MIRNAS IN

DIAGNOSTICS AND
PROGNOSTICS
Genomics, Proteomics, Metabolomics assignment.

SYED M. ZAKI HAIDER, M. FIDA UR REHMAN

SUBMITTED TO DR. SABA IRSHAD
School of Biochemistry and Biotechnology, University of the Punjab
 MIRNAS IN DIAGNOSTICS AND PROGNOSTICS

What are MiRNAs?

MiRNAs (micro RNAs) are one of the members of a large class of non-coding RNAs, which means
that they do not need to get translated into proteins to perform their function (Bavelloni et al.,
2017). They are endogenous (already produced in cells), approx. 19-24nt in length(Bhaskaran &
Mohan, 2014) and they act as gene expression regulators at post-transcriptional level by interacting
with the coding mRNA (messenger RNA). MiRNA are coded by DNA sequences into primary miRNAs
(pri-miRNAs) that are further rendered to precursor-miRNA (pre-miRNA) and finally into mature
miRNAs. They are known to regulate the expression of approx. 60% of total mammalian
genes(Makarova et al., 2016). We all know that all the cells of our body share the same genetic
material, but how come each type has different functions and biology? Its due to the fact that not all
genes are expressed, some are silenced, either temporarily accordingly to the physiological condition
of the body, or permanently. This is where we owe to these short RNAs. In majority of cases, miRNAs
are seen to suppress gene expression by binding with the 3’UTR (untranslated region) of the
particular mRNAs(Ha & Kim, 2014). However, other interactions of miRNAs for the purpose of gene
silencing includes the 5’UTR, the complementary coding sequence itself, or the gene promoters a
well (Broughton et al., 2016). Interestingly, miRNAs also seemed to activate gene expression as well
by direct or indirect means in certain circumstances(Vasudevan, 2012).

Travelling back to time, discovery of miRNA:

MiRNA was first discovered in 1993 by Victor Ambros and Gary Ruvkon and colleagues while they
were working on the genetic regulation of development in the reproductive system of the
roundworm Caenorhabditis elegans. The first miRNA, encoded by the lin-4 gene was identified as a
noncoding RNA responsible for regulation of expression of lin-14 protein, whose downregulation
was found to be crucial for transition from first larval stage (L1) to L2 in C.elegans. It was found that
lin-14 gene was post-transcriptionally downregulated by its 3’UTR region, to which lin-4 was
complementary to(Lee et al., 1993). The transcribed lin-4 was not translated into proteins, instead it
produced to 2 small RNAs (21 and 61nt) where the longer one formed a stem looped structure which
acted as a precursor for the shorter RNA(Bhaskaran & Mohan, 2014). Seven years later (in 2000), it
was found that miRNA let-7 was playing a vital role in progression from later larval stage to adult in
C. elegans by downregulation of lin-41 gene(Reinhart et al., 2000, p. 7). Since then, a number
miRNAs were discovered in different species of animals and plants. Many of them were seen to be
highly conserved across species and showed cell type specificity. A miRNA registry with the name of
miRBase is working as the primary online repository for all potential miRNA sequences, annotation,
nomenclature, and target prediction information since 2002(Bhaskaran & Mohan, 2014).

Biogenesis and working of miRNAs:

The biogenesis of miRNAs starts with the production of their transcripts by RNA pol ll/lll. Approx.
50% of miRNA express from the non-protein coding transcripts in animals, the rest either originate
from the introns of protein encoding transcripts or the exons (which another vast area of research,
adding to the complexity of miRNA biogenesis and maturation)(Bhaskaran & Mohan, 2014). At first
the miRNA gene is transcribed into a longer primary miRNA (pri-miRNA) which has a 5’guanosine
cap and 3’ polyadenylated tail (post-transcriptional modifications)(Peng & Croce, 2016). This hair-
looped pri-miRNA is further cleaved at the base of hairpin to form a precursor miRNA (pre-miRNA)
approx. 70-120nt in length by a complex known as microprocessor.(O’Brien et al., 2018). This
processor complex is composed of a 160kDa RNase lll enzyme called drosha and a double stranded
RNA (dsRNA) binding protein called DGCR8(Bhaskaran & Mohan, 2014). 2nt 3’ overhangs and 5’
phosphate are present in this precursor. The pre-miRNA is then exported outside of nucleus to
cytoplasm by RAN-GTPase dependent nuclear transport receptor protein exporting 5 (EXP-5)(Peng
& Croce, 2016) (RAN is basically a GTP binding transport protein involved in transportation in and
out of nucleus). In cytoplasm, further processing by another RNase lll enzyme DICER-1 (in a complex

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 MIRNAS IN DIAGNOSTICS AND PROGNOSTICS

with dsRNA-binding proteins, protein kinase RNA activator and transactivation response RNA
binding protein) results in the formation of mature 20-22nt miRNA duplex(Bavelloni et al., 2017)
where the terminal loop is excised from the duplex structure. This duplex is composed of 2 strands,
the guide strand and a passenger strand. The duplex is unwound and the guide strand (acting as
final miRNA) is incorporated with the RNA induced silencing complex (RISC) to perform its function
of gene silencing. This complex comprises of the above mentioned dsRNA binding proteins, DICER1
and Argonaut (AGO) proteins (AGO1-4 in case of humans)(O’Brien et al., 2018). The guide strand
guides the RISC to the target site on mRNA where it shows complementarity. Partial
complementarity leads to translation inhibition and perfect complementarity leads to degradation of
mRNA as RISC chops the transcript at target site(Bhaskaran & Mohan, 2014). Thus, miRNAs regulate
gene expression by binding to the mRNA of the target genes and direct their post-transcriptional
repression(Bavelloni et al., 2017). However, interestingly research also showed that miRNA can also
act as translational activators under certain conditions such as in case of a cell cycle
arrest((Vasudevan et al., 2007).

MiRNAs in diagnosis and prognosis:

According to Merriam-Webster, “A  diagnosis  is an identification of a disease via examination. What
follows is a  prognosis, which is a prediction of the course of the disease as well as the treatment and
results”. The fact that MiRNAs are regulating gene expression and the resulting proteins, miRNAs are
vital for a variety of body functions like differentiation, metabolism, growth and development,
apoptosis etc. there is a ton of research evidence supporting the fact that as miRNA are involved in
gene regulation, then definitely they can prove to be possible diagnostic and prognostic biomarkers
of various diseases and genetic disorders. A biomarker is a biological indicator of a biological

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condition. Unlike the protein based biomarkers (like enzymes or antigens), miRNAs have great
potential to act as biomarkers with higher specificity, accuracy and senstivity(Faruq & Vecchione,
2015). Stable miRNAs have been found in biological fluids like as plasma, serum, saliva, milk,
cerebrospinal fluids(Faruq & Vecchione, 2015) and their upregulation or downregulation is shown to
be linked with cancers, autoimmune diseases and other maladies(Faruq & Vecchione, 2015). Now
we shall look at some most recent updates regarding the use of miRNAs in diagnostic and
prognostics.

Current updates:
1. Hypomethylation and downregulation of miR-23b-3p are associated
with upregulated PLAU: a diagnostic and prognostic biomarker in head
and neck squamous cell carcinoma (October, 2021):
HNSCC (head and neck squamous cell carcinoma) is a cancer that comprises of a heterogeneous
group of tumors arising from the squamous epithelium of the oral cavity, oropharynx, larynx and
hypopharynx, and characterized by low survival, high recurrence rate with observable lymph node
development(Huo et al., 2021). With more than 800,000 patients being diagnosed and 40,000 dying
per year(Huo et al., 2021), it sixth most common malignancy in the world(Huo et al., 2021).
Through use of a variety of bioinformatics tools and cancer databases like The Genome Cancer Atlas
(TGCA) along with clinical samples from HNSCC patients,the upregulation and overexpression of
plasminogen activator urokinase (PLAU) was concluded to be an important characteristic of HNSCC
tissues(Huo et al., 2021). The expression of PLAU and the clinical stage were important prognostic
elements for patients with HNSCC(Huo et al., 2021).
It was further observed that PLAU hypomethylation was positively correlated with its
overexpression, irrespective of mRNA expression of PLAU(Huo et al., 2021). Thus, PLAU
hypomethylation proved to be another important prognostic biomarker(Huo et al., 2021).
The downregulation of miR-23b-3p was also an important factor associated with metastasis and
PLAU overexpression(Huo et al., 2021). Being PLAU target miRNA, it meant that upregulation of
miR-23b-3p suppressed the malignant properties of PLAU and that combined with hypermethylation
would nullify the oncogenic role of PLAU in HNSCC(Huo et al., 2021).
2. MiR-4454 Promotes Hepatic Carcinoma Progression by Targeting Vps4A and
Rab27A (November, 2021):
Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer (starting from
liver). HCC malignancy the most widespread in China, with more than 50% of new cases and deaths
worldwide(Lin et al., 2021). At present main HCC therapy remains surgery but survival rates are low.
In case surgery does not remain an option, a multikinase inhibitor Sorafenib is an approved drug for
treating HCC but it has loads of side effects and relatively slow curative effect(Lin et al., 2021). Thus,
the understanding of disease at cellular and genetic level is very important to come up with a cure
that is efficient, accurate and with less side effects. Recent research showed that miR-4454 actually
promotes the development of HCC(Lin et al., 2021).
Exosomes are bilayer coated vesicles that cells release under different physiological and pathological
conditions(Lin et al., 2021). These exosomes are found to be rich in micro-RNAs and these vesicles
can actually regulate the tumor phenotype and behvaiour, further study of which will be helpful in
devising ways for timely diagnosis, accurate prognosis and efficient treatment of the malady
afterwards(Lin et al., 2021).
The effect of different stress conditions such as heat shock, doxorubicin (a chemotherapeutic drug),
transforming growth factor beta one (TGF-β1, a multifunctional cytokine), hypoxia and
reoxygenation was observed on the HepG2 cells (a human liver cancer cell line) that what type of
exosomes were released and what kind of miRNA expression they exhibited(Lin et al., 2021).

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It was revealed that exosomes exhibited different morphologies under different conditions and the
expression of miR-4454 was upregulated in HCC(Lin et al., 2021). The target genes for this miRNA
turned out to be Vps4A and Rab27A whose expression was suppressed(Lin et al., 2021). On contrary
the inhibition of miR-4454 suppressed the proliferation, accelerated cycle arrest, enhanced
apoptosis, and lowered oxidative stress resistance, migration and invasion ability of HepG2 cells(Lin
et al., 2021).
3. Let 7 microRNAs in the diagnosis, treatment and prognosis of leukemia
(November, 2021):
Leukemia is the cancer of blood which is produced due to increase the number of white blood cells
(leukocytes) in the bone marrow and blood circulation. Radiation exposure is the major cause of
blood cancer. It can be acute and chronic have four major types including acute myelogenous (AML),
acute lymphoblastic (ALL), chronic myelogenous (CML) and chronic lymphocytic leukemia (CLL)(Chen
et al., 2022).
As leukemia is the malignant disease its pathogenesis include numerous epigenetic factors. Among
various epigenetic factors, Let-7 miRNA is an important molecule which play major role in the
differentiation, development and proliferation.(Chen et al., 2022) and can be used as potential
biomarker for the diagnosis of blood cancer(leukemia)(Chen et al., 2022).
The oncomiRs or miRNAs target the cancer genes like oncogenes or tumor repressor genes which
are linked with carcinogenesis(Chen et al., 2022). The Let-7 family of miRNAs is the major example
of oncomiRs which act as tumor suppressor gene in cancer cells. The Let-7 in cancer development is
considered to be the occurrence of leukemia(Chen et al., 2022). Let 7 physiologically regulates
several target genes related to cell cycle progression, proliferation, differentiation and cell signaling
pathways. There are a variety of mature Let 7 subtypes, and nine important members have been
identified as Let 7a, Let 7b, Let7c, Let 7d, Let 7e, Let 7f, Let 7g, Let 7i and miR 98(Chen et al., 2022).
Let 7 may regulate the characteristics of leukemia stem cells by inhibiting self-renewal and
promoting differentiation. These findings suggest that Let 7 is a key regulator of cell cycle
progression and differentiation(Chen et al., 2022).
The miR-125 family, including miR-125a/miR-99b/Let-7e, miR-125b-2/miR-99a/Let-7c-1 and
miR-125b-1/miR100/Let-7a-2, are located on human chromosomes 19, 21 and 11(Chen et al., 2022).
Overexpression of miR-125 will promote the development of a myeloproliferative neoplasm-like
phenotype, which will then develop into AML(Chen et al., 2022). As a member of the miR-125 family,
Let-7 can bind to target mRNA and cause its degradation or interrupt its transcription. Let -7 was
found to lead to defects of cell proliferation and differentiation of AML cells.Let-7a-2-3p expression
could be used alone as a biomarker for the prognosis of patients with AML.(Chen et al., 2022) It is
also observed that high Let-7a-2-3p expression could be used as a good prognostic marker in
patients with CN-AML independently or in combination with other risk factors, particularly in
patients after stem cell transplantation(Chen et al., 2022). Therefore, Let-7a-2-3p could be used not
only as a prognostic biomarker but also evaluate treatment outcomes too. Let-7a-2-3p was
upregulated in primary cells of patients with AML, and that it could promote the transformation of
normal hematopoietic cells into malignant cells in vivo or in vitro(Chen et al., 2022).
ALL accounts for 75% of childhood leukemias and 30% of childhood malignant tumors. Although
intensive combination therapy can improve the therapeutic outcomes, 15-20% of patients still fail to
respond appropriately, and relapse is the primary cause of treatment failure(Chen et al., 2022).
Let-7b was one of 27 miRNAs differentially expressed between ALL and AML, and certain important
miRNAs could distinguish ALL from AML more accurately. (Chen et al., 2022) Although Let-7b is
primarily used as one of the miRNA biomarkers for distinguishing ALL and AML(Chen et al., 2022).
Let-7b-5p can be used as a potential biomarker for the diagnosis and prognosis of CML. This strong
evidence showed the central role of Let-7 expression in tumorigenesis, and confirmed the regular

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characteristics of miRNAs in leukemia. (Chen et al., 2022)The discovery of non-coding RNA Let-7
opens up novel prospects for the diagnosis, prognosis and treatment of CML(Chen et al., 2022).
APL is a type of acute leukemia with rapid onset symptoms, insufficiency of intravascular
coagulation, a poor response to chemotherapy and a high mortality rate miRNAs were involved in
the occurrence of APL by regulating the expression of certain genes(Chen et al., 2022). For example,
the proto-oncogene c-Myc is known to promote cell viability and proliferation, and plays a key role
in maintaining the cell cycle in the majority of hematopoietic cell lines. In several malignant tumors
of the hematopoietic system, the high expression of c-Myc is associated with a poor prognosis, and
c-Myc is often activated by PML/RARα in APL(Chen et al., 2022). Failure to downregulate c-Myc in
transgenic mice can lead to myeloid leukemia, which is characterized by blocked differentiation,

indicating that the inhibition of c-Myc is a key event for cells to commit to the differentiation
pathway(Chen et al., 2022).
4. Diagnostic and prognostic value of serum miR-9-5p and miR-128-3p levels in
early-stage acute ischemic stroke (November, 2021):
Acute ischemic stroke (AIS) accounts for 70% of all stroke cases and is associated with markedly high
patient disability and mortality rates.(Wang et al., 2021) Early diagnosis and timely intervention
would significantly reduce these rates and improve the prognostic outcomes and quality of life of
patients with AIS.(Wang et al., 2021) Currently, AIS is diagnosed using computed tomography
(CT).However in the early stages of the disease, CT cannot identify any abnormalities in
approximately 40–50% of the patients with AIS(Wang et al., 2021). Although interleukin-6, neuron-
specific enolase (NSE) glial fibrillary acidic protein, and 25-hydroxyvitamin D are associated with the
onset of AIS, additional markers are needed owing to the low specificity and sensitivity of these
markers(Wang et al., 2021a).
MiRNAs have been detected in the cerebrospinal fluid, urine, serum, and plasma samples of patients
with AIS, suggesting their diagnostic value .(Wang et al., 2021b) Their expression levels change
significantly during the pathogenesis of AIS, a process that involves platelet aggregation, endothelial
dysfunction, and neuronal injury. (Wang et al., 2021b) Previous studies have found that mir-424 and
miRNA-15a are closely correlated with the occurrence of stroke. Indeed, the development of AIS has
been found to correlate with changes in miRNA levels in the peripheral blood of patients. (Wang et
al., 2021b) The miRNA miR-9-5p is likely involved in the development of AIS.(Wang et al., 2021b) It
has been shown to attenuate ischemic stroke by directly targeting endoplasmic reticulum
metallopeptidase1 (ERMP1) mediated endoplasmic reticulum stress. (Wang et al., 2021b) However,
other invivo and in vitro studies have revealed that blocking of miR-9-5p and miR-128-3p activity
could result in reduced ischemic stroke induced neuronal cell death and infract volume. The

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relationship between AIS and serum miR-9-5p and miR-128-3p levels has not yet been fully
explored(Wang et al., 2021b).
MiR-9-5p was found to be upregulated during AIS pathogenesis. By targeting B-cell lymphoma 2-like
11, miR-9-5p can inhibit neuronal apoptosis in AIS, and the upregulation of miR-9-5p promotes
neuronal self-repair.(Wang et al., 2021b) In the early stages of stroke, detection of miR-9-5p in the
blood clarify the diagnosis of early brain injury in stroke, and provide insights for developing efficient
treatment strategies for AIS.(Wang et al., 2021b) It has been reported that miR-128b is significantly
upregulated in the plasma of patients with ischemic stroke.(Wang et al., 2021b) In the early stages of
stroke, the detection of high serum miR-128-3p levels can also be used as an important criterion for
predicting the occurrence of neuronal apoptosis in patients, thus providing clues for the early
diagnosis of ischemic stroke.(Wang et al., 2021b) In serum miR-9-5p and miR-128-3p levels exhibit
clinical utility in the early diagnosis of acute cerebral infarction and can therefore be used as
potential markers for the diagnosis and treatment of AIS(Wang et al., 2021b).
5. MiR-124 Is Downregulated in Serum of Acute Cerebral Infarct Patients and
Shows Diagnostic and Prognostic Value (October, 2021):
Acute cerebral infarction (ACI) refers to the disruption of blood supply to brain tissues caused by
intracranial and extracranial artery stenosis or occlusion, resulting in ischemic necrosis or
cerebromalacia because of focal tissue ischemia and hypoxia.(Zhou & Qi, 2021) Without rapid clinical
decision-making, ACI may result in severe outcomes, such as disability or even death consequently,
early diagnosis of ACI is crucial for the improvement of curative effects and the reduction of
potential sequelae and mortality. (Zhou & Qi, 2021) The blood sample is the most readily available
material in monitoring brain changes in acute ischemic stroke and rehabilitation of ischemic
stroke(Zhou & Qi, 2021).
Accumulating studies have unveiled the aberrant expression of miRNAs in the brain tissues and
peripheral blood of ACI patients, implying that miRNAs are implicated in the pathogenesis,
diagnosis, and prognosis of ACI(Zhou & Qi, 2021).
2,7,8 miR-124 is identified as one of the most abundant miRNAs in the human brain that modulates
variant biological functions of the central nervous system (CNS), and dysregulated miR-124 is tightly
associated with stroke.(Zhou & Qi, 2021) The elevated miR-124 expression has the property to
reduce the infarct size and facilitate the recovery of neurological functions post-ischemic stroke.
MiR-124 participates in the pathophysiological process of ischemic brain injury, and plasma miR-124
is identified as a potential biomarker for the time-sensitive diagnosis of ACI(Zhou & Qi, 2021).
6. MicroRNAs as Regulators of immunological reactions in hepatocellular
carcinoma (October 2021):
Hepatocellular carcinoma (HCC) is the cancer which start in liver and is considered the third major
cause of cancer mortality.(Hepatocellular Carcinoma, n.d.) In the early stage if it is diagnosed than it
can be cured through surgery or transplant. In the cases which are more advanced it can’t be cured
but treatment and support help patients to live longer and better. (Hepatocellular Carcinoma,
n.d.)Exact causes of Hepatocellular Carcinoma (HCC) is not to be sure however Hepatitis B ,Hepatitis
C ,Obesity, Increased iron storage and more alcoholic drink consumption can increase the risk of
Hepatocellular carcinoma(Hepatocellular Carcinoma, n.d.-a).
MiRNAs are small non-coding RNAs which are the regulators of gene expression by mRNA
degradation or through translational inhibition.(Gupta et al., 2021) MiRNA s have regulative impact
on both innate and adaptive cells, control activities and all developmental stages of immune system.
If there is any inexpression caused by miRNA s leads to cause various cancer types including
HCC(Gupta et al., 2021).
The miRNAs played important role to regulate the gene expression of the whole immune system and
also essential in activation, differentiation and development of T lymphocytes. They are also
important in cellular cytokine expression.(Gupta et al., 2021)

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MiRNAs act as oncogenes, suppressors or tumor in cancer associated pathways by regulating

numerous immunological reactions.(Gupta et al., 2021)
MiRNAs in Hepatocellular Carcinoma (HCC) target several signaling hubs and regulate innate and
adaptive immune cell responses.(Gupta et al., 2021)
Human leukocyte antigen (HLA-G) is crucial marker to immune suppression and tolerance bind with
immune suppressive receptors of natural killer (NK) and T cells and generate immunosuppressive
signaling. Infection of with Hepatitis B (HBV) downregulate the expression of mir-152(a HLA-G
targeted miRNA) and upregulate the HLA-G. Natural killer (NK) cells inhibited due to high expression
of HLA-G which shows that mir-152 is involved in HBV-induced Hepatocellular carcinoma.
(Hepatocellular Carcinoma, n.d.-b)

Mir-615-5p shows cell and disease specific functions in both NK cells of HCC patients and HCC cell
lines. It causes the suppression of signaling in both cells and showed anti-cytotoxic effect in NK cells
and tumor suppressor effect in HCC cell lines.(Gupta et al., 2021)
Mir-122 down regulation is also considered as a prognostic marker in HCC and the expression is
inversely proportional to the levels of toll like receptors4 in normal liver cells and HCC cells.miR-122
mediated blockage of TLR4 modulates the innate immunity of host in hepatoma cells(Gupta et al.,
2021).

Conclusion:
MiRNAs are non-coding RNAs and important regulators of gene expression at post transcriptional
stage by either cleaving the target sequence or suppressing protein formation. Their upregulation or
downregulation is an important factor in various physiological and pathological conditions and this
can prove to be a diagnostic and prognostic biomarker in various cancers, autoimmune diseases and
other genetic conditions. Further study of their mechanism of gene expression regulation, their
interaction with different other molecules and signaling pathways, whether or not they can
accurately target specific cells under physiological conditions, how different cells take up miRNAs
and how they can be transported for the purpose of gene regulation will definitely help in better
diagnosis and prognosis of diseases. This will usher us in a new era in fields of molecular biology,

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genomics, proteomics, metabolomics, health biotechnology and other life science disciplines. The
journey towards betterment will be always continued on the road of science.

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SYED M. ZAKI HAIDER, M. FIDA UR REHMAN 9