The Expression And Role Of Deubiquitinating Enzyme Ataxin-3in Gastric Cancer

Investigated backgroundGastric cancer (GC) is one of the most common malignant tumor with thehighest mortality rate. China is one of the high incidence area of gastric cancer,it is23.2%about the death number for GC vs. all kinds of cancers. Theprognosis for patients with advanced gastric cancer is poor, and the overall5-year survival rate is less than30%after surgery. Treatment about combinedof multiple targets for advanced gastric cancer has become an important strategyof human against gastric cancer. Because the molecular mechanisms ofmalignant tumor are complex and diverse, the molecular mechanism of gastriccancer is still not very clear, so the effective therapeutic targets still faces tohuge challenges, more researchs remains to be done.The ubiquitin proteasome system (UPS) is one of the protein degradation andthe quality control primary ways. UPS plays important role in cell quantitativecontrol by degradate the degener ation, misfolded,abnormal accumulation andtranslation of damaged proteins. In some cases, the amount of abnormal proteinsto be degraded exceeds the degrading capacity of the UPS, which can result inthe abnormal accumulation of ubiquitinated proteins and may eventually cause cell dysfunction, cells death, or possibly major diseases, such as cancer.Deubiquitinating enzymes (DUB) are an important part of the UPS, and functionin the deubiquitination of proteins by recognizing specific sequences in ubiquitin(Ub) and proteasome or substrates. Involve the regulation of malignant tumorcell function is mainly manifested in the following aspects:1) Regulation of cellsignaling pathway.2) Control is associated with survival and cell cycle of cells.3) Involving in regulation of transcription. The dysregulation of ubiquitinationand deubiquitination is closely related to the occurrence of tumor. As adeubiquitinating enzyme, Ataxin-3has many similarity functions to the DUBsrelated to cancer. So it is worthy of further study on its role in the developmentof malignant tumor.Ataxin-3is a polyglutamine protein (polyQ) and a kind of important DUBs.That was initially considered for the core etiology of spinocerebellar ataxin type3and Machado-Joseph disease (SCA3/MJD). The human Ataxin-3protein isencoded by the ATXN3gene located on chromosome14q21, and is expressedby cells throughout the body. The present reporte, human Ataxin-3proteinincluded12transcription subtypes, the longest subtype is about376amino acidresidues, and the molecular weight is42kDa. The Ataxin-3protein contains aglobular, N-terminal Josephin domain (JD) with deubiquitination function, theC-terminal tail has2~3basic models of ubiquitin-interacting motifs (UIM)that is ubiquitin binding structure and polyQ region. The main functions include:1)Interact with ubiquitin, polyubiquitin chain, ubiquitin like proteins (such asNedd8) and ubiquitinated proteins.2) Edit the polyubiquitin chain with it’s DUBactivity.3) Interaction with proteasome subunit.4) Interaction with compositemolecular (such as VCP/p97). The main cell and biochemical processes wereincluding: influence the degradation of proteins for DUB’ activity, transcription regulation, participation in cell apoptosis and death pathways, someorganizations’ cell differentiation and formation of cytoskeleton, etc. As adeubiquitinating enzyme, Ataxin-3has many similarity functions to the DUBsrelated to cancer. At present, the research on Ataxin-3is very limited, especiallyrare on its role or statu of the cell’s malignant transformation and tumors’development. Recently, application an unbiased siRNA screen in non-small-celllung cancer cells to identify deubiquitylases (DUBs) that have an impact onPI3K signaling by regulating the abundance of PTEN. That found PTENexpression was mostly induced by depleting DUBs——Ataxin-3. In A549cells, while ANTX3gene function defects, the transcription of PTEN enhanced,enough down regulation of Akt phosphorylation and activation of PI3K pathway,then the cell viability decreased.TP53tumor suppressor gene is a gene of the uppermost relationship tohuman tumors. The wild and mutant types of Ataxin-3can all enhance p53transcriptional activity. The chaperone-associated ubiquitin ligase CHIP (Cterminus of Hsc70-interacting protein, also called STUB1or STIP1) is animportant molecular chaperone dependent ubiquitin ligase E3. The researchshowed that CHIP can target both wild-type and mutant p53for degradation. Asa deubiquitinating enzyme (DUB), Ataxin-3participates in initiating, regulatingand terminating the CHIP ubiquitination cycle by limitting the length ofubiquitin chains on CHIP or substrates. But whether there is difference aboutthe expression of Ataxin-3in gastric cancer tissues? Does Ataxin-3involve incancer cell growth and apoptosis control process in GC? Dose Similar to thenetwork about CHIP and p53interaction with Ataxin-3exist in the gastriccancer? There are not reported.Becaue, there is no information on the exact that the expression and role of Ataxin-3in gastric carcinoma. This study will be designed to investigate theexpression of Ataxin-3in gastric cancer, preliminary exploration for the relevantfunction and mechanism.The content of this study includes the following three parts:Part1Expression and significance of Ataxin-3in gastric carcinomaObjectiveDetected the expression of Ataxin-3protein in GC, intraepithelial neoplasiaand control gastric mucosal tissues adjacent GC. Investigated the Ataxin-3expression relation with clinical and pathological features of patients withgastric cancer, and conducted single factor and multi factor analysis; Detectedthe expression of Ataxin-3mRNA in gastric cancer tissues and control mucosaltissues, in patients with gastric cancer, Ataxin-3whether there are differences inexpression at the transcriptional level; analyze whether there are differences ontranscription about Ataxin-3in gastric cancer; Detected the expression ofAtaxin-3protein and mRNA in human gastric mucosal epithelial cells (GES-1),gastric cancer cells (SGC7901) and (MKN45), analyze the difference ofAtaxin-3expression in gastric cancer cells. The implementation of this studywill provide experimental evidence for the role and status of Ataxin-3in gastriccancer, especially in the carcinogenesis and development.Methods1. Immunohistochemical detected the expression of Ataxin-3protein in gastriccancer (536cases), intraepithelial neoplasia (low grade86cases, high levels of75cases) and the control of gastric mucosa (312cases). Combined with the theclinical and pathological characteristics of gastric cancer patients, analyzed therelationship with gastric intraepithelial neoplasia, the clinical pathologyfeatures of gastric cancer and survival factors. 2. Application of real-time fluorescent quantitative PCR (Real-time PCR) forthe detection of Ataxin-3mRNA in gastric cancer (14cases) and control gastricmucosa (14cases), to analyze whether there are differences in expression at thetranscriptional level.3. Application of Real-time PCR and WB（Western blot） to detect theexpression of Ataxin-3protein and mRNA in human gastric mucosal epithelialcells (GES-1), gastric cancer cells (SGC7901) and (MKN45), analyze thedifference of Ataxin-3expression in gastric cancer cells.Results1. Immunohistochemistry showed the expression of Ataxin-3was100%of all ofthe control gastric mucosa(312cases). The positive rate of Ataxin-3in lowgrade intraepithelial neoplasia (86cases), high grade intraepithelial neoplasia(75cases) and gastric cancer (536Cases) were58.1%,88%and72.76%respectively. Comparison of expression intensity of each group, gastric cancerand high grade intraepithelial neoplasia group has no difference (P>0.05), andthe other groups were significantly different (P <0.001).2. The expression of Ataxin-3have relevant with the tumor size, histologicaltype (Lauren), histological differentiation and mutation P53protein (P <0.05),the rank correlation coefficients were:-0.087(P <0.05),-0.384(P <0),-0.230(P <0) and+0.108(P <0.05) respectively. But there were no relationship withage, gender, tumor location, Bormann classification, depth of invasion, lymphnode metastasis, distant metastasis and clinical staging of TNM independently(P>0.05).3. The median survival time was32.73months and the1,3,5-year cumulativesurvival rates were71%,46%and33%.4. Univariate analysis showed that5-year survival rate had relation with the location of the tumor size, histological type, Lauren, depth, lymph nodemetastasis, infiltration of the number of tumor metastasis, TNM staging andmutation p53protein expression (P <0.05). However,Age, gender, pathologicaltype, histological differentiation, invasion, the expression of Ataxin-3protein,smoking and drinking history not found relation with prognosis (P>0.05).5. Multivariate analysis showed that tumor site, histological type Lauren,histological differentiation, clinical stage and mutation of TNM P53proteinexpression is an independent prognostic factor of gastric cancer (P <0.05); andnot suggest that age, gender, tumor size, Bormann type, smoking and drinking isto have independent prognostic significance (P>0.05).6. The expression of Ataxin-3mRNA in gastric cancer tissues and cell lineswere lower than that in the control of gastric mucosal tissues and cell lines (P <0.05); the expression in MKN45was minimum.7. The results by WB suggested that Ataxin-3protein expression was higher inGES-1than in SGC7901,and at least in MKN45.Conclusion1. Down regulation the expression of Ataxin-3had closely relation with gastriccancer (especially in diffuse type gastric carcinoma) and tumor celldifferentiation state distant metastasis. It was suggested that Ataxin-3might berole in the carcinogenesis and development in gastric cancer.2. There was no direct correlation between the prognosis of GC patients andAtaxin-3, suggesting that it might be play a synergistic role. Ataxin-3mightbecome a candidate factor for the multi targets treatment of gastric cancer.Part2The function of Ataxin-3in gastric cancer cellObjectivepLV-EF1a-EGFP-2A-ATXN3plasmid was transfected into SGC7901cell. Over expression of Ataxin-3effected on SGC791cell growth, apoptosis, theexpression of p21, Bax, P53mRNA, and the expression of the CHIP protein.Further analyzed the mechanism of Ataxin-3in gastric cance from the view offunction.MethodsThe pLV-EF1a-EGFP-2A plasmid was transiently transfected into theSGC7901cells. There were a blank control group (SGC7901), ATXN3overexpression and vector control group. Through the CCK8method to detect cellproliferation, flow cytometry to detect cell apoptosis, Real-time PCR detect themRNA expression of p21, Bax, P53,which were cell cycle and apoptosis relatedfactors, and WB to detect the expression of CHIP protein.Result1.Over expression of ATXN3in SGC7901showed significant growth inhibitionto cells.2. The apoptosis rate was obviously increased in cells with overexpression ofATXN3.3. The mRNA expression of p53was the lowest, and Bax was the highest (P <0.05) in the cells with over expression of ATXN3; and p21mRNA expressionhad no significant difference in the three cells (P>0.05).4. The Western blot assay showed that overexpression of Ataxin-3significantlyrelated with the downregulation of CHIP protein.Conclusion:Overexpression of Ataxin-3can inhibit the growth of gastric cancer cells,promote cell apoptosis and affect the expression of CHIP, which can involved ingastric carcinogenesis and development. It further imply Ataxin-3may be thecandidate target for GC therapy. Part3Construction of ATXN3-EGFP/SGC7901cell modelObjectiveTo further explore the function and mechanism of Ataxin-3in GC, weconstructed lentiviral eukaryotic expression vector carrying Ataxin-3gene,using lentiviral vector(LV) system Ataxin-3, which was transfected intoSGC7901cells. The establishment of stable SGC7901cell model,which wasover expressing ATXN3gene (ATXN3-EGFP/SGC7901), and provideexperimental platform of stably transfected cell lines for the next biologyresearch.MethodsThe full-length cDNA ATXN3gene was cloned into the lentiviral transfervector pLV-EF1a-EGFP-2A, construct the lentivirus vector system canindependently express GFP protein and ATXN3protein. The competent bacteriacomplete clonal amplification of plasmid and extraction plasmid. Sequenced theDNA of ATXN3gene that was inserted to the plasmid. After lentiviruspackaging in293T cells, and transfected into SGC7901cells, the stablytransfected SGC7901cells can be sorted by the flow cytometry andfluorescence. At last, detected protein expression of ATXN3by Western-blot inthe stability transgenic lines.Result1. Verified the sequence of gene inserted into the plasmid was correct sequencefor ATXN3.2. LV system consisted of a vector plasmid pLV-EF1a-EGFP-2A, coatedplasmid PL1, PL2and packaging plasmid pMD2.G. And finish packaged in293T cells.3. LV was transfected into SGC7901cells, transfected cells were observed with the green fluorescence (issued by GFP protein). The flow cytometry throughtfluorescence sorted stably transfected SGC7901cell, with fluorescenceseparation rate was higher than50%. Finally screen the GC cell line stable overexpression of Ataxin-3, pLV-ATXN3-EGFP/SGC7901. Western blot showedhigh expression of ATXN3-EGFP/SGC7901in group Ataxin-3protein.ConclusionWe successed construction of Ataxin-3over expression vector in vitro, andscreened the stably over expression of Ataxin-3in transfected SGC7901. Itprovide experimental platform of stably transfected cell lines for the nextbiology research about Ataxin-3.