Overview
The goal of our project is to design and develop a screening approach
that can evaluate cancer risk in post myocardial infarction (MI)
patients by detecting the expression of cancer-promoting miRNA in these
patients. Accordingly, our experimental design consists of the following
parts: verification of effectiveness of established-LIRA system,
identification of certain miRNA as ideal biomarkers, design and
development of LIRA to target these miRNAs, and establishment of a
cell-free detection system.
As shown in Figure 1, we verified the effectiveness of established
Loop-Initiated RNA Activator (LIRA) system that was designed to detect
viral RNA [1]. Next, we identified our biomarkers through
bioinformatic methods in dry lab and cell experiments in wet lab.
Subsequently, we designed and made our own LIRA that specifically target
miR-210-3p and miR-142-3p, two miRNA that were both highly expressed in
MI and capable of promoting cancer. Finally, we combined LIRA with a
reporter gene LacZ and tested the effectiveness of this LIRA in a
cell-free system to prepare our LIRA-based screening approach for
further application.
Figure 1. Schematic Representation of our Experimental Design
Verification of effectiveness of established-LIRA system
In this part, we verified whether LIRA could detect its target RNA
effectively by conducting experiment on an well-established LIRA system
to explore the best experiment setting for LIRA system. We chose H01 and
H05, two LIRA that has been reported with excellent performance to
conduct our verification experiments [1].
To fully evaluate the function of LIRA accurately, we included the
following experiments in our evaluation procedure. (Figure 2)
We transformed LIRA plasmids with or without Input RNA into BL21-DE3 for
protein expression and verified successful transformation through
antibiotic resistance selection and endonuclease digestion.
We applied IPTG induction to activate T7 promotor and control the
reaction time.
We got protein sample from IPTG-induced BL21-DE3 through ultrasonic
disruption.
We conducted Western Blot and SDS Page after ultrasonic disruption to
test whether the protein gene EGFP downstream of LIRA could be
expressed.
We measured the fluorescence from EGFP protein to evaluate the
effectiveness of LIRA quantitatively.
Figure 2. Schematic Representation of LIRA testing process
We used On/Off ratio to evaluate the effectiveness of LIRA.The On/Off
ratios of LIRA refers to on-state reporter gene expression, which is
LIRA-mediated downstram gene expression in the presence of the
corresponding input RNA, divided by off-state reporter gene expression,
which is LIRA-mediated downstram gene expression in the absence of the
corresponding input RNA. Finally we analized the result through t-test in
graphpad.
Identification of target miRNA
According to studies of reverse cardio-oncology, certain miRNA that are
highly expressed in cardiovascular disease could promote cancer. Through
bioinformatic method, we find that miR-210-3p and miR-142-3p were highly
expressed in post-MI patients. To verify whether miR-210-3p and
miR-142-3p could promote cancer progression, we conducted cell
experiments in human renal clear cell carcinoma cells (786-o cells).
We conducted Transwell assay to evaluate whether invasion level could be
affected by miR-142-3p and miR-210-3p.
We conducted Wound Healing assay to evaluate whether migration level
could be affected by miR-142-3p and miR-210-3p.
Test of LIRAs that target miR-142-3p and miR-210-3p
After identifying miR-142-3p and miR-210-3p as ideal biomarkers, we
designed and developed LIRA that target these two miRNAs. To figure out
whether stem-loop ratio of recognition region of LIRA affect the
function of LIRA, we tested the effectiveness of our designed single-arm
LIRA with different stem-loop ratios. Next, we tested the effectiveness
of 3 double-arm LIRA that target both miR-210-3p and miR-142-3p.
In this stage, we evaluated the function of LIRA following the procedure
described in Figure 2.
LIRA sequences were linked by EGFP gene as downstream reporter.
We transformed LIRA plasmid with Input plasmids into BL21-DE3 and
verified successful transformation through antibiotic resistance
selection and endonuclease digestion.
We examined the LIRA-mediated protein expression by fluorescence
measurement after IPTG induction.
Establishment of a cell-free detection system
To prepare for future application, we combined our double-arm LIRA
with a LacZ reporter gene in a cell-free system to enhance the
feasibility of conducting our screening approach without lab
equipment, i.e. Multi-mode Microplate Reader, incubator etc.
We linked a lacZ coding sequence to the LIRA as reporter gene.
We prepared double arm LIRA plasmid for the expression of LIRA in
cell-free system.
We add CPRG (yellow) as substrate, which could be converted to CPR
(purple) by lacZ enzyme.
Please check our
Protocols
for exact reaction conditions and other details in each step of our
experiment.
For details of Identification of biomarkers, please view Model page.
For specific data, image and detailed analysis please check our Result page.
For details of Identification of biomarkers, please view Model page.
For specific data, image and detailed analysis please check our Result page.
Reference
[1] Ma D, Li Y, Wu K, et al. Multi-arm RNA junctions encoding
molecular logic unconstrained by input sequence for versatile
cell-free diagnostics. Nat Biomed Eng. 2022;6(3):298-309.