Effect Of NGX6 Gene On The Protein Exprssion Profiles In Colon Cancer Cells

BackgroundColon cancer is one of the most common digestive tract cancersand poses a serious threat to human health in the world.The incidence and mortality of colon cancer has been rising every year.Col--on cancer is a multi-gene and multi-stage evolution of the pathological process and abnormal inactive of the suppressive gene and the abnormal active of the oncogene perform the significant role in the development,proliferation and the metastasis of the colon cancer.It has become a critical subject to reveal the pathogenesis of col--on cancer through identifying novel tumor suppressor genes or sus--ceptible genes. In addition,it is critical important to establish valid molecular prognostic methods to cure and prevent from colon cancer.Molecular genetics laboratory of Cancer Research Institute cloned a novel candidate tumor suppressor gene(gene access Number:AFI188239)which contains 2.1kb located on the region of chromosome 9p21-22.From the analysis of bioinformatics,NGX6 gene encodes transmembrane proteins which contain 338 amino,two trans-membrane structure,a EGF-Like domain, several glycosylation sites and Tyrosine kinase phosphorylation sites. Loss of heterozygosity in the 9p21-22 region is the common phenomenon in various carcinomas.Pr--evious researches show that the expression of the NGX6 is downregulated to different extents in the different clinic stages of the colon cancer.Particularly,the rate of down-regulate of the NGX6 in the colon cancer with metastasis is 93%.The results of the cell cycle,Cell growth curve,MTT colorimetric,Soft agar colony-formi--ng,Nude mice inoculated show that NGX6 can reverse the maligna--nt phenotype of colon cancer.Flow Cytometry experiment. Result finds NGX6 can delay the process from G0/G1 period to S period and induce the apoptosis of the colon cancer.Invasion and metasta--sis.In vitro experiments and Scrape-loading and dye experiment results find NGX6 can inhibit the proliferation and metastasis of the colon cancer cell. Biological effects of NGX6 mentioned above demonstrate that NGX6 performs as the suppressive gene in colon cancer.Furthermore,we use cDNA microarray to analyze how NGX6 affects the gene expression profile of the colon cancer.The result shows that NGX6 can inhibit the proliferation and metastasis of colon cancer through negatively regulating Wnt/β-catenin signal pathway and Jnk pathway mediated by EGFR. EMSA result showesthat NGX6 can induce the apoptosis of colon cancer through inhibiting the activation of the NF-KB.Immun oprecipitation techniques detections find NGX6 can combine with ezrin protein which acts as a "bridge" protein that can connect membrane protein with the cytoskeleton.They plays an important role in cell motility,signal transduction pathway.Thus,It strongly suggests NGX6 plays an important role in regulating the Cell adhesion and motility.All studies suggest that NGX6 plays as a suppressive gene in colon cancer.The research on the gene level suggests NGX6 may be combined with ezrin which negatively regulates signaling pathway activity mediated by EGFR to play its biological functions as a suppressive gene.ObjectsThe ultimate implementation of life is the protein.How NGX6gene regulates the protein profile of colon cancer?,And no relevantresearch results have been reported yet.Based on previous studies,Our study is to analyze the different protein expression profiles in colon cancer cells before and after transfected with NGX6 gene.Thus,We can illuminate tumor suppressive role of NGX6 gene in protein level for main mechanism.MethodsWe use differrential in-gel electrophoresisi DIGE and MALD1 TOF-MS(Matrix-assisted laser desorption/ionization time of flight mass spectrometry) to compare the protein expression profiles of colon cancer HT-29 cells before and after transfected with NGX6 gene,Specific methods as following:1) cell proteins are extracted and then treated with Clean-up kit.we carry on 2D-DIGE gel elec--trophoresis after cell proteins concentrations are measured by Quantitative assay kit.Typhone scanner are used to obtain the protein mapps and Labscan scanning software are used for mapping analysis.2) After mapping analysis, different proteins are selected to dig,Enzymolysis,different proteins are identified by mass spectrometry.We look for function of different xppression proteins by bioinformatics to clear the suppressive role of NGX6 in protein level in colon cancer.ResultsThere are 12 down-regulated(fold＞1.8) proteins are obtained inour study,such as:nucleolin,90kDa heat shock protein,Tubulin,HNRPH1, AnnexinⅡ,HCRTR1 protein,microtubule-associated protein 6 isoform 1, Guanine nucleotide-binding protein,beta-2 subunit,Chain A,Crystal Structure Of A Soluble Dimeric Form OfOxidised Clic1,Chain A,Molecular Basis For The Recognition Of Phosphorylated And Phosphoacetylated Histone H3 By,Chain A,Molecular Basis For The Local Confomational Rearrangement Of Human Phosphoserine Phosphata,and 4 proteins are up-regulated such as Aldehyde dehydrogenase 1,Chain A,X-Ray Structure Of A Deletion Variant Of Human Glucose 6-Phosphate Dehydrogenase Complexe,phosphoglycerate mutase 1(brain) and fatty acid binding protein 5(psoriasis-associated).And furthermore,we use method of Western-blot validate the expression of HSP90 and AnnexinⅡ.The Western-blot analysis of HSP90 and AnnexinⅡis consistent with theresult of MS result.After bioinformatic analysis of the function of these proteins we find they closely relate with the incidence and development of tumor.These proteins classified by function as following: cell signal transduction,cell cycle,apoptosis,cytoskeleton,cell metabolism and motility.These genes involve in tumor proliferation, apoptosis and metastasis-associated signal transduction pathways,such as Jnk/SAPK signal transduction pathway,Raf-1/MAPKsignal pathway, IKK/NF-κB signaling pathway,PI3K-Akt signaling pathway,Apoptotic Bcl-2 family signaling pathway,as well as the Fas apoptosis signaling pathway and so on.These pathways form the complex network systems through the"crucial molecules".ConclusionsTherefore,we may speculate NGX6 regulates these proteins to regulate Cellular signal transduction,cell cycle,apoptosis,cytoskele--ton, cell movement and metabolism-related protein molecule expression.It may regulate MAPK,IKK/NF-κB,PI3K-Akt and Apoptosis pathway activity to play its anti-tumor role in proliferation,metastasis and promote apoptosis of tumor cells.Our further work will deeply reveal how NGX6 gene regulate mentioned signaling pathways to perform its biological effects.