Pin1 Is Overexpressed And Associated With Proliferation And Apoptosis By Up-regulating Cyclin D1 In Cervical Cancer

Phosphorylation of serine or threonine residue preceding proline (Ser/Thr-Pro) is a key regulatory mechanism involving proliferation and transformation. The conformation of certain phosphorylated Ser/Thr-Pro bonds is regulated specifically by the prolyl isomerase Pin1. Such conformational changes following phosphorylation are a novel signaling mechanism. Its imbalance would induce oncogenesis. Recent work indicates that Pin1 is strikingly overexpressed in many human cancers and cooperates with many oncogenesis signaling. As a catalyst, Pin1 plays a pivotal role in oncogenesis in solid cancers such as breast and prostate cancer. Cervical cancer is the most common in gynecologic cancers and belongs to solid cancer. It's one of the leading causes of cancer-related death in women. Although the detailed molecular mechanism remains to be elucidated, studies have now confirmed that HPV is tightly associated with cervical cancer. In HPV oncogenesis signaling, many factors might be enhanced by Pin1 because they contain Ser/Thr-Pro. Among of them, cyclin D1 is essential for neoplastic transformation induced by HPV E₆/E₇ in normal cells. E₆/E₇ fail to induce neoplastic transformation of cyclin D1(-/-) cells in vivo and in vitro. The abnormal of cyclin D1 plays an important role in tumorigenesis malignancy in cervical cancer. However, it is not clear why cyclin D1 is deregulated. Interestingly, Pin1 positively regulates cyclin D1 function not only at the transcriptional level but also through post-translational stabilization in breast cancer. This ensures cyclin D1 accumulation in cell nucleus and being active persistently. We supposed that Pin1 might be a catalyst and contribute to cervical cancer by regulating cyclin D1. Pin1 overexpression might contribute to the disruption of the cellular signaling balance and enhance oncogenesis signaling by up-regulating cyclin D1. This might be a pivotal risk factor for neoplastic transformation and uncontrolled proliferation induced by HPV E₆/E₇ in normal cervical epithelium. However, the analysis of Pin1 and cyclin D1 expression has not been demonstrated, and it is not known whether Pin1 depletion would affect actual cellular growth or would block tumorigenic properties in human cervical cancer. This study was to elucidate these questions. OBJECTIVE:This study was to investigate the expression and clinical significance of Pin1 and cyclin D1 in cervical cancer tissues; to elucidate the effects of manipulating Pin1 on cyclin D1 expression in HeLa cells; to explore the potential of Pin1-targeted gene silencing in inhibiting cellular growth and tumorigenicity in cervical cancer. METHODS:A. Pin1, cyclin D1 and Ki67 were detected by immunohistochemistry in 88 samples of cervical tissues involving 10 normal cervixes, 21 cervical intraepithelial neoplasia (CIN) and 57 invasive cervical cancers. Their clinical significance was analyzed in cervical cancer. Amplification of Pin1 gene was examined by RT-PCR in 15 invasive cervical cancers and 5 normal cervixes among of the above them.B. Amplification of Pin1 gene was examined by RT-PCR, and the expression of Pin1 protein was examined by immunoblotting and immunocytochemistry in HeLa, Siha, C33a and Caski cell lines.C. A sense Pin1 recombinant plasmid was constructed and transfected to up-regulate Pin1 in cervical cancer cells, and then Pin1, cyclin D1 were detected by RT-PCR and immunoblotting. The proliferation and apoptosis after transfection were examined by MTT assay, colony formation in soft agar and flow cytometry (FCM).D. A new technique, RNA interference (RNAi) was used to silencing Pin1 specifically. The effect of Pin1 depletion on cyclin D1 expression was detected by immunoblotting. The proliferation and apoptosis after silencing Pin1 in HeLa cells were examined by MTT assay, colony formation in soft agar and flow cytometry (FCM).RESULTS:A. Compared with normal cervical epithelium, Pin1 mRNA and protein were differently overexpressed in cervical cancer tissues (p<0.05). The expression of Pin1 increased progressively along with the disease process from normal cervix to CIN and to invasive cervical cancer (p<0.05). No significant difference of Pin1 expression was found with clinicopathologic parameters, such as FIGO stage, pathological grade and lymph node metastasis (p>0.05). But the expression of Pin1 was significantly higher in adenocarcinoma than those in squamous carcinoma of the uterine cervix (p<0.05). In cervical cancer tissues, the over-expression of Pin1 was positively correlated with that of cyclin D1 and Ki67 (p<0.05).B. Comparing with normal cervical epithelium, Pin1 mRNA and protein were differently overexpressed in HeLa, Siha, C33a and Caski cell lines (p<0.05).C. Up-regulated Pin1 led to the elevation of the endogenous cyclin D1 protein in HeLa cells. In contrast, Pin1 deletion specifically with RNAi technique obviously suppressed the expression of cyclin D1.D. Specific silencing of Pin1 with RNAi blocked the proliferation and induced the apoptosis of cervical cancer cells (p<0.05). Exogenously increased Pin1 had no significantly effects on the proliferation and the apoptosis of cervical cancer cells (p>0.05).Conclusion:A. Pin1 was strikingly over-expressed in cervical cancer tissues as well as cell lines compared with the normal cervical tissues. Its level was tightly correlated with cyclin D1 and Ki67. Pin1 may be involved in cervical oncogenesis as a pivotal catalyst.B. Pin1 overexpression might promote transformation and uncontrolled proliferation of cervical epithelial cells. One of mechanisms of Pin1 is to regulate the endogenous cyclin D1 expression.C. The over-expressed Pin1 may act as a novel molecular marker for diagnostics and a specific target for therapeutics in human cervical cancer. Pin1-targeted RNAi might be useful to control this disease.