The Initial Study Of Silence Ec9706 Cells Cd147 Expression

Background and objectivesCD147 is one member of immunoglobulin super family discovered in 1982. Initially,people think CD 147 was one kind of tumor surface protein which can induce matrix metalloproteinases to be expressed in fibroblasts,which initially was named tumor cell collagenase stimulatory factor(TCSF).When it's purified,TCSF was defined as a kind of crossing membrane glycoprotein which was congenetic with the protein of immunoglobulin superfamily(IgSF),and could induce theproduce creation of matrix metalloproteinases(MMP).Research showed that CD147was highly expressed in a wide variety of tumors,and closely related with the invasion and metastasis.But its expression is not limited within in the tumor cells.CD147 is one kind of effective moleculars,CD147 plays an important role on fetuses grown-up and function of the retina,and has been confirmed the function during the development of thymus T cells.Because it expressed widely and had multi-effective molecular,It is very important to study the interaction among proteins and adjust the toward treatment strategies of tumor intervention in CD147.Therefore,this experiment designed to construct siRNA gene expression vector for CD147,liposomes wrapped was transfected into human EC9706 cell,To test CD147 gene expression inhibiting effect in this cell,we preliminarily observe and analyze the effect of which inhibition of CD147 of gene expression takes the invasion and metastasis of EC9706 cells.We applied the promega siRNA target sequence analysis and design system to scann human CD147 cDNA coding sequence(NM₀01728),according to the designed principle of sequence of siRNA target,and BLAST homology analysis system to choose 3 siRNA target sequences of the CD147.Synthesize 3 groups hairclip-like DNA single chains of CD147 targeted siRNA sequence respectively, annealing them into double-chains,to be restructured into siRNA pGeneClip expression vector,Utilize to screen and identify pGeneClip-si257,pGeneClip-si943,pGeneClip-sil203;Taking PstⅠenzyme-cut;With to take DNA sequence analysis to inserting sequence of recon with M13+/M13-primers.We convert siRNA expression vector pGeneClip-si257,pGeneClip-si943 and pGeneClip-si1203 into human EC9706 cells with liposome package,to set pGeneclip control group(transfection of pGeneClip) and control group(blank transfection),to test expression level of CD147 mRNA and protein in each group of EC cells with fluorescence quantitative RT-PCR and Western -Blot,to test the levels of MMP-9 in each group of cells with ELISA,to (?) cell cycle and invasion and metastasis in each group of cells with flow cytometry and Matrigel Invasion Assay.Results:1 By preliminary screaming we got 14 siRNA target segments of candidates, through the homologous sequence retrieval and further optimization,eventually (?)termined 257-275 sites(GATCCAGTGGTGGTTTGAA),943-961 sites GGT CAGA(GCTA CACATTGA) and 1203-1221 sites(GGGCAGCACCAGAATG ACA) to be target sequence section.2 After the annealing siRNA hairpin DNA,we could see three bright bands through clectrophoresis,(3 annealed products si257,si943 and si1203) located near the '00bp sites in concert with the initial dsigns.3 We got recons pGeneClip-si257,pGeneClip-si943 and pGeneClip-si1203 through PstⅠenzyme-cut screening,in analysizing its inserting sequence was concerted with our designs.4.Compare the experimental groups(pGeneClip-si257,pGeneClip-si943 and pGeneClip-si1203)with the two controls(pGeneclip and blank) EC9706 cells CD147gene's mRNA expressions are decreasing obviously.The differentials are ignificant.(P＜0.05) Among them pGeneClip-sil203 is of of strongest effects of inhibition.5.There are obvious imprinting bands around 60KD in the blank controls and pGeneClip.The three experimentals are poor colors obviously.The third group (transfection pGeneClip-si1203)are of the most shallow imprinting bands.It corresponds to the results of the quantitative fluorescence RT-PCR.6.Among experimental 1,2,3 groups,pGeneClip and blank controls,there are no large difference at cell proliferation and proportion between the phase of G₀～G₁,G₂～M and S.7.Two controls(pGeneClip and blanks),MMP-9 levels are of no significant differences(P＞0.05);With the three experimental comparison,there are a large difference.(P＞0.05).It demonstrate that inhibiting the expression of esophageal cancer EC9706 cells CD 147 can restrain MMP-9.8.Compare experimental 1,2,3 groups with pGeneClip and blanks,the numbers of cells of the penetration to the artificial basement membrane are decreasing significantly.The heterogeneities are different obviously in inhibiting effect of the penetration to artificial basement membrane.The experiments:number three group(transfection pGeneClip-si1203) are stronger than number 2 and 1.Conclusions1 CD147 gene 1203-1221(GGGCAGCACCAGAATGACA) is the siRNA effective target sequence of silence expression.The siRNA pGeneClip-si1203 of construction and the transfection esophageal cancer EC9706 cells can significantly reduce the expressions of CD147 m-RNA and proteins.2 CD147 gene's expression in silence esophageal cancer EC9706 cells can effectively restrain the secretion of MMP-9 and invasion or metastasis of EC9706 cells.