Study On The Function And Regulatory Mechanism Of SH3GL2 Gene In Laryngeal Carcinoma

Study on the function and regulatory mechanism of SH3GL2 gene in laryngeal carcinomaLaryngeal cancer is a common head-and-neck tumor and 98% of laryngeal cancer belongs to laryngeal squamous cell carcinoma (LSCC), which affects the health of the LSCC patients mentally and physically. The incidence of LSCC has increased since past decades in China, especially in Northeast region. The pathological and molecular mechanism of LSCC is still not clear. It is important to discuss molecular basis of LSCC for early diagnosis and treatment of the tumor. In our previous study, we analyzed LSCC genomic DNA through CGH(comparative genomic hybridization) firstly, and discover high frequent loss at chromosome 9p22-23. Meanwhile, we find lots of differentially expressed genes in LSCC by eDNA microarray, among which SH3GL2 gene, which locates at chromosome 9p22, show significant down-regulation at different development stages of LSCC. Our RT-PCR, immunohistochemistry and Western blot results indicate that there exists relationship between SH3GL2 and LSCC. The data above imply that SH3GL2 participates in the carcinogenesis and development of laryngeal carcinoma and SH3GL2 maybe a new TSG(tumor suppressor gene) of LSCC. In this study, we explore the function and regulatory mechanism of SH3GL2 in laryngeal carcinoma, which helps to disclose the molecular mechnism of SH3GL2 in laryngeal carcinoma and find a new molecular marker of LSCC.MethodsThe eukaryotic expression vector and specific siRNA of SH3GL2 are constructed to assay the effect of abnormal SH3GL2 gene expression on cell biological feature, such as cell proliferation ability, apoptosis and in vitro invalibility, to further understand the probability role of SH3GL2 in laryngeal carcinogenesis. The mouse fused with SH3GL2 gene is constructed to research it further. Luciferase Reporter Gene Assay analyzes the 5'-flanking sequence of SH3GL2 to identify promoter and elements within it. And EMSA and co-immunoprecipation are used to research the regulatory mechanism and position of SH3GL2 in the signal transduction pathway.Results1. Obtaining of the overexpression Hep2 cell modelWe had successfully obtained the overexpression Hep2 cell model by transfecting eukryotic expression vector of SH3GL2 into Hep2 cells. The model was tested by RT-PCR ans western blot methods.2. Obtaining of the lowexpression Hep2 cell modelWe had successfully obtained the lowexpression Hep2 cell model by transfecting SH3GL2 specific siRNA into Hep2 cells. The model was tested by RT-PCR arts western blot methods.3. Effect of SH3GL2 on Cell proliferation abilityThe MTT assay showed that Hep2 cell proliferation ability was reduced significantly in the experiment group of SH3GL2 overexpression model (P＜0.05) and increased in that of lowexpression model (P＜0.05).4. Effect of SH3GL2 on apoptosisThe apoptosis of SH3GL2 overexpression and lowexpression model was detected by flowcytometry. Our results showed that the apoptosis of experment group in overexpression model was significantly increased (P＜0.05); while the apoptotic cells were decreased at some extend in the lowexpression model (P＜0.05).5. In vitro invalibility assayThe in vitro invalibility of Hep2 cells were finished by Transwell Chamber. In our study, the invalibility of experiment group showed significant decreased in overexpression model (P＜0.05) and no obvious alteration in lowexpression model (P＞0.05).6. Constucting and identifing of the mouse fused with SH3GL2 gene The mouse fused with SH3GL2 gene is constructed, seven F0 mouse were identified by PCR and Southern blot.7. Cloning and activity analysis of SH3GL2 gene promoterSH3GL2 gene 5'-flanking sequence was cloned. The region of SH3GL2 gene promoter was identified by Luciferase Reporter Gene Assay and two obvious modulating regions were found in this region. EMSA result displayed that NF-κB could combine 5'-flanking sequence of SH3GL2 gene.8. Identification of SH3GL2 interaction proteins by immunoprecipationTwo new interaction proteins of SH3GL2 were found by immunoprecipation, which were SP100 and hypothesis protein XP₉41880, respectively. 9. The expression change ofETS-1 gene followed those of SP100 gene and SH3GL2 geneWith the decreased expression of SP100 and increased expression of SH2GL2, the expression of ETS-1 gene also decreased (P＜0.05).Conclusion1. For the first time, we confirmed SH3GL2 gene, which was downregulated in laryngeal tissues, participated in laryngeal carcinogenesis and development,.2. For the first time, we verified SH3GL2 gene may be a potential tumor suppressor gene and regulated many biological effect of laryngeal carcinoma, such as cell proliferation, apoptosis and metastasis.3. We Constucted and identifed F0 mouse fused with SH3GL2 gene.4. For the first time, we identified the promoter of SH3GL2 gene.5. For the first time, we confirmed NF-κB can bind 5'-sequence of SH3GL2 gene。6. For the first time, we found SH3GL2 interacted with SP100 in laryngeal squamous carcinoma cells.7. Our result implies that SH2GL2 gene might regulate ETS-1 gene through SP100 gene.