Relation Between HPV16/18 And Pin1, CyclinD1 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 huron 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.Pin1,CyclinD1 was overexpressed in cervical cancer and cervical intraepithelial neoplasia(CINⅠ,Ⅱ,Ⅲ)compared with it in normal cervical tissue.So detection of Pin1,CyclinD1 protein and HPV16/18 in cervical exfoliated cells or tissue is very important for diagnosis at cervical cancer and precancerous lesion.In our study,we detect HPV16/18 by the hybridization in situ and Pin1,CyclinD1 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)specimen570 cervical liquid-based cells remained samples were obtained from the Department of gynecology in the First Affiliated Hospital of China Medical University, during Nov 2006 to Nov 2007.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 CINⅠwere 21,with CINⅡ\Ⅲwere 19. The average age is 48.4(range,26～72 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 Pin1,CyclinD1 protein was friendly presented by Jin woo Kim professor in Korea.S-P Kit and DAB Kit were obtained from MAIXIN biological technology company in Fuzhou.2.Methods(1)Detection of Pin1,CyclinD1 by Immuohistochemical analysis S-P methods. Resultdetermination:The overexpressed staining was in the cytoplasm.The number of staining cells were graded as follows:10-24%(+);25-49%(++);50-74%(+++);≥75% (++++).(2)Detection 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)Detection of HPV 16/18 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 supernatant 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. (4)Statistical analysisAll the datas were analysis with x~2 test and Fisher's test,P=0.05.The relation between HPV 16/18,Pin1 and CyclinD1 was analysis with spearman analyses,P=0.05.Results1.Expressions of Pin1,CyclinD1 in cervical exfoliated cells and cervical tissue by Immuohistochemical analysis S-P methods.Pin1 was overexpressed in cervical cancer85%(34/40),CINⅡ/Ⅲ68.4%(13/19),CINⅠ57.1%(12/21)compared with that in normal cervical exfoliated cells and tissues 7.7%(1/13)(p＜0.05).The expression of Pin1 in cervical cancer and precancerous lesion correlated well with that in cervical neoplasia(CIN).It's consistent both in cervical exfoliated cell and in cervical tissue.2.Expression of HPV 16/18 by Hybridization in situ.HPV16/18 was overexpressed in cervical cancer82.5%(33/40)and CINⅡ/Ⅲ57.9%(11/19),CLNⅠ42.9%(9/21)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.Expression of HPV16/18 bywesternHPV16/18 was overexpressed in cervical cancer85%(34/40)and CINⅡ/Ⅲ68.4%(13/19),CINⅠ57.1%(12/21)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.4.The correlation of HPV16/18 and Pin1,CyclinD1Overexpression of HPV16/18 and Pin1 showed a significantly positivecorrelation, (r_s=1,P＜0.05.)Overexpression of HPV16/18 and CyclinD1 showed a low correlation, (r_s=0.6,P＜0.05.) Conclusions1,In CIN and CC,The positive rates of Pin1,CyclinD1 and HPV16/18 increase with the aggravation of cervix affection.2,In CIN and CC,the expression rate of Pin1,CyclinD1 and HPV16/18 showed a positive correlation,It will be more accurate for screening CIN and CC to detect them together.3,Testing HPV16/18 by western has high sensitivity,especially in CIN.It is suitable for questioned case by this way.It helps testing HPV16/184,The possible role of either Pin1,CyclinD1 or 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.