Short Communication
Rationale for Using of microRNA as Markers for Screening of Colon Cancer
Farid E Ahmed*
Department of Biotechnology, Institute for Research in Biotechnology, USA
*Corresponding author: Farid E Ahmed, Institute for Research in Biotechnology, USA
Published: 11 Mar, 2018
Cite this article as: Ahmed FE. Rationale for Using of
microRNA as Markers for Screening
of Colon Cancer. Clin Oncol. 2018; 3:
1421.
Short Communication
Colon cancer (CC) could be cured at early disease stage; therefore diagnostic screening potentially
reduces mortality. Early CC detection is desirable, and would decrease mortality. Immunological
fecal occult blood test (FOBTi) screening method lacks sensitivity and requires dietary restriction,
which decreases compliance. Colonoscopy, considered as the “Gold Standard” for colorectal
cancer (CRC) screening is invasive, and in certain cases could lead to mortality. Compared to the
FOBT test, a noninvasive sensitive screen that does not require dietary restriction would be a more
convenient test. Although colonoscopy screening is a reliable method to screen for colon cancer, it
is an invasive test, is often accompanied by abdominal pain, and has potential for complications and
is not economical; all are factors that have hampered its worldwide application.
On the other hand, a screening approach that uses the relatively stable and nondegradable
micro(mi)RNA molecules when extracted from either the noninvasive human stool, or from the
semi-invasive blood samples by available commercial kits and manipulated thereafter, would be
more preferable than a transcriptomic messenger (m)RNA-, a mutation DNA-, or an epigenetic- or
a proteomic-based test. The proposed quantitative miRNA approach utilizes reverse transcriptase
(RT), followed by a modified quantitative real-time polymerase chain reaction (qPCR). To
compensate for exosomal miRNAs that would not be measured, a parallel test on stool or plasma's
total RNAs needs to be carried out, and corrections for exosomal loss are made to ascertain accurate
results. Ultimately, a chip need to be developed to facilitate diagnosis, as has been carried out for
the quantification of genetically modified organisms (GMOs) in foods. If laboratory performance
criteria are met, a miRNA test in human stool or blood samples based on high throughput automated
technologies and quantitative expression measurements currently employed in the diagnostic
clinical laboratory, would eventually be advanced to the clinical setting, making a noticeable impact
on the prevention of colon cancer.
Stool testing has several advantages over other colon cancer screening tests because: it is
noninvasive and requires no unpleasant cathartic preparation, formal health care visits, or time
away from work or routine activities. Unlike sigmoidoscopy, it reflects the full length of the
colorectum and samples can be taken in a way that represents both the right and left side colon.
Because colonocytes are released continuously and abundantly into the fecal stream, contrary to
situation in blood --where it is released intermittently as in FOBT-- and transformed colonoctes
produce more RNA than normal ones; therefore, this natural enrichment phenomenon in stool
partially obviate for the need to use a laboratory technique to enrich for tumorigenic colonocytes.
Furthermore, because testing can be performed on mail-in-specimens, geographic access to stool
screening is therefore unimpeded, which makes it prepherable to screening in blood.
It should be emphasized that although not all of the shed cells in stool are derived from a tumor,
data indicate that diagnostic miRNA gene expression profiles are associated with adequate number
of exfoliated cancerous cells, and enough transformed RNA is released in the stool. Moreover, the
availability of measurable amount of circulating miRNA genes in stool or blood (either cellular or
extracellular), by a sensitive quantitative technique such as PCR in spite of the presence of bacterial
DNA, non-transformed RNA and other interfering substances, makes accurate quantification of
miRNAs feasible, because of the high specificity of PCR primers that are employed in this method,
will overcomes screening obstacles; hence, the number of abnormally-shed colonocytes in stool, or
total RNA present in stool, plasma or serum becomes unlimiting.
A test that employs miRNA in stool or blood could also result in a robust screen because of
the durability of the miRNA molecules. Moreover, an approach utilizing miRNA genes is more
comprehensive and encompassing than a test that is based on the fragile messenger (m) RNA,
because it is based on mechanisms at a higher level of control. I believe
that ultimately the final noninvasive test in stool or blood will include
testing of several miRNA genes that show increased and decreased
expression, and eventually a chip containing a combination of these
stable molecules will be produced to simplify testing.
For mature miRNAs testing, there are currently available
commercial preparations that save time and provide the advantage
of manufacturer's established validation and QC standards. Small
noncoding RNAs that exhibit little variation in different cell types
(e.g., snoRNAs and snRNAs) are polyadenylated and are reverse
transcribed (RT) in the same way as the small miRNAs, and thereby
could serve as controls for variability in sample loading and realtime
RT-PCR efficiency. They are, however, not suited for data
normalization in miRNA profiling in blood because they are not well
expressed in serum and plasma samples. Therefore, normalization in
blood by a plate mean (i.e., mean CT value of all the miRNA targets
on the plate), or by using a commonly expressed miRNA targets (i.e.,
only the targets that are expressed in all samples are used to calculate
the mean value) would be needed for a proper normalization of the
amplification reaction.
As colon cancer-specific miRNAs are identified in stool
colonocytes or blood by microarrays- and qPCR-based approaches,
the validation of novel miRNA/mRNA target pairs within pathways
of interest could lead to discovery of cellular functions collectively
targeted by differentially expressed miRNAs.
Unlike screening for large numbers of mRNA genes, a modest
number of miRNAs is used to differentiate cancer from normal, and
unlike mRNA, miRNAs in stool and blood remain largely intact and
stable for detection. Therefore, miRNAs are better molecules to use for
developing a reliable noninvasive diagnostic screen for colon cancer,
since: 1) the presence of Escherichia coli does not hinder detection
of miRNA by a sensitive technique such as qPCR, as the primers
employed are selected to amplify human and not bacterial miRNA
genes, and 2) the miRNA expression patterns are the same in primary
tumor, or in diseased tissue, as in stool and blood samples. Although
exosomal RNA will be missed when using restricted extraction of
total RNA from blood or stool, a parallel test could also be run on
the small total RNA obtained from noninvasive stool or seminvasive
blood samples, and the appropriate corrections for exosomal loss can
then be made after the tests are carried out.
To be able to screen several miRNA genes using PCR technology,
a sequence-specific stem-loop RT primers designed to anneal to the
3’-end of a mature miRNA, followed by a SYBR Green®-based realtime
qPCR analysis “TaqMan® PCR” method is often employed using
an appropriate normalization standard. A “reference” housekeeping
internal standard gene (e.g., endogenous reference genes RNU6
genes RNU6A and RNU6B, SNORD genes SNORD43, SNORD44,
SNORD48, SNORA74A) or miRNA normalizers (e.g., miRNA 16,
miRNA-191), or in some cases against several standards because the
total input amount may vary from sample to sample. To ensure that
miRNA quantification is not affected by the technical variability that
may be introduced at different analysis steps, synthetic nonhuman
spike-in miRNA can be used to monitor RNA purification and RT
efficiencies. In PCR reactions, QC procedure, as Minimum Information
for Publication of Quantitative Real-Time PCR expression (MIQUE)
is implemented to ensure uniformity, reproducibility and reliability
of the PCR reaction and data integrity. Statistical analysis is used
for data interpretation, and bioinformatics may be employed to
correlated miRNAs with mRNAs data. Our group demonstrated
using melt curve analysis (MCA) for interpretation of mild
nutrogenomic miRNA expression data in a recent three week dietary
intervention trial, by measuring the magnitude of the expression of
key miRNA molecules in stool of healthy human adults as molecular
markers, following the intake of Pomegranate juice (PGJ), functional
fermented sobya (FS), rich in potential probiotic lactobacilli, or their
combination, in which total small RNA was isolated from stool of
25 volunteers before and following dietary intake. Expression of 88
miRNA genes was evaluated, using Qiagen's 96 well plate RT2 miRNA
qPCR arrays. Employing parallel coordinate’s plots, no observed
significant separation was observed for the gene expression (Cq)
values, using Roche 480® PCR Light Cycler instrument, and none of
the miRNAs showed significant statistical expression after controlling
for the false discovery rate. On the other hand, melting temperature
profiles produced during PCR amplification run, identified seven
significant genes (miR-184, miR-203, miR-373, miR-124, miR-
96, miR-373 and miR-301a), which separated candidate miRNAs.
These miRNAs could thus function as novel molecular markers of
relevance to oxidative stress and immunoglobulin function, for the
intake of polyphenol (PP)-rich, functional fermented foods rich
in lactobacilli (FS), or their combination see (Figure 1), which is a
graphical representation of the parallel plot coordinates of the studies
miRNA genes. The genes were ordered using the p-values of a one
way ANOVA based on groups. Genes with the smallest p-values are
presented first. (Figure 1A) below shows control genes, while in
(Figures 1B and C) five miRNA genes show separation.