Molecular Mechanisms Of EMT Elicited By HMGA2in Gastric Cancer

BACKGROUND AND OBJECTIVES: Gastric cancer mortalityhas declined markedly around the world over the last20years. Nonetheless,it still estimated to one of the most dangerous disease in our country andprobably is the second leading cause of cancer death. The death of gastriccancer is mainly caused by invasiveness and metastasis. Over the last fewyears, more and more evidence has indicated that EMT plays an importantrole in cancer progression and metastasis. In EMT process, cancer cells lostits epithelial properties and gain mesenchymal properties therebyenhancing their abilities of migration and invasiveness. However, theunderlying molecular mechanisms of these conditions are poorlyunderstood. Therefore, exploring the underlying molecular mechanisms ofEMT may provide a new treatment strategy for gastric cancer.High mobility group protein A2(HMGA2) is an architecturaltranscription factor that plays an important role in development andprogression of malignant neoplasias. Recently, some studies reported thatHMGA2is also implicated in epithelial-mesenchymal transitions (EMT)and cancer stem cells. In this study, at first, we investigate the relationship between theexpression of HMGA2and clinical parameter in gastric cancer. Secondly,we construct HMGA2overexpression lentiviral vectors and HMGA2siRNA. Then, we examined the changes of molecular and cellular biologyin gastric cancer cells with or without HMGA2overexpression. At last, wefind out target genes of HMGA2and validate them in vitro thereby exploremolecular mechanism of EMT elicited by HMGA2in gastric cancer.METHODS:(1) Immunohistochemistry was performed to examinethe HMGA2expression and investigated the relationship between theexpression of HMGA2and clinical parameter in gastric cancer.(2) Comparison of the expression of HMGA2in different gastricepithelial origin cell lines by western blot. Via homologus recombinationbetween pIRES2-EGFP and pLV-UbC-IRES2-EGFP, obtained lentiviralvectors plasmids for overexpressing HMGA2and then packaged intolentivirus particles. Inducted HMGA2gene in MKN28and GES-1celllines by above lentivirus particles and then analyses growth rates, migration,invasion and anchorage-independent growth by MTT, millicell and softagar assays after MKN28and GES-1cell lines were infected. Transienttransfected of HMGA2targeting siRNA into MKN45cells. Western blotwas performed to detect expression of E-cadherin, N-cadherin, Vimentin ingastric cancer cells with or without HMGA2overexpression.Immunohistochemistry were performed to detect expression of E-cadherin, N-cadherin, Vimentin in subcutaneous xenograft model of nude mouse.(3) Global mapping of HMGA2potential transcription factor bindingsites was identified by promoter microarray in these cells and the dateobtained from the microarrays were validated via chromatinimmunoprecipitation-PCR (ChIP-PCR) and qPCR. HMGA2potentialtarget genes were classified in KEGG database analyses. ChIP-qPCR，Western-blot，qPCR and immunofluorescence were performed to validateabove results from microarray.RESULTS:(1) The expression of HMGA2was correlated with TNMstage and lymph node metastasis.(2) Lentiviral vectors plasmids for overexpressing HMGA2wasconstructed successfully. Exogenous HMGA2expression was observed tolocate in the nucleus by immunofluorescence analysis. Overexpression ofHMGA2enhances oncogenic properties and improves migration andinvasion in MKN28and GES-1cell lines but reduced their growth rates.Furthermore, overexpression of HMGA2increased N-cadherin andVimentin expression and decreased E-cadherin expression in MKN28andGES-1cells. This condition could be reversed by transient transfected ofHMGA2targeting siRNA in MKN45cells. Overexpression of HMGA2inMKN28cells had a higher tumor formation rates but a smaller tumor sizein subcutaneous xenograft model of nude mouse.(3)1366genes were identified to be potential targets of HMGA2by ChIP on chip and then the target genes were analyzed for their roles inbiological functions and pathways with the KEGG databases.144of themwere clustered to35pathways potential in involved in cancer. TWIST1andAXIN1were singled out because they are related to EMT. HMGA2overexpression increased TWIST1expression and decreased AXIN1expression. β-catenin was observed to translocate from the usualmembrane/cytoplasm-bound site observed in MKN28without HMGA2expression to the nucleus in MKN28with HMGA2expression byimmunofluorescence.CONCLUSIONS: The expression of HMGA2was correlated withprogression of gastric cancer. HMGA2is an important contributor to EMTin gastric epithelial origin cell lines. HMGA2expression enhancedoncogenic properties of gastric cancer in vivo and in vitro. The molecularmechanism of EMT elicited by HMGA2is that HMGA2could activateWnt/β-catenin pathway via regulating expression of TWIST1and AXIN1.