Introduction
Cardiovascular diseases (CVD) and cancer are two major causes of global
mortality[1], and cancer and cardiovascular disease often coexist. The
emerging field of Reverse Cardio-Oncology suggests a correlation that
cardiovascular disease may accelerate the onset and progression of
cancer. In recent years, the overall upward tendency in the incidence of
myocardial infarction has sparked public concern[10], but the potential
cancer risk of patients with myocardial infarction has not been widely
recognized. In response to this new finding and the critical knowledge
gap, we designed a diagnostic kit named Heartecho which provides a rapid
and simple way to warn myocardial infarction patients of their risk of
cancer in order to refine the prognosis and extend the survival duration
of them.
Figure 1 Top 10 causes of death globally in 2000, 2019, 2020 and 2021
Meaning of “Heartecho”
Heartecho means "listening to the echo of the heart", which not only
indicates that heart disease may facilitate the occurrence and
development of cancer through signal molecules, but also implies that
the occurrence of heart disease can serve as a signal for the onset of
other diseases.
Background
Reverse Cardio-Oncology
Over the past several decades, CVDs and cancer have been regarded as two
distinct separate conditions without any direct connection. The
establishment of Cardio-Oncology highlighted that tumor could induce
CVDs through multiple pathways. However, the impact of cardiovascular
diseases on cancer has been hardly noticed.
Reverse Cardio-oncology, as a burgeoning star in the cross discipline of
cardiovascular and cancer, is devoted to studying the role and mechanism
of cardiovascular disease in the occurrence and development of cancer.
Figure 2 Graphic depiction of the hypothesis of secreted proteins by the
failing heart that enhance tumorigenesis
In recent years, an increasing number of research and discoveries have
also spurred the prosperity and advancement of this field and offered
more potent scientific evidence.
Figure 3 Timeline of Reverse Cardio-Oncology
So far, Reverse Cardio-Oncology has witnessed remarkable development.
The nearly decade-long research has considerably enhanced our
comprehension of the connection between CVDs and cancer, two leading
global causes of death. We believe that the advancement of Reverse
Cardio-Oncology will provide better cancer prevention or treatment
strategies for cardiovascular disease patients in the future.
Inspiration
Analyses from large databases suggest an increased incidence of cancer
in patients with myocardial infarction, particularly within the first
six months post-admission. However, the extent to which this
represents an underlying biological link remains unclear [8]. A recent
retrospective analysis of clinical data indicated that within the
first three months after STEMI, the incidence of cancer is
significantly higher in STEMI patients compared to that of the general
population (HR 2.45, 95% CI 1.13–5.30) (Figure 4) [9]. Similar reports
have been published by other researches, for example Rinde's team in
2017 identified the increased early and late cancer risks of
post-myocardial infarction (Figure 5) [10]. Which have mentioned above
highly leads us to focus on the potential cancer risk of patients
suffered from myocardial infarction.
Figure 4 Relative risk and cancer risk indicators for STEMI patients
post-STEMI[9]
Figure 5 Gender-stratified incidence and risk ratio of cancer after
myocardial infarction[10]
Myocardial Infarction
Myocardial infarction (MI) refers to ischemic necrosis of the
myocardium. Pathologically, MI can be classified into ST-elevation
myocardial infarction (STEMI) and non-ST-elevation myocardial
infarction (NSTEMI). Clinically, MI can be divided into five subtypes,
with Type 1 and Type 2 MI being the most common. Type 1 refers to
spontaneous MI associated with ischemia due to primary coronary events
such as plaque erosion and/or rupture, fissuring, or dissection. Type
2 MI is secondary to ischemia caused by conditions such as coronary
artery spasm, coronary embolism, anemia, arrhythmias, hypertension, or
hypotension [11-12]. Clinically, MI primarily manifests as chest pain,
chest tightness, palpitations, and sweating.
Figure 6 Myocardial infarction
Ischemic heart disease (IHD) is the leading cause of death worldwide,
presenting clinically as myocardial infarction and ischemic
cardiomyopathy. According to the results of global epidemiological
studies on IHD based on the Global Burden of Disease research, the
incidence of IHD is on a significant upward trend. Additionally, the
prevalence increases with age, continuing to rise up to 89 years old.
Figure 7 Age distribution of global ischemic heart disease prevalence
(A) and incidence (B) in 2017 [13]
MI is primarily found in developed countries but is also common in
developing countries. Despite a global decline in MI-related
mortality, the mortality and morbidity associated with MI-related
heart failure remain high [14-15].
Figure 8 The upper line represents the cumulative incidence of
all-cause mortality or hospitalization due to MI-related heart
failure, while the lower line represents only the hospitalization rate
due to MI-related heart failure [15]
The condition of myocardial infarction is highly dangerous and it has
caused a considerable health burden to the global community. If
patients suffer from cancer subsequent to myocardial infarction, the
prognosis will be much more severe, which will pose a tremendous
threat to the lives of patients.
Therefore, early detection and early diagnosis of cancer is extremely
crucial for the quality of survival life of patients.
Our Solution
Heartecho employs a Loop-Initiated RNA Activator (LIRA)-based AND logic
gate system which can detect the biomarker microRNA (miRNA) and
integrates with a cell-free system to show results.
MiRNA
Numerous studies have shown that the expression of various secretory
factors in failing hearts is aberrant, and these abnormal secretory
factors are closely related to the occurrence and development of cancer
[16] (Figure 9).
Figure 9 Cardiac secretory factors promote tumor development
miRNAs are a class of small non-coding RNAs, typically 21-23 nucleotides
in length, which play a crucial role in gene regulation. MiRNAs
downregulate gene expression by base-pairing with the 3' UTR of their
target mRNAs[17], thereby controlling cellular processes in eukaryotes
[18] (Figure 10).
Figure 10 Function mechanism of miRNA
Current experimental studies have demonstrated that exosomes containing
miR-22-3p secreted by ischemic myocardial cells enter the plasma and are
absorbed by cancer cells, leading to the inhibition of apoptosis in
cancer cells and thus promoting cancer progression [19] (Figure 11).
Therefore, we propose a solution: by detecting upregulated miRNAs in the
plasma of post-myocardial infarction patients that can promote cancer
progression, we can identify high-risk cancer populations among these
patients, achieving early detection, diagnosis, and treatment of cancer.
Figure 11 Ischemic myocardial cells secrete exosomes containing
miR-22-3p to promote tumor development
LIRA Detection Technology
The Loop-Initiated Isothermal RNA Activator (LIRA) is a novel RNA
detection technology based on RNA secondary structure[20]. The
single-arm structure of LIRA consists of a loop and complementary paired
stem, with its recognition domain divided into two parts located on the
stem (b*) and the loop (a*). LIRA hides the ribosome binding site (RBS)
sequence and the start codon AUG necessary for RNA translation within
the stem-loop structure. When the target miRNA is present, it binds to
the LIRA recognition domain, disrupting its original structure and
exposing the RBS and AUG, thereby initiating the translation of
downstream reporter genes.
Figure 12 Working principle of single-arm LIRA
In the AND gate structure of LIRA, the secondary structure is designed
with two recognition arms, A* and B*. The B* recognition arm is
thermodynamically stable, and only when miRNA-A binds to the A* arm and
destabilize it, can the B* arm be opened by miRNA-B, exposing the RBS
and AUG to initiate downstream gene translation.
Figure 13 Working principle of double-arm LIRA
Chromogenic Method
To promote our detection system more widely, we aim to indicate results visibly. We identified an enzyme, LacZ, which can react with CPRG to produce a purple substance. We replaced the downstream gene of LIRA with the LacZ and added CPRG substrate. When the translation of LacZ in the LIRA system is initiated by the two miRNAs, the LacZ reacts with CPRG to produce a purple substance.
Figure 14 Color development principle of double-arm LIRA combined with LacZ enzyme.
Future Application
The supernatant was collected after centrifugation and incubated with cell-free system containing LIRA system and CPRG at 37℃ for 1 to 2 hours. If there are two target miRNAs in the sample simultaneously, the complementary bases on the LIRA system's arms and door stem will open, exposing the RBS sequence and AUG to start the translation of the downstream LacZ gene. LacZ holoenzyme will digest CPRG to produce a purple substance, turning the system purple; if only one target miRNA or no target miRNA is in the sample, the LIRA system won't start and the system remains yellow.
Figure 15 Application process of Heartecho
Project Innovation
Based on the epidemiological discovery of clinical problems, we brought in a new discipline, namely Reverse Cardio-Oncology, into the iGEM community for the first time, and creatively combines the double-arm LIRA and the simplicity of cell-free system to screen high-risk tumor individuals among patients after myocardial infarction which will be extremely beneficial for patients in the clinical settings.
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