| Cervical carcinoma is the second most common malignant disease among women, with nearly 80% of cases in less developed countries. Cervical cancer will develop in about 500,000 women yearly worldwide, and it is the most common cause of cancer death and years of life lost owing to cancer. The primary cause in development of cervical cancer is high-risk human papillomavirus (HR-HPV). Infection of HR-HPV and persistent expression of viral oncogenes E6 and E7 are causally linked to the cell abnormal transformation and proliferation, which is essential for cervical carcinoma occurrence. Recent studies indicated that cell transformation and proliferation are necessary but insufficient for complete transformation of human epithelial cells in vivo; the cell dedifferentiation may play key roles in canceration.Relative longer interval from HR-HPV infection to cancer occurrence and slower progression and extension of cancer make early diagnosis and early treatment of cervical carcinoma possible. Radical hysterectomy and pelvic lymphadenectomy are initial treatment for early stage disease. Some clinical pathological parameters, regarded as factors that are able to predict good or poor prognosis of the patients who underwent surgery, are usually used as guidance of subsequent treatment following surgery, such as radiation therapy. The clinical stage, status of the lymph nodes, size of tumor and tumor cell differentiation are the most affirmative prognostic factors of cervical cancer. Many studies have reported that the patients with poor celldifferentiation have lower survival rate. The tumor cell differentiation may be one of the most important prognostic factors in cervical carcinoma.It is well known there must be a dynamic balance between cell proliferation and differentiation in normal embryo development and normal tissues. However, the key molecular mechanism involved in those procedures has not been clarified up to date. Many of the transcriptional factors may constitute an intricate network in regulating human embryonic stem cell (hESC) differentiation, such as LIF-BMP, OCT3/4, Wnt, Notch-Hes signal pathways. The Notch-Hes pathway is the most important in regulating the cell differentiation. Notch1 is a transmemberance protein, determines the stem cell fate in embryonic development and inhibits the cell differentiation via the Notch-Hes pathway. The bHLH gene Hes1 and Hes5 are candidate target genes for mammalian Notch pathway, highly expressed in epithelia in the process of embryogenesis or in neural stem cells. Hes1/Hes5 directly binds to the N-box (CACNAG) and actively represses the gene expression in association with TEL/Grg or forms a non-functional heterodimer with some bHLH transcription activators (such as Hash1, E47, Myo D) with E-box (CANNTG) which activates the gene expression, then inhibits the activators' activity and represses the transcription.The roles of Notch-Hes pathway in regulating the cell differentiation and proliferation indicates that it may participate the occurrence and the development of tumor. The abnormal expression of Notch1 has been detected in many human solid tumors (such as cervical carcinoma, endometrial carcinoma, renal carcinoma, breast carcinoma, pleural endothelioma, et al) and hematological system tumors. As effectors of Notch1, the roles of Hes1 and Hes5 in tumorigenesis was not clear, the association of Hesl/Hes5 and Notch-Hes pathway with the tumorigenesis and development of cervical carcinoma has not been reported up to now, to our knowledge.In our study, we detected the expression of Hes1 and Hes5 in normal cervical epithelia, CIN and SCC; analyzed the correlation with the prognosis of early-stage cervical carcinoma to investigate the relationship between Hes1/Hes5 and the tumorigenesis, prognosis of cervical carcinoma. Then knockouted the Hes1 and Hes5gene in SiHa cells respectively by RNAi, examined the expression of differentiation and proliferation associated protein in SiHa cells after RNAi, elucidated the roles of Hes1/Hes5 and the signal pathway in regulating cervical carcinoma cell differentiation.Part IExpression of Differentiation Associated Protein Hes1 and Hes5in Cervical Squamous Carcinoma and its PrecursorsObjective Detected the expression of Hes1 and Hes5 in normal cervical epithelia, CIN and squamous cervical carcinoma cells to investigate the relationship between Hes1/Hes5 and the tumorigenesis, prognosis of cervical carcinoma.Methods The specimens (n=295) were obtained from the Pathological Department of Women's Hospital, School of Medicine, Zhejiang University from October 2004 to June 2005, and comprised 78 normal cervical epithelia, 31 mild dysplasia (C1NI), 77 moderate-severe dysplasia (CIN II-III) and 109 squamous cervical carcinomas (SCC). All the patients were not treated with chemotherapy, immunotherapy, or radiotherapy prior to specimen collection. Among the 109 SCC patients, 73 were diagnosed as early-stage (including 10 Ia, 59 Ib, and 4 IIa) and underwent radical hysterectomy and pelvic lymphadenectomy. The complete clinical pathological parameters were collected, including (1) age of the patients, (2) clinical stage, (3) differentiation of tumor cells, (4) tumor size, (5) invasion depth of cervical stroma, (6) status of lymph nodes, (7) involvement of lymph-vascular space in parametrial tissue. The expression of Hes1 and Hes5 were detected by immunohistochemistry.Results 1. There were significant differences of expression of Hes1 and Hes5 among normal, CIN and SCC groups (P=0.000). Expression of both Hes1 and Hes5 in SCC were higher than that in normal and CIN, as well as higher in CIN than in normal. 2. The expression of Hes1 and Hes5 were significantly increased in both larger than 2cm of lesion size and deep stroma invasion group. In addition, Hes5 was also significantly more expressed in relative later clinical stage, poorer cell differentiation, and lymph node metastasis.Conclusions1. Hes1 and Hes5 may be involved in carcinogenesis and progression of cervical carcinoma.2. Overexpression of Hes1 and Hes5 are probably variables to predict poor prognosis of the patients with early-stage cervical carcinoma.Part IIRegulating Roles of Hes1/Hes5 in Differentiation and Proliferation inCervical Carcinoma CellsObjective To investigate the roles of Hes1/Hes5 and Notch-Hes pathway in regulating proliferation and differentiation in cervical carcinoma cells; elucidate the roles of Hes1/Hes5 and its signal pathway in cervical carcinoma cell differentiation.Methods Silenced the Hes1 and Hes5 gene respectively in SiHa cells by RNAi, detected the expression of Hash1 (Hes1/Hes5 downstream protein), Notch1 (Hes1/Hes5 downstream protein), differentiation and proliferation associated protein in SiHa cells by immunocytochemistry and western blot after gene silence. The ability of cell proliferation was evaluated by MTT.Results 1. The expression levels of Hes1/Hes5 mRNA and protein in experiment group were significantly decreased compared with the blank and negative control group after gene silence (P=0.000). 2. After Hes1/Hes5 RNAi, the expression of their downstream protein Hash1 was significantly enhanced, while the expression of their upstream protein Notch1 was not significantly changed; and the expression of differentiation associated protein Nanog, SSEA-4, TRA-1-60 were significantly decreased in experiment group compared with the blank and negative control group (p<0.05) after RNAi; 3. The expression of Ki-67 and PCNA in SiHa cells after Hes1 and Hes5 gene silence were significantly decreased (P<0.05) in experiment group compared with blank and negative control, and the ability of cell proliferation was significantly weaker. There was no different between negative control group and blank control group.Conclusions1. RNAi is able to silence Hes1/Hes5 gene successfully in cervical carcinoma cells.2. Hesl/Hes5 may inhibit the cervical carcinoma cell differentiation, in which Hash1 is involved as a signal pathway.3. Downregulated Hes1/Hes5 expressions may inhibit the proliferation of cervical carcinoma cells. |
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