Expeirmental Studies On Silencing Of Gabl By RNA Interference Inhibiting Angiogcnesis In Vitro

OBJECTIVETo investigate the silencing expression of Gab1by RNAi, inhibitingangiogenesis in vitro, to lay a foundation for the further research in the inhibition oftumor angiogenesis and other vascular disease, and to provide an experimentalevidence for gene therapy in tumor and other vascular disease.METHODS1、EA.hy926cells were cultured with DMEM containing10%fetal bovineserum. The3-7generation of them were used as research subjects.2、Using the mRNAs of Gab1as the target sequences, we constructed lentiviralRNA interference (RNAi) vectors of Gab1(LV-Gab1-siRNA)，then transfectedEA.hy926cells to get Gab1-siRNA interfering EA.hy926cells.3、constructing Three-dimensional vascular-like structure culture system in vitro,we added matrix adhesive liquid confrigurated to every hole in24hole plate amountto200~250ul/cm~2. EA.hy926cells of the fourth passage were obtained and dividedinto three groups(n=8)，In the lentivirus interfering group and the negative controlgroup, LV-Gab1-siRNA and LV-Scr-siRNA were transfected into EA.hy926cellsrespectively. No lentivirus RNAi vector was added in the blank control group. Thethree-group cells were inoculated in the model of three-dimensional culturerespectively.4、The24hole plate was put in incubator, The number and lenth of vascular-likestructure of in vitro three-dimensional culture model was observed On the24hour.5、A portion of the EA.hy926cells above three groups was analyzed by reversetranscription-polymerase chain reaction (RT-PCR), Western blotting, Tanswell cellmigration and FACS (fluorescence-activated cell sorting), including the mRNA andprotein expression of Gab1gene, the cell migration ability and apoptosis rate.RESULTS1、After transfection for3d, the expression of green fluorescent protein (GFP)was significantly increased. 2、In the lentivirus interfering group, the cell morphology observed underinverted microscopy was distinctly different, by long fusiform became a star orpolygon, but negative and blank were a normal long fusiform.3、RT-PCR revealed that the mRNA expression of Gab1gene in the lentivirusinterfering group was inhibited obviously (P<0.05), but the expression level has notsignificant difference (P>0.05) between negative and blank.4、Western blotting displayed that the protein expression of Gab1gene in thelentivirus interfering group was inhibited obviously (P<0.05), but the expression levelhas not significant difference between negative and blank (P>0.05).5、Tanswell cell migration indicated that the cell migration quantity in lentivirusinterfering group was lower than in the negative and the blank control groups(P<0.05), but it for negative and blank was basically the same (P>0.05).6、FACS apoptosis experiment revealed that the cell apoptosis rat in lentivirusinterfering group was higher than in the negative and the blank control groups(P<0.05), but there was no significant difference on apoptosis rat between negativeand blank (P>0.05).7、The number and lenth of vascular-like structure of in vitro three-dimensionalculture model have a significant decrease after EA.hy926cells transfected withLV-Gab1-siRNA (P<0.05), It was obvious that angiogenesis was inhibited in vitro.CONCLUSIONGab1-siRNA can inhibit the expression of Gab1protein and mRNA, slow thecapability of cell migration, accelerate cell apoptosis and at last inhibit angiogenesisin vitro model of three-dimensional culture. It is suggest that Gab1gene plays animportant in angiogenesis.