Studies Of Cervical Carcinogenesis And Metastasis Associated With Pinl-induced Epithelial-mesenthymal Transition(EMT)and The Regulation Of Gene Expression

Objective:In this study,we will focus on cervical cancer in Uyghur women and study the association cervical carcinogenesis,invasion and metastasis with the expression of peptidyl-prolyl cis-trans isomerase 1(Pinl)that might induce epithelial-mesenchymal transition(EMT)by regulation of candidate proteins specific to epithelium and mesenchyma in tumor tissues and cancer cell lines,for revealing the mechanism of cervical carcinogenesis and metastasis related to EMT and identify molecular markers for early diagnosos and monitoring the treatment of cervical carcinoma.Methods:(1)We collected 189 cases of cervical lesions,including 116 cases of cervical squamous cell carcinoma(CSCC),32 cases of cervical intraepithelial neoplasia(CIN)Ⅱ-Ⅲ and 41 cases of normal controls.The protein expression of Pin 1,E-cadherin,N-cadherin.β-catenin,Snail1 and Vimentin was detected by immunohistochemical staining,and the association of clinical parameters of cervical carcinoma with changes in the expression of these proteins was further analyzed.(2)The mRNA and protein expression of Pin 1 was determined in HeLa,SiHa and C33a cervical carcinoma cells by real-time fluorescent quantitative RT-PCR.The SiHa cervical carcinoma cells were transfected with the RNAi vector expressing shRNA fragment corresponding to coding mRNA of Pin 1 gene and established as a cell model by determination of mRNA and protein expression of Pin 1 by quantitative RT-PCR and western blotting.The changes in mRNA and protein expression of E-cadherin,β-catenin,N-cadherin,Snail1 and Vimentin were also detected before and after transfection.(3)The effect of Pin 1 on the cell proliferation.invasion,and migration as well as cellular apoptosis and cell cycle check point was analyzed using tetramethylthiazolate(MTT)method,cell scratch test and flow cytometry.Results:(1)Immunohistochemical analyses showed that the development of cervical carcinoma and its precursor lesions was accompanied with the significantly increased expression of Pinl and epithelial-specific proteins N-cadherin and Snaill(P<0.05),and the significantly decreased expression of mesenchima-specific proteins E-cadherin and P-catenin(P<0.05),in addition to relatively high clinical stage,median and low differentiation state and lymph node metastasis of cervical carcinoma that were also accompanied with significant changes in the expression of these proteins(P<0.05),which might be manifested in the trend of Pinl mediated epithelial-mesenchymal transition(EMT),malignant transformation and metastasis of cervical carcinoma.(2)Pin1 expression was detected in several cervical carcinoma cells,such as HeLa、SiHa and C33a cell lines,with the relatively high expression in SiHa cervical carcinoma cells.After transfection with sh-RNA specific to Pinl mRNA,Pinl transcription and protein expression were significantly decreased(P<0.05),and this was accompanied with significant downregulation of epithelial-specific genes E-cadherin and β-catenin(P<0.05),and the upregulation of mesenchymal-specific genes N-cadherin,Snaill and Vimentin(P<0.05),presenting a change in the expression pattern of EMT related genes regulated by Pin 1 expression.The cell biological analyses showed that the cell proliferation and migration were decreased and cellular apoptosis increased with the transfection of Pinl specific sh-RNA,presenting a typical EMT transformation phenotype.Conclusion:(1)In accordance with the studies focusing on patients,the development of cervical carcinoma,malignant transformation,median-and low differential state and metastasis might be accompanied with the upregulation of Pinl and mesenchyma-specific proteins,and the downpregulation of epithelium-specific proteins,presenting manifestations of typical EMT transformation.(2)With the inhibition of Pinl expression by RNAi cell model,the expression pattern of EMT related genes and cell phenotype were reversely regulated,which indicates that Pinl may induce EMT transformation of the cervical epithelium via regulation of epithelium-and mesenchyma-specific gene expression.