Trabajo para biología molecular desarrollado por Antonia Londoño Pérez. Tercer semestre. Medicina. Universidad Pontificia Bolivariana Medellin. 2019

1. Antonia Londoño Pérez
ID: 000364149
3rd semester
Medicine
Universidad Pontificia Bolivariana
Medellin
2019

2. INTRODUCT
ION
•Pancreatic ductal adenocarcionoma (PDAC) in the
second leading cause of cancer-related deaths
worldwide, due to it’s resistance to conventional
treatments prognosis is very poor, with a les than
a 5-year survial rate.
•PD-L1 (programmed death ligand 1) is expressed
in tumor cells and its expression and the
mechanism of resistance in PDAC to antiPD-L1 is
associated with poor diagnosis. It has been shown
that the expresión of PD-L1 is key in
inmmunotherapy efficacy.

3. INTRODUCT
ION
•RGFP966, the specific inhibitor of HDAC3 (histone
deacetylase 3) decreases the protein’s expression and
rRNA level of PD-L1in pancreatic cancer cells.
•The study intends to show that HDAC3 is critical for
PD-L1 regulation and positively correlated with PD-L1
in PDAC patients.

4. GENERAL
OBJECTIVE
Explore the regulatory mechanism
of PD-L1 and search for the small
molecular inhibitions that suppress
the expression of PD-L1 in
pancreatic cancer cells.

5. MATERIALS
AND
METHODS
WESTERN BLOT
Western blot is a laboratory technique used to
detect a specific protein in a blood or tissue
sample.
The method involves the use of gel
electrophoresis to separate the proteins from the
sample. The separated proteins are transferred
from the gel to the surface of a membrane. The
membrane is exposed to a specific antibody
against the protein under study. The binding of
the antibody is detected using a radioactive or
chemical label.
A Western Blot is sometimes used to diagnose
diseases. It makes possible to estimate the size of
a protein, confirm the presence of post-
translational modifications such as
phosphorylation, and be used to quantitatively
compare protein levels between samples
https://www.genome.gov/glossarys/index.cfm?id=207
http://www.ecogen.com/upfiles/A56009.pdf

6. MATERIALS
AND
METHODS
REAL-TIME RT-PCR
Enables reliable detection and
measurement of products generated
during each cycle of PCR process. This
technique became possible after
introduction of an oligonucleotide probe
which was designed to hybridize within
the target sequence.
Cleavage of the probe during PCR because
of the 5' nuclease activity of Taq
polymerase can be used to detect
amplification of the target-specific
product.
https://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/

7. MATERIALS
AND
METHODS
IMMUNOHISTOCHEMISTRY
Immunohistochemistry is a powerful
microscopy-based technique for
visualizing cellular components, for
instance proteins or other macromolecules
in tissue samples.
The strength of IHC is the intuitive visual
output that reveals the existence and
localization of the target-protein in the
context of different cell types, biological
states, and/or subcellular localization
within complex tissues.
It’s used as an important tool in health
care and pathology for diagnostic purposes
or to stratify patients for optimized
treatment regimes, is also widely used in
research where molecules of interest are
analyzed to study their roles in both
healthy and diseased cells.
https://www.proteinatlas.org/learn/method/immunohistoc
hemistry

8. MATERIALS
AND
METHODS
RNA INTERFERENCE
Double-stranded RNA-mediated interference
(RNAi) is a simple and rapid method of
silencing gene expression in a range of
organisms. The silencing of a gene is a
consequence of degradation of RNA into
short RNAs that activate ribonucleases to
target homologous mRNA. The resulting
phenotypes either are identical to those of
genetic null mutants or resemble an allelic
series of mutants.
RNAi is used in functional genomics and
developing therapies for the treatment of
viral infection, dominant disorders,
neurological disorders, and many types of
cancers (in vivo inactivation of gene products
linked to human disease progression and
pathology).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC309050/
https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/

9. RESULTS
Cells treated with DMSO
and without RGFP966
(inhibitor) showed higher
expression of PD-L1.
Between the two cell lines,
the BxPC-3 showed more
resistance to the inhibitor
than MIA PaCa-2 wich
showed less expression to
gradual increase of
RGFP966 through a period
of 48 hours.

10. RESULTS
The silencing of HDAC3
with short hairpin led to
decrease in the
expression of PD-L1
mRNA, the decrease was
more evident in BxPC-3
The overexpression of
HDAC3 increases the
appereance of the PD-L1
in both cell lines.

11. RESULTS
Consequence of higher
expression of PD-L1 is a
notorious decrease of
tissue due to the
immunosupresing action
of the protein.

12. RESULTS
The silencing of STAT3
reduces the expression of
HDAC3 and therefore, of
PD-L1.
Ectopic HDCA3 increases
expression of PD-L1, this
is evident when there’s
silencing of STAT3

13. DISCUSSION
AUTHOR STATEMENT CONFIRMATION
Winograd R, Byrne KT,
Evans RA, Odorizzi PM,
Meyer AR, Bajor DL
A growing body of evidence
suggests that the expression
level of PD-L1 in pancreatic
cancer is critical for the
efficacy of anti-PD-L1
therapy.
It was confirmed
Booth L, Roberts JL,
Poklepovic A, Kirkwood J,
Dent P
HDAC1 and HDAC2 silencing
in other studies hasn’t
decreased the PD-L1 level.
No. Only states that
silencing HDAC3
decreases the protein
level
Eddekaoui M, Chheda C,
Soufi B, Zayou F, Hu RW,
Ramanujan VK
HDAC inhibition inproves the
immune response and
outcome of pancreatic cancer
in mice.
Confirmed.

14. CONCLUSIO
NS
•As PD-L1 works supressing the immune system,
favors proliferation and cell survival, it’s
expression in cancer cells increases the resistance
of this type of cancer, the higher the expression of
this protein, the more places the anti-PD-L1can
work in, so that the cellular death can take place.
•Managing to silence STAT3 and therefore reducing
levels of expression of HDAC3 and PD-L1in cells
may lead to the development of new
immunotherapy treatments.