAIXIN biological technology company in Fuzhou,antibody of E-cadherin,β-catenin protein was friendly presented by Jin woo Kim professor in Korea.Sub-type HPV-DNA detection kit purchased from Cape company.S-P Kit and DAB Kit were obtained from MAIXIN biological technology company in Fuzhou.2.Methods(1) ImmunohistochemistryDetection of E-cadherin,β-catenin by Immuohistochemical analysis S-P methods. Result determination:The overexpressed staining was in the membrane.The number of staining cells were graded as follows:10-24%(+);25-49%(++);50-74%(+++);≥75% (++++).(2) In situ hybridizationDetection of HPV16/18 by Hybridization in situ.The masculine staining was in the nucleus.The overexpressed staining was in the cytoplasm.The number of staining cells were graded as follows:10-24%(+);25-49%(++);50-74%(+++);≥75%(++++). (3) HPV-DNA hybridization diversion type detectionDetection of HPV-DNA expression in the various subtypes.Results in a corresponding subtype of the template on a solid dot the region is the emergence of positive expression of the subtypes(4)Detection of HPV16/18 by western-blot.Stamples were incubated on ice for 30 min in an appropriate RIPA buffer(Solarbio) with 1%PMSF(Solarbio).The stamples was centrifuged at 15000g for 45 min at 4℃, and the supematant was fractionated by electrophoresis on a 15%PAGE and transferred to a nitrocellulose membrane.After blocking with 5%skimmed milk,the membrane was incubated with the appropriate primary antibody overnight at 4℃,and with labeled secondary antibodies(1:2000,Chemicon,USA) for 2h at 37℃.Proteins were visualized by DAB(Perking Applygen Co.,Ltd.) according to the manufacturer's protocol.(5) Statistical analysisThe results of the various groups usingχ2 test and Fisher's exact probability test,P <0.05 statistical significance.Results1.Expressions of E-cadherin,β-cateninExpressions of E-cadherin,β-catenin in cervical exfoliated cells and cervical tissue by Immuohistochemical analysis S-P methods.Membrane E-cadherin,in normal cervical exfoliated cells and high expression in cervical tissue,cervical lesions in each group there are different levels of expression:E-cadherin,in normal,CIN,invasive carcinoma of the expression rates were 84.6%(11/13),72.5%(29/40),27.5%(11/40),β-catenin in normal,CIN,invasive carcinoma of the expression rates were 76.9% (10/13),67.5%(27/40),32.5%(13/40).As the extent of cervical intraepithelial lesions showed a downward trend to increase2.Expression of HPV16/18 by Hybridization in situ. HPV16/18 was overexpressed in cervical cancer82.5%(33/40) and CIN 50.0%(20/40) compared with it in normal15.4%(2/13),P<0.05.The expression of HPV16/18 in cervical cancer and precancerous lesion correlated well with cervical neoplasia(CIN).It's consistent both in cervical exfoliated cell and in cervical tissue.3.HPV-DNA hybridization diversion type detection to detect the expression of HPV subtypesHPV-DNA test results of the subtypes in the corresponding subtype of the template region appear solid dots,which is positive for the subtype.HPV-DNA of various subtypes in normal cervical exfoliated cells are not expressed or weakly expressed in cervical lesions in each group there are different levels of expression:600 patients were positive for HPV detection were 245 cases,the positive rate was 40.83%(245/600),including the composite infection,specific infections shown in Table 1.Table 2.HPV21 subtypes,in addition to HPV43,HPV44 There are 19 kinds of subtypes have been detected,the highest rate of infection is HPV16(87/245),other common type for the HPV58(36/245),HPV6(33/2-45), HPV53(28/245),HPV18(20/245),HPV31(19/245),HPV52(19/245),cp8304(16/245) and HPV11(15/245).HPV infection in women can be seen in our province is the main subtype of HPV 16,HPV58 and HPV6.4.Expression of HPV 16/18 by westernHPV16/18 was overexpressed in cervical cancer85%(34/40) and CIN62.5%(25/40) compared with it in normal23.1%(3/13),P<0.05.The expression of HPV16/18 in cervical cancer and precancerous lesion correlated well with cervical neoplasia(CIN). It's consistent both in cervical exfoliated cell and in cervical tissue.Conclusions1 In CIN and CC,The positive rates of E-cadherin,β-catenin decrease with the aggravation of cervix affection. 2 In CIN and CC,The positive rates of HPV16/18 increase with the aggravation of cervix affection.3 In CIN and CC,the expression rate of E-cadherin,β-catenin and HPV16/18 showed a negative correlation,It will be more accurate for screening CIN and CC to detect them together.4 Testing HPV16/18 by western has high sensitivity,especially in CIN.It is suitable for questioned case by this way.It helps testing HPV16/185 The possible role of HPV16/18 in the remained samples of cervical liquid-based cells is coincident with that in cervical tissues.There is great feasibility in screening cervical affection by utilization of the remained samples of cervical liquid-based cells.6 HPV-DNA hybridization diversion typing detected 600 eases of women in our province of the positive rate of HPV infection to 40.83%(245/600),infection is the main subtype of HPV16,HPV58 and HPV6.