Effection Of ECGF1 SiRNA On Colon Cancer

objective:the role of the ECGF1 including,raise the synthesis of DNA,increasethe ability of migration of the endothelio cells,stimulate the angiopoiesis and tumorgrowth .the publicated literature show the cell that transfected the correspondingplussense and anti sense RNA to mRNA can degrade the mRNA specifically.moreversilence the corresponding gene.such post-transcriptional gene silencing was known asthe RNA interference.in the recent year,RNAi technology has been paid moreattention in mammalian cell and become the new important method on the genefunction.to investigate the effect of the ECGF1 on the tumor ,differentiation,migrationof the bladder carcinoma,we adopt the gene silence method—RNA interfencesuppress the ECGF1 expression of the colon carcinoma cell.according to the gene orderof the ECGF1,construct the vector plasmid that can form the hairpin structure with thetranscription vector pTZU6+l containing the RNA polymerase promotor III,transfect itto colon carcinoma cell LOVO and observe the effect on the expression of theECGFl.to observe if it can silence the ECGF1 gene specifically though the in vitroexprement and bearing cancer naked mouse exprement in vivo as well as investigate theeffect on the bioactivity of colon carcinoma cell and the suppress extent on the tumor invivo,morever establish the basis of progress and development of colon carcinoma.Method: the expression of ECGF1 in colon carcinoma lineslovo wereconfirmed by immunochemistry method, look for the target sequence fittingthe designing characteristics from the downstream of start codon of ECGF1 geneaccording the siRNA desigen principle.design the specific siRNA aimed the humanECGF1 gene sequence.such sequence come from NCBI and compare with humanEST database,confirm no-integrate loci with other gene.the integrate loci of siRNA isa pair of justo-and anti-sense nucleotide chain 5'-GCAGCTTGTGGACAAGCAT -3 '(sequence A) aimed 687-708 nucleotide base pair.design the negative control5'-AAGGCTCTGACAGGTGGTT-3 (sequence B).analyze the homologizationsequence to ensure such short chain RNA sequence have no homologization. HindIHand BamHl enzyme cut the pRNAT-U6/Neo plasmid that be retrived ,breed andpreserve,the made of competence germ,transfection of plasmid, amplification, link thepurified vector, pRNAT-U6/Neo and the DNA fraction cut by Hind III andBamHl.then transform the recombinant plasmid,culture cell,cell transfection bycalcium acid phosphate methods,appraise the function aftertransfection .detected the expression of ECGF1 protein byimmunochemistry, the expression of ECGFlmRNA by RT-PCR aftertransfection to ensure the success of transfection.then divided the cellsinto four groups,control group, pRNAT -U6/Neo siRNA 1 group;pSIECGFl-A group;pSIECGFl-B group, observe the expression of EGFR under the fluorescentmicroscope,the P53, caspase-3 protein were detected by Western blot and flowcytometry method 48h after transfection. The cell cycle and cell apoptosis rateinduced by DDP were decteced by flow cytometry .the cell migration rate wasdetected by scratch assay,the cell apoptosis was detected by fluorescent staining,theDNA break was detected by agar sugar gel electrophoresis,the cell survive rate andsuppress rate were detected by MTT.the apoptosis and bearing cancer were detectedby bearing cancer subcutaneouly of athymic mouse after transfection.Results :the expression of ECGF1 in the human colon carcinoma high afterimmunochemistry staining .the staining located in intracy-toplasm. the level ofECGFlmRNA decreased confirmed by RT-PCR in transfected pSIECGFl-A LOVOcell.but the level of ECGFlmRNA have no significant change in other groups.the levelof ECGF1 protein decreased obviously in LOVO cell transfected by pSIECGFl-A buthave no change in other groups.such expriment confirmed success of transfection ofECGFlsiRNA.the tumor cell volume transfected by ECGFlsiRNA became smallerthan cells in other groups.nucleus appear sublobe forms,refraction in cell boundaryweak,the fluorescence weaken in siRNA2 group compared to other groups.theexpression of caspase-3, P53 protein did not increased detected by western blot andflow cytometry .the dsRNAECGFl can suppress the proliferation of cell confirmed bycell count and colony formation experiment,the rate of suppress is 42.78%.cell cycleshow the percent of G1-G2 stage decrease to 10.13% compared to controlgroup.scrach assay illustrate the dsRNAECGFl can suppressthe migration rate ofLOVO ,the apoptosis of cell decreased by MTT assay.it appear typical ladder strap inagarose electrophoresis.it explain the dsRNAECGFl can increase the apoptosisrate.adding the DDP after transfection can increase the suppress rate compared othergroups(P<0.05). the most notable on 48h.the form rate of tumor id only 50% in 21stday after inoculation of LOVO cell in RNAi2 group though bearing tumor exprimentin vitro.the rate of form tumor is 100% in other groups, tumorous growth curve showsthe growth velocity is slower in RNAi2 than other groups.we calculated the rate ofsuppress on the tumor through the tumor weight on the 30th day,the rate of suppressin idle vector group ,RNAil group is 2.05% and 6.93%.the rate of suppress is 56.43%in RNAi2 group . the expression of ECGF1 protein is lowest in RNAi2 group .Conclusions:the ECGF1 express higher in malignant colon cancer cell thaninnocent tumor ,we may get out and amplificate purpose gene make use of suchmethods.RNA interference techonology can suppress the corresponding gene,ECGFlsiRNA can suppress the expression of ECGF1. DsRNAECGFl can suppressthe tumor growth by promoting cell apoptosis but no increasing the caspase-3, P53gene .transfecting dsRNAECGFl can increase the sensitivity to the antitumormedicine DDP.