Study On The Expression And Biological Function Of SP100 In Laryngeal Carcinoma

Study on the expression and biological function of SP100 in laryngeal carcinomaIntroductionLaryngeal carcinoma is one of the most common head and neck cancers, whose incidence rates 1-5%of all human malignancies, and the incidence of laryngeal cancer has been rising over past decades:over 13,000 new cases were detected and diagnosed in 2007 in the Untied States, while laryngeal cancer has become the second most common reSPiratory system cancer in China and the northeast part of is the high incidence region of laryngeal carcinoma. DeSPite the quality of life for early stage cancer patients has improved in recent years due to organ preservation approaches, most late stage cancer is still fatal and the survival rate remains unchanged in the past 30 years. Like other malignancies, the initiation and progression of laryngeal cancer involve multi-factors and multi-steps. Thus, study on the molecular basis of tumorigenesis may greatly help make early diagnosis, develop target therapeutic strategies, and improve clinical outcomes including both survival rate and quality of life in the patient population.SP100 protein belongs to a permanent ProMyelocytic Leukaemian nuclear bodies (PML NBs) associated proteins, whose name comes from its SPeckled appearance pattern in the nucleus with approximately 100kDa molecular mass. SP100 protein family comprise of four isoforms:SP100HMG, SP100A, SP100B and SPOOC. SP100 protein is found mainly located in PML NB domain in the nucleus, and proved to be a multi-functional protein. SP100 participates in different cellular biological processes, such as regulation of transcriptional activity, viral infection defense, apoptosis, and so on. Study on participation of SP100 in human malignancies has not been reported before.In this study, SP100 expression levels were explored in different cancer cell lines and in laryngeal cancer samples as well. The biological function of SP100HMG and its interaction with SH3GL2 were studied. The aim of this study is to clarify the molecular mechanism of SP100 in malignant tumor, and to explore the potential diagnosis and treatment of SP1OOHMG as target in laryngeal cancer.Materials and MethodsHep2, Hela, Bel, SGC7901, BGC823, KPN, HEK293 and GES-1 cell lines were used in this study, and the SPecimens from 96 patients with laryngeal cancinoma together with the clinicopathological data were also analyzed. SP100 gene expression was detected using real-time RT-PCR and semi-quantitative RT-PCR in cell lines, and was detected by RT-PCR, Western blot and immunohisotological staining in clinical SPecimens.SP1 OOHMG transcript expression was evaluated using emi-quantitative RT-PCR in different cells lines. The biological changes of Hep2 cells after transfected with RFP-SP1 OOHMG vector were detected by MTT, flow cytometry, transwell, RT-PCR and Western blot. Co-immunoprecipitation was used to explore the interaction of SP100HMG and SH3GL2, and the possible binding site of two proteins predicted by bioinformatics software was confirmed by co-immunopecipitation.Result1. Real-time and semi-quantitative RT-PCR results showed SP100 mRNA levels were significantly lower in all the cancer cell lines than those in the normal mucosa cell lines.2. RT-PCR and Western blot results showed that both SP100 mRNA and protein levels were coherently down-regulated in laryngeal carcinoma compared to those in the adjacent mucosa tissues.3. Immunohistochemical staining results showed that SP100 protein was expressed in the nucleus and plasma, and expression level correlated with cancer cell differentiation. SP100 protein was predominantly located at plasma in low expression cancer cells.4. RT-PCR results showed that SPIOOHMG mRNA levels were significantly lower in all the cancer cell lines than those in the normal mucosa cell lines.5. Western blot results showed that SP100 protein level increased significantly in the RFP-SP100HMG transfected Hep2 cells, and RT-PCR results showed that SPIOOHMG expression was upregulated.6. Influence of SPIOOHMG transfection on biological characteristics of Hep2 cells:compared to the control group, the abilities of invasiveness were significantly lower, and the rate of apoptotic cells was significantly higher in Hep2 cells.7. Co-immunoprecipitation results showed that SP100HMG interacted with SH3GL2, and immunofluorescent staining indicated that the interaction site was located in the nucleus.8. The docking site of SP100HMG was located in the SAND-HMG, which was predicted by bioinformatics software and confirmed by co-immunoprecipitation.Conclusion1. Low expression of SP100 participates in the genesis and development of malignant tumor.2. SP100 might be a potential laryngeal cancer repressor.3. SP100 exerts different biofunction among different stages of laryngeal cancer.4. SP100HMG participates in the genesis and development of malignant tumor.5. SPIOOHMG can inhibit the invasiveness and promote the apoptosis of laryngeal cells, which indicates SPIOOHMG might be an potential tumor supressor gene..6. SPIOOHMG binds with SH3GL2 by SAND-HMG domain, which suggests that SP100HMG-SH3GL2 complex modulates downstream gene activity.