Effection Of Differential Proteins Expression In Pancreatic Cancer Cells Transfected With Ad.CMV.eGFP.hSSTR2 By Proteome Analysis And Construction And Identification Of Lv-VIM-shRNA

PartⅠEffection of Differential Proteins Expression in PancreaticCancer Cells Transfected with Ad.CMV.eGFP.hSSTR2 by ProteomeAnalysisObjective To study the effection of differential proteins expression in pancreatic cancercells Panc-1 transfered hSSTR2 gene in vitro and search new sensitive therapeutic targetsof pancreatic cancer.Methods The full length hSSTR2 cDNA was introduced into pancreatic cancer cell linePanc-1 by adenovirus mediated transfection and stable expression of RNA and protein ofhSSTR2 were detected by RT-PCR and Westen blot.Comprehensive analyses of proteinswere focused on total protein spots exhibiting statistical alternations among theexperimental group,vector control and mock control by 2D-DIGE analysis.Proteinidentification was done by peptide mass finger printing with matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry(MALDI—TOF/TOF).Western blotwas performed to verify the differential expression of vimentin,PKM2 and SEPT11.Immunohistochemistry was performed to detect the expression of vimentin,PKM2 andSEPT11 in pancreatinc carcer tissues.Results hSSTR2 gene was transfected into Panc-1 pancreatic cancer cells in vitro successfully,and fluorescence difference protein expression patterns were establishedbetween hSSTR2 negative and positive expression of pancreatic cancer cells Panc-1.ByDeCyder v6.5 software analysis,a total of 21 have been statistically significant differencesin protein spots.There had the difference 18 points (greater then 1.3-fold) and these proteinspots was identified by mass spectrometry for 13 proteins.Proteins found to have lowerabundance levels in pancreatic cancer cells Panc-1 with hSSTR2 positive expressioncompared to negative expression included GMP synthase,Stress induced phosphoprotein1,Glutamate dehydrogenase 1,Septin-11,vimentin,Isocitrate dehydrogenase [NAD] subunitalpha,Import inner membrane translocase subunit TIM50.Proteins found to have increasedabundance levels in Panc-1 pancreatic cancer cells with hSSTR2 negative expressioncompared to positive expression included Elongation factor 1-alpha 1,Isoform M2 ofPyruvate kinase isozymes M1/M2,Enoyl-CoA hydratase tripartite motif-containing 28protein,Isoform 1 of Mitochondrial inner membrane protein,Heat shock protein 105 kDa.Conclusion The function of screening difference proteins involved in the process ofmetabolism of sugar,fat and nucleic acid,and the regulation of cell growth.It was beenhelpful for revealing the molecular mechanisms of growth,invasion and metastasis ofpancreatic cancer cell which missed hSSTR2 gene expression to further validate theirfunctions.PartⅡConstruction and Identification of Lv-VIM-shRNAObjective To construct a lentiviral expression vector for RNA interference (RNAi) ofhuman VIM gene;and assess its gene silencing effect in pancreatic cancer cell line Panc-1.Methods Three pairs of human VIM gene short hairpin RNA (shRNA) sequences weredesigned following the procedure of a software available on-line and one pair came fromdocument.After synthesis and annealing,four double-stranded oligonucleotides (dsOligo) were cloned into the pGCL-GFP/U6 plasmid,which were subsequently confirmed by PCRand DNA sequencing analysis.Real-time PCR and Western Blot were used to screen theeffective pGCL-GFP-shRNA plasmid in 293T cells,then the most effective one was packedinto the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells.The titer of lentivirus was determined by hole-by-dilutiontiter assay.The silencing effect of Lv-V1M-shRNA in Panc-1 cells was validated byreal-time PCR and Western Blot.Results An effective Lv-VIM-shRNA was successfully constructed.The titer of lentiviruswas determined on 2×10~9TU/ml.The expression of VIM mRNA and vimentin wasdown-regluated in the Panc-1 cells infected with Lv-VIM-shRNA.Conclusion An effective Lv-VIM-shRNA could inhibit the expression of VIM gene inPanc-1 cells in vitro,which provides a tool for investigating the role of VIM gene in thesignaling pathway involved in tumorigenesis and progression of pancreatic cancer andsearching new therapitic targets.