| Part One Expression and Clinical Pathology Significance of SIPA1Protein in Cervical CancerObjectiveTo explore the expression and clinical significance of signal induced proliferation associated protein1(SIPA1) in cervical cancer.MethodsIn paraffin embeded cervical tissues, including244cervical cancers and40normal cervix. The expression of SIPA1in these speciments was detected by immunohistochemistry. The correlations between expression of SIPA1and clinicopathological features of patients were analyzed.Results1. SIPA1was expressed in87.5%of normal cervix and58.2%of cervical cancer tissues. The difference was significant (P<0.01).2. In patients with versus without lymph node metastasis, the positive rate of SIPA1was17.0%and68.0%respectively (P<0.001). The risk of lymph nodal metastasis for patients with negative SIPA1expression in tumor was higher than SIPA1positive patients (OR=10.4,95%CI=4.578～23.484). There was a negative correlation between SIPA1and lymph node metastasis in cervical cancer (r=-0.331, P<0.001). However, SIPA1expression was not correlated with age at diagnosis, pathological type, FIGO stage or histological grading.ConclusionSIPA1is down-regulated in cervical cancer and associated with lymph node metastasis, suggesting it is a promising predictor for lymph node metastasis of cervical cancer. Part Two Relation of SIPA1Expression to HPV16/18E6and Ras-Raf-ERK pathway in cervical CancerObjectiveTo explore the relation of SIPA1expression to HPV16/18E6and Ras-Raf-ERK pathway in cervical cancer.Methods1. The expression of SIPA1and HPV16/18E6protein were detected in cervical cancer cell lines C33a, SiHa, HeLa and Caski by western blot.2. The expression of HPV16/18E6protein was detected in174in paraffin embedded cervical cancer tissues by immunohistochemistry.3. The recombinant plasmid pEGFP-N1-SIPA1-EGFP was constructed and then stably transfected into HeLa and SiHa cells by Lipofectamine2000. The cells were then screened by G418.4. Western blot was applied to determine the expression of SIPA1, ERK1/2, pERK1/2and integrin β1in HeLa and SiHa cell lines. 5. The expression of SIPA1, ERK1/2, pERK1/2and intergin β1in10in paraffin embeded cervical speciments was detected by immunohistochemistry.6. F-actin was observed under microscopy after immunofluorescent cytochemical staining.7. The cell proliferation activity was determined by MTT assay.8. The migration ability of cells was assessed by monolayer wound healing assay and transwell migration assay.Results1. The expression of SIPA1protein was negatively correlated with E6in cell lines.2. HPV16/18E6was expressed in79.3%of cervical cancers. E6expression in cancers with lymph nodal metastasis was significantly higher than that in those without lymph nodal metastasis (97.6%vs.73.4%, P<0.001). Additionally, odds ratio (OR) analysis suggested that the probability of metastasis in HPV16/18E6positive cases were14.79-folds (95%confidence interval [CI]=1.96～111.63) higher than negative cases. There was a negative correlation between SIPA1and HPV16/18E6in cervical cancer (r=-0.249, P<0.001).3. Western blot results showed that the expression of p-ERK1/2was decreased, and integrin β1was increased in HeLa SIPA1+and SiHaSIPA1+cells.4. HeLaSIPA1+and SiHaSIPA1+cells tended to round slightly and their pseudopods were decreased, and F-actin became filaceous and surrounding membrane, when compared with HeLavector and SiHavector cells.5.There were no significant effects on the proliferation of cervical cancer HeLa SIPA1+and SiHaSIPA1+cells.6. HeLasIPA1+and SiHaSIPAI+cells exhibited poorer migration ability compared with control cells in migration assay and wound healing assay (P<0.05).ConclusionThe down regulation of SIPA1in cervical cancer is associated with human papillomavirus infection, and SIPA1participates the regulation of Raf-MEK-ERK signal pathway. Re-expression of SIPA1in cervical cancer cells can inhibite cell migration, suggesting SIPA1is involved in cervical cancer invation by regulating Ras/Raf/ERK signal pathway.
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