INVESTIGATION OF THE EFFECT OF VEGFshRNAs CLONED INTO SILENCING PLASMIDS ON VEGF EXPRESSION IN ENDOTHELIAL CELL


Creative Commons License

Şalva E. , Yeşilada E.

International Multidisciplinary Symposium on Drug Research & Development (DRD 2019), vol.1, no.1, pp.313-316, 2019 (International Conference Book)

  • Publication Type: Article / Article
  • Volume: 1 Issue: 1
  • Publication Date: 2019
  • Title of Journal : International Multidisciplinary Symposium on Drug Research & Development (DRD 2019)
  • Page Numbers: pp.313-316

Abstract

INTRODUCTION Angiogenesis is a highly complex process that is tightly controlled by many stimulator and inhibitor growth factors [1]. Vascular endothelial growth factor (VEGF) is the major growth factor in the formation of new blood capillaries in angiogenic transformation. VEGF, which is highly potent in angiogenesis, plays an important role in both physiological and pathophysiological processes [2]. It increases the growth rate of the tumor and accelerates metastasis, as it enables the transfer of nutrient and oxygen to the tumor. In addition, conversion to an angiogenic phenotype helps the development of a malignant tumor [3]. VEGF is a family of six different homologous factors (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PIGF)). Among these, VEGF-A is a growth factor that is highly effective in the growth of blood vessels [4]. As an anticancer strategy, angiogenesis can be prevented by reducing new tumor vascularity. RNA interference (RNAi) is a gene-specific gene silencing technology that enables effective and potent silencing of overexpressed genes in many diseases [5]. Vectors containing the target-specific sequence express shRNA when introduced into mammalian cells. shRNAs are stem loop RNAs. Expressed long RNA hairpin transcripts are passed to the cytoplasm for intracellular processing and then cut with Dicer to form siRNAs. These siRNAs participate in RNA induced silencing complex (RISC) activity in the cytoplasm and thus target mRNAs are degraded [6]. The aim of this study was to investigate of anti-angiogenic effects in endothelial cells by cloning of two different VEGF-A sequences into the silencing plasmid. MATERIALS AND METHODS Materials psiRNA-DUO plasmid was purchased from InvivoGen. HUVEC cell line was gift from Marmara University. DMEM medium, trypsin/EDTA and penicillin/streptomycin for cell culture were purchased from Gibco. Method Competent Cell Preparation CaCl2 method was used for the bacterial transformation. The propagated E.coli GT115 bacteria in the LB medium was inoculated on petri dish using loop and incubated at 37°C. The following day, single colony bacteria were inoculated into liquid LB medium and incubated until OD 0,4-0,6. The cold CaCl2 solution was added to bacterial pellet obtained by centrifugation and then centrifuged again after waiting 20-30 minutes. The cold CaCl2 solution was added to bacterial pellet again and aliquoted and stored at -80°C. Transformation and Isolation of psiRNADUO and Control Studies The competent E.coli GT115 cells were used to transformation of psiRNA-DUO. The appropriate number of competent cells were placed on ice and allowed the cells to thaw on ice for 2-5 minutes. Supercoiled psiRNA in pre-chilled tubes was added to competent cells and incubated the tubes in ice for 30 minutes.