Expression Of Maspin In Cervical Squamous Cell Carcinoma And Its Correlation With Tumor Lymphangiogenesis

Backgroud: Cervical cancer is the commonest malignant tumor of gynecological cancers. Although the obviously therapeutic effect on invasive cervical carcinoma have been made, fifty thousand female die of it every year. Now the data shows that the patients are making more youthful. Lymph node metastasis is the key route of distant metastasis, also a vital prognostic factor in cervical carcinoma and now the function of tumor lymphatic microvessel in the lymph node metastasis is being focused on. Being lack of specific marker of micro-lymphatic vessel for long term, study about tumor lymphatic vessel fell behind tumor vessel research. Podoplanin is a 43kd membrane protein and it was proved a qualified specific marker of lymphatic vessel. Research about the tumor lymphatic vessel is promoted vigorously by podoplanin.Maspin is a new member of the serpin (serine protease inhibitors) superfamily. Numerous researches of maspin revealed that it may be a multifunctional tumor suppressor for its notablely ability of inducing tumor cells apoptosis and anti-angiogenesis. A question that if there was correlation between maspin and the tumor lymphangiogenesis rose from genesis homology of the microvessel and the micro-lymphatic vessel.Objective: The study was performed to detect the expression of maspin, podoplanin-a marker of micro-lymphatic vessel, in squamous cell cervical carcinoma(SCC). To evaluate the effect of over-expressive maspin on cell proliferation ability in vitro and the tumor growth in vivo and investigate the expression of maspin, podoplanin in the transplanted tumor of the nude mouse. The aim of this study was to investigate the role of maspin in SCC development and the tumor lymphangiogenesis.Methods: The study was composed of three parts. 1. Immunohistochemistry (IHC) was performed to detect the expression of maspin, podoplanin in formalin-fixed, paraffin-embedded specimens of normal cervixes (n=13), cervical intraepithelial neoplasia-grade III(CINIH) (n=15), SCC(n=62) and lymph node with tumor cell positive(n=13).Density of lymphatic microvessel (LMVD) marked with podoplanin was counted. Correlations between expression of maspin and clinicopathologic parameters of SCC, LMVD were statistically analyzed. 2. The recombinant plasmid pcDNA3-maspin was stable transfected into human SCC cell line siha. Maspin mRNA was determined by RT-PCR and the maspin protein was detected by Western Blot and IHC methods. 3. The proliferation activity of the cells was measured with MTT method. The apoptosis rate and cell cycle distribution were detected by Flow cytometry to understand the changes of the cell bionomics characteristics. 4. Cells transfected by plasmid pcDNA3-maspin were seeded subcutaneously on female nude mice. Tumor growth, the expression of the maspin and LMVD were detected to evaluate the effect of maspin on tumor growth and the tumor lymphangiogenesis in vivo. Results: 1. (1) The maspin expressed both in SCC cytoplasm and nucleus and the maspin level was obviously reduced compared to normal cervix and CINIII (P<0.05) . Maspin in SCC II cells nucleus is obviously lower than SCC I b (P<0.05 ), however maspin in cytoplasm had no difference(P>0.05). A significant decrease in maspin scores was noted between normal cervix, CINIII, SCC vs tumor emboli in. lymph node (P<0.05) . (2) Significant correlation was noted between reduced maspin scores of SCC nucleus and later SCC stage, lymph node metastasis (P<0.05) , and there was no same relationship with other clinical parameters such as infiltration depth and histological grade (P>0.05) . Reduced cytoplasm maspin scores was only correlated with lymph node metastasis significantly (P<0.05) . Significant correlation was also noted between increased LMVD and later stage, lymph node metastasis and higher histological grade (P<0.05) . (3) There was significant negative correlation between maspin scores of SCC nucleus and clinical stage, LMVD (P<0.05) . Maspin scores of SCC cytoplasm was onlycorrelated with LMVD negatively (P<0.05) . Significant positive correlation was demonstrated between LMVD and clinical stage (P<0.05) . 2. The recombinant plasmid pcDNA3-maspin was stably transfected into human SCC cell line siha and the strengthened expression of maspin gene in siha cell was confirmed by RT-PCR, Western Blot and immunocytochemistry. Obviously suppressed proliferation activity and increased apoptosis rate of siha-m vs siha and siha-vector cell (siha-pc3) was disclosed by MTT and Flow cytometry methods (P<0.05) , and the latter two had no statistic significance (P>0.05) . 3. The growth of tumor were notablely inhibited by recombinant plasmid pcDNA3-maspin in vivo. Althouth not statistically significant, maspin scores of siha-m group nude mice tumor were higher compared to that siha and siha-pc3 group. LMVD of siha-m group nude mice tumor was markedly lower compared to siha and siha-pc3 group (P<0.05) , and the latter two had no difference.Conclusion: The expression of maspin in SCC was down-regulated and the decreased maspin at nucleus was significantly correlated with later clinical stage, lymph node metastasis and increased LMVD. The decreased maspin at cytoplasm was significantly correlated with lymph node metastasis and increased LMVD. The raised LMVD showed obviously correlation with later clinical stage, lymph node metastasis and higher histological grade. The tumor lymphatic microvessel was possiblely involved in pelvis lymph node metastasis. SCC cell line and the tumor growth were significantly suppressed by mapspin and especially the tumor lymphangiogenesis could be inhibited effectively. The research about anti-lymphangiogenesis of maspin would be a new focus and the maspin may be a new molecular target in the gene therapy of SCC.