Research Abstract: CHLA 2015

Abstract Title
miR-186 silences Myc-N, a key oncogene in Neuroblastoma

First Author: Ramirez, Angel; Neviani, Paolo, PhD; Wise, Petra,

PhD
Last Author: Fabbri, Muller, MD, PhD
Key Words
Neuroblastoma, miR-186, Myc-N.
Background
Neuroblastoma is the most frequent solid cancer present in children
outside of the skull, with over 600 new cases emerging each year. Despite
all existing treatments, 40% of children diagnosed with Neuroblastoma
die. Myc-N helps to define a specific subgroup in Neuroblastoma
characterized by a high recurrence rate, poor response to therapy, and
poor clinical outcome. MiR-186, a type of noncoding RNA is
downregulated in Neuroblastoma and has Myc-N as a potential target,
therefore harboring the potential to downregulate Myc-N expression. By
silencing Myc-N in Neuroblastoma, miR-186 might exert an anti-tumoral
effect and could represent a new anti-cancer therapeutic tool. My goal is
to answer the question: Is it possible to silence Myc-N by using a
lentiviral vector that induces stable miR-186 expression in
Neuroblastoma cell lines?
Hypothesis
Increased expression of Mir-186 will inhibit Myc-N expression in
Neuroblastoma cell lines.
Methods
QPCR
check
miR-186
expressio

Maxi- Prep
Vector: pmiR
DoubleDigest
PCR Insert
(miR-186)

Ligation

Bacterial
Transformation

Sequencing

Infect
CHLA136

Transfect
293tn
cells

Citofluorimetry

Results
I was able to successfully generate a clone that contained our miR-186. I

Western
Blot

Clones

Colony PCR
was also able to see the expression of miR 186 in our 293tn cells. My
Neuroblastoma cell lines (CHLA-136, and Lan-5) expressed miR-186 when
infected by such lentiviral particles, as shown in our results generatedQPCR
by
using Citofluorimetry. By western blotting, I was able to show that
miR- of miRExpression
186 downregulated the expression of Myc-N in CHLA-136 and LAN-5186 in
Neuroblastoma cell lines.
293tn Cells

GFP
Positive
Control
CHLA 136

Mir-186
CHLA 136
Myc-N Expression

Loading Control

Conclusions
I was able to successfully clone miR-186 in a lentivirus plasmid, generate
lentiviral particles expressing miR-186, and conclude that Myc-N
expression can be reduced by the increased expression of miR-186 in two
Neuroblastoma cell lines (CHLA-136 and LAN-5).
The cell lines that I generated (CHLA-136 and LAN-5 expressing miR186) will be used to assess the effects of miR-186 in MYC-N targeting and
in Neuroblastoma growth both in-vitro and in-vivo.