The Significance Of Expression Of SLP-2 Protein In Several Malignant Cancers And The Primary Research On Its Function

Introduction:To study the function of a novel gene SLP-2 in the process of cancer genesis and development,make clear its location, research its protein function preliminaryly and consider the bioinformaticanalysis to speculate which signal transduction passageway SLP-2 may anticipate to go on thefurther study.PartⅠAims:To study the expression of SLP-2 protein in many malignant cancers, analyse the correlationbetween SLP-2 expression and clinicopathologic parameters and research the significance of theSLP-2 expression during the process of esophageal epithelial tumorigenesis.Methods:Use the technique of RT-PCR, Western blot and IHC to detect the expression of SLP-2 mRNAand protein in tissues of ESCC (220 cases), LSCC (104 cases), invasive breast cancer (263cases) and their normal counterparts, respectively. Analyse the association between SLP-2expression and clinicopathologic parameters and the variation of SLP-2 expression in differentprogress stages of esophageal epithelial transformation.Results:1. Compared with normal epithelium, ESCC tissues show higher expression of SLP-2 on thelevel of mRNA (74% cases) and protein (72%), IHC analysis on ESCC TMA reveal that thehigh expression of SLP-2 correlates with the extent of ESCC invasion (P=0.033).w2. Similarly, LSCC tissues show higher exprssion of SLP-2 than in their normal counterparts onthe level of mRNA (83% cases) and protein (70% cases), LSCC TMA analysis find thatSLP-2 high expression associates with advanced clinical stage (P＜0.001) and the metastasisof lymph node (P=0.003).3. Study on TMA of invasive breast cancer demonstrates that about 52.5% cases show SLP-2high expression and it can relate with tumor size (P=0.020), lymph node metastasis(P＜0.001), advanced clinical stage (P＜0.001), distant metastasis (P=0.002) and HER2/neuprotein expression (P=0.037). Survival analysis suggest that SLP-2 high expression canobviously decrease the overall survival of the patients. 4. IHC staining results show that SLP-2 protein can express in the early stage of atypicalhyperplasia of esophageal epithelium, with the consistent expression level in ESCC tissue.SLP-2 protein mainly locate near basal membrane in early stage of atypical hyperplasia,whereas it can concentrate on the periphery of mass of invasive cancer cells. Also singlecancer cell can be detected by SLP-2 staining.Conclusion:1. SLP-2 can be a novel cancer related gene, can play an important role in tumorigenesis ofmulticancer.2. The high expression of SLP-2 can associate with cancer invasion and metastasis.3. SLP-2 protein expresses in the early stage of epithelial malignant transformation, itdemonstrates that SLP-2 can be of great value to early diagnosis of cancer.PartⅡAims:To study the location of SLP-2 protein from cellular level to molecular level, make clear itsfunction and research its interactive protein.Methods:Use ultracentrifugation to separate the special component of cell membrane-lipid raft and detectwhether it can contain SLP-2. Use immunoelectromicroscope staining and immunofluorescencestaining to observe where SLP-2 protein can locate and whether SLP-2 protein can co-locatewith cytoskeleton proteinα-actin orβ-tubulin, respectively. Co-IP and GST pull down assays canstudy whether SLP-2 protein can interact withα-actin from in vivo and in vitro.Results:1. After ultracentrifugation, the cellular component can be confirmed by caveolin-1 antibody tobe lipid raft, which can contain the SLP-2 protein detected by SLP-2 antibody.2. Under immunoelectromicroscope observation, colloid gold marked SLP-2 can be detectedand mainly focused on cytoskeleton fibre, their binding are stable and specific.3. From immunofluorescence staining observation, under different moving state, SLP-2 proteincan co-locate with cytoskeleton proteinα-actin.4. Co-IP assay shows that SLP-2 can interact with cytoskeleton proteinα-actin, but GST pulldown assay does not support it.Conclusion:1. SLP-2 protein can locate in lipid raft, it may possesses biomolecular activity to participate the signal transduction in the membrane region.2. SLP-2 protein can bind cytoskeleton protein specificly, maybe it can act as cytoskeletonprotein or cytoskeleton related protein to regulate cell movement.3. SLP-2 protein can co-locate with cytoskeleton proteinα-actin, they can act together-tocontrol cell movement.4. Initial result shows SLP-2 protein can interact with cytoskeleton proteinα-actin, co-regulatecell movement, control the process of invasion and metastasis of cancer cell. It also needsfurther research to confirm.PartⅢAims:To verify the function of cytosketeton related protein SLP-2, research whether, directed inhibitionof the expression of SLP-2 protein can change the ability of cell invasion and migration andobserve whether TPA can affect SLP-2 expression. To infer which signal transductionpassageway SLP-2 can join in.Methods:Use the technique of RNAi can directed inhibit SLP-2 protein expression. After it, transwellassay can be done to compare the ability of cell invasion and migration. Use TPA to stimulatecell to study whether the expression of SLP-2 protein can be changed. Use bioinformatic analysisto infer which signal transduction passageway SLP-2 can join in.Results:1. Transfecting with Hs-STOML2-2-siRNA, after 72h, SLP-2 protein can be inhibited directlyand completely.2. Transwell assay show that the cells with SLP-2-siRNA transfection have obviously decreasedinvasive ability, but show no variation of migration ability.3. Use TPA to stimulate cell, the expression of SLP-2 in EC9706 cell have no change, but showhigher expression in Hela and KYSE510 cell.4. Bioinformatic analysis reveal that the structure domains of SLP-2 protein and Rho familyprotein may exist interaction.Conclusion:1. RNAi technique can effectively inhibit certain protein expression.2. SLP-2 can only affect cell invasion ability rather than cell migration ability.3. TPA can induce the varable expression of SLP-2 protein, but show difference on cell type,effective time and change intensity. 4. Bioinformatic analysis reveal that SLP-2 protein may interact with Rho family protein toanticipate Rho signal transduction passageway to regulate cell invasion.