International Multidisciplinary Symposium on Drug Research & Development (DRD 2019), vol.1, no.1, pp.313-316, 2019 (International Conference Book)
Angiogenesis is a highly complex process that
is tightly controlled by many stimulator and
inhibitor growth factors . 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 . 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 . 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 . 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 . 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 .
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
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.
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.