Inhibition of vascular calcification by microRNA-155-5p is accompanied by the inactivation of TGF-β1/Smad2/3 signaling pathway

1. Por: Julián Mauricio Jiménez Álvarez y Juan David Jiménez Ramírez.
Tercer semestre, Universidad Pontificia Bolivariana, Medellín.

2. Introduction
● MicroRNA. Kind of non coding and single stranded of RNA molecule
containing 19 to 25 molecules of nucleotides and can bind to 3´-UTR of
target microRNA, the effects are translational represion or target
degradation
● article studies the relation between the miRNA.155-5p,
TGF-beta1/Smad2/3 signaling pathways and VC (vascular calcification) on
rat aorta abdominalis and calcified-vascular smooth muscle cells (VSMCs)
● signaling pathway is a group of molecules that transmits chain signals to
control one or a variety of functions of the cell.

3. Introducción
TGF β1: The Transforming Growth Factor β1 is a protein that triggers
many chemical signals that work together to regulate many processes
like cells growth, proliferation, differentiation, motility and apoptosis; it’s
involved in the blood vessels formation, development of muscular tissue
and body fat, inflammatory processes in the immune system and
prevention of tumor growth.
Smad2/3: The Smad are proteins that work like signal transducers and
transcriptional modulators in many pathways. This protein is recruited
to the TGF-beta receptors through its interaction with the SMAD anchor
for receptor activation (SARA) protein.

4. Introducción

5. General Objective
Exhibit the inhibition of vascular calcification (VC) and
the inactivation of TGF-beta1/Smad2/3 signaling
pathway by the miR-155-5p in calcified-vascular smooth
muscle cells (VSMCs) and rat aorta abdominalis.

6. Methods
Immunofluorescence staining: It’s a technique that uses the
specificity of the antibodies to their antigen to mark with specific
dyes specific biomolecules targets inside of the cell. Later allows
the visualization of the distribution of the target biomolecule
through the sample with the help of a fluorescence microscope.
In this study were used antibodies against α-smooth muscle actin, smooth
muscle protein 22a and Cy3- labeled goat anti-rabbit.

7. Methods
Real-time PCR: Also known as quantitative Polymerase Chain
Reaction, is a technique used to monitor the amplification of
targeted DNA molecule while the procedure and not at the end.
The real time PCR is made in a thermal cycler that illuminates each
sample with a beam of light with specific wavelength and detect
the fluorescence emitted by a excited fluorophore.
In the study was used to monitor the cDNA, produced by the M-MLV
reverse transcriptase .

8. Methods
Western Blot
Is a analytical technique used for detecting specific proteins in a determined
sample
It uses a gel electrophoresis to separate the proteins of the sample, the
proteins go to a membrane exposed to a specific antibody against the studied
protein
The union of the antibody is detected using a radioactive or chemical traser

9. direct indirect

10. Methods
Luciferase Assay
A luciferase assay is used to determine if a protein can activate or repress the expression of a
target gene.
The luciferase assay is able to establish a functional connection between the presence of the
protein and the amount of gene product that is produced. Luciferase is an enzyme used for
bioluminescence.
The scientist produces a construct in which the regulatory region of a target gene is fused with
the DNA coding sequence for luciferase
If the protein is able to upregulate transcription of the target gene, the cells will express
luciferase. If the protein downregulates transcription, the cells will express less luciferase than
normal

11. Results
“Cell identification results showed that
α-SMA and SM22a were well-expressed in
rVSMCs. The overexpression efficiency of
miR-155-5p was verified by real-time PCR
analysis in cells .
The procedures confirm that the presence
of miR-155-5p in rVSMCs is the first step
to avoid the VC.

12. Results Electrophoresis D
when cells are undergoing calcification, with the
calcification medium (CM). this was in vitro
Leves of the proteins were detected by western blot.
beta actin is the reference protein. With the CM and
negative control values are the same, we see
overexpression of pSmad-3, this is the active form of
the Smad-3 because it's phosphorylated and
downexpression of SM22a. Also there is
overexpression of ALP, TGF beta-1, pSmad-2, Smad-2.
this actions are inhibited by the expression of
miR-155-5p.
With the expression of the miR 155-5p we see that
there could be a overexpression of SM22a undergoing
calcification

13. Results
Subsequently, miR-155-5p was knocked down in
rVSMCs. By alizarin red staining assay,
miR-155-5p inhibitor transfected VSMCs showed
a higher calcification degree in the CM-cultured
cells at day 7. By further ALP assay, the
CM-mediated increase of ALP activity in rVSMCs
was promoted by knockdown miR-155-5p . All
these results indicate that miR-155-5p knockdown
can promote a higher rVSMC calcification in
comparison to the rVSMCs with miR-155-5p
overexpression.

14. Results Electrophoresis G
This was in vivo in the animal model, the
vascular calcification was induced by the
CaCl2.
With the CaCl2 and NC group we see
overexpression of TGF-beta1 pSmad-2,
Smad2, pSmad-3, Smad-3, inducing the VC.
Those alterations caused by CaCl2 were
supressed by the expresion of miR-155-5p
inhibiting VC

15. Discussion
Zheng et al., 2016.
“MiRs have been reported to be
associated with a variety of diseases,
including cardio-vascular diseases,
cancers, and autoimmune diseases, but,
the role of miRs in VC has been not
extensively investigated”
Metzinger-Le Meuth et al.,
2017.
“… miR-155-5p was implicated in
VC.”
Xu et al., 2019.
“Mechanism studies showed that
overexpression of miR-155-5p decreased the
level of TGF-β1, pSmad2 and pSmad3,
combined with the direct combination of
miR-155-5p and Smad2, indicating that
miR-155-5p may inhibit VC by regulating TGF-
β1/Smad2/3 signaling pathway. In addition, VC
is regulated by other signaling pathway”.

16. Conclusions
● Molecular Biology is very important in medicine because of the
relationship with the genetic material that is the form to save the
genetic information of a form of organic live.
● Molecular Biology is important to detect genetic diseases especially in
fields of medicine such as infectious diseases and cancer.
● Molecular Biology is important to provide a treatment for diseases
from a genetic point of view.

17. Mapa Conceptual-Juan David Jimenez

18. Conceptual map: Julián Jiménez