3.1. Rheumatoid Arthritis RA is a heterogeneous, enfeebling, systemic AD. This chronic ailment
pertaining to musculoskeletal pain, prolonged morning stiffness,
inflammatory alterations of the tissues of synovial joints, cartilage
and bones, often resulting in erosion of bones and subsequently,
complete destruction of joints, if not treated on time . The internal
extra-articular organs like skin, eyes, lungs, heart, kidney, and blood
vessels are also affected by this symmetrical AD .
People afflicted with RA range from 0.24% to 1% globally . It is
predominant in women compared to men and has increased prevalence in the
age group of 50-65 years . The emergence of non-communicable diseases
have become a leading cause of mortality worldwide and RA is one of
them. Both developed and low-middle income countries (LMIC) are severely
affected by these diseases . RA being a complex AD, the pathophysiology
is regulated by several inflammatory markers and immunogenic proteins. B
lymphocytes, T lymphocytes and macrophages predominantly contribute to
the progression of the disease . These autoreactive B cells induce the
secretion of cytokines like tumor necrosis factor-α (TNF-α),
interleukin-6 (IL-6), IL-12, IL-23, and IL-1α in the diseased tissue.
Another cytokine, receptor activator of nuclear factor κB ligand (RANKL)
has been found to show enhanced production from memory B lymphocytes in
peripheral blood, synovial fluid and affected tissue of patients. This
cytokine is responsible for bone resorption as it binds to its receptor,
stimulates differentiation and activation of osteoclasts . B lymphocytes
are also responsible for the secretion of rheumatoid factors (RF) and
anti-citrullinated protein antibodies (ACPA) .Figure 3 - Detailed immune regulation and miRNA networks
involved in RA proliferation. Illustrative representation of
multifaceted roles of mast cells secreting several proinflammatory
interleukins like IL-6, IL-1 and tumor necrosis factors leading to
osteoclast maturation and subsequent bone degradationThe activation of T lymphocytes are stimulated by both B cells and
macrophages. Synovial T cells have been reported to secrete cytokines
like, IL-15, IL-7, IL-13, IL-4, IL-2, basic fibroblast growth factor,
epidermal growth factor and these cytokines have a major contribution in
the etiology and pathogenesis of the ailment . Macrophages, in the early
stages of the disease, are known to stimulate T cells and enhance the
production of IL-1α, IL-1β, and Matrix metalloproteinases (MMPs) . The
inflammatory mediators secreted by the autoreactive immune cells
interact with neurons, and neuroglia and result in neurogenic
inflammation . RA patients are often broadly classified into
ACPA-positive and ACPA-negative. These two patient types have very
different pathophysiology, ACPA-positive being a more clinically
aggressive-phenotype. Citrullination is categorised as a
post-translational modification which results in the synthesis of a
polar (neutral) citrulline from a positively charged arginine and is
also aided by Ca2+-requiring enzyme peptidyl arginine-deiminase (PAD).
The genetic and epigenetic factors interact and trigger the production
of ACPA in diseased individuals. Environmental factors like smoking,
inhalation of nanosized silica or silica dust may trigger mucosal-TLRs
which activates Ca2+-dependent PADs and also antigen presenting cells
(APCs) like dendritic cells and B lymphocytes .
miRNA-117-5p expression is seen to be reduced in chronic RA. This miRNA
specifically targets the JAK-STAT pathway and downregulates the IL6
expression. Similarly, several miRNAs acts as a biomarker and can be
detected from the synovial fluid or tissue in RA patients. miRNA-34a is
regulated by NF-kB family of cell signaling molecules, which are further
activated by inflammatory cytokine molecules . miRNA-29a acts as an
anti-inflammatory marker in RA fibroblast-like synoviocytes (RA-FLSs).
The drop in miR-199a-3p expression contributes to local hyperplasia.
miRNA-375 in RA-FLSs exhibits a positive effect on synovial pathogenesis
that occurred as a result of canonical Wnt signaling inactivation,
thereby influencing the progression of the disease and severity of the
condition . Exosome secreted miRNAs are important biomarkers and are
vital targets for developing therapies to treat RA. According to a study
by Cunningham et al. eight serum miRNAs viz., miRNA-126-3p, let-7d-5p,
miRNA-431-3p, miRNA-221-3p, miRNA-24-3p, miRNA-130a-3p, miRNA-339-5p,
let-7i-5p are found to have enhanced expression in RA afflicted
individuals compared to that of healthy people . Exosome derived
miRNA-486-5p from synovial tissue is associated with tumor initiation or
suppression in a number of cancers and also pulmonary fibrosis.
miRNA-486-5p secreted from the exosomes of RA Fibroblast-like
synoviocytes (RA-FLS-Exo) has been reported to promote osteoblast
differentiation in RA patients by decreasing the expression level of
Tob1 gene and triggering the bone morphogenetic protein (BMP)/Smad
pathway . Fibroblast-like synoviocytes are known to be responsible for
cartilage destruction and maintenance and progression of inflammation in
RA patients. Mesenchymal stem cell (MSC)-derived exosomes are evident to
exhibit immunosuppressive effects like suppressing B and T lymphocytes.
miRNA-320a is found to have very low expression levels in RA and is
known to have anti-proliferative effect and increases the apoptotic
death of FLSs by downregulating MAPK/ERK1/2 signaling pathway . There
are
CD4+CD25+Foxp3+ T
cells also called regulatory T lymphocytes or Treg cells. These cells
are known to inhibit the autoimmune response found in RA. Patients
non-respondent to disease-modifying anti-rheumatic drugs (DMARDs, or
Methotrexate) are often treated with biological DMARDs or immunotherapy.
Increasing the number of Treg cells has demonstrated a reduction in the
pro-inflammatory responses. Stimulation of Treg cell-specific targeted
gene proliferation induced more and more production of Treg cell which
slowed the disease progression and proved itself to be an important
treatment for ADs .3.2. Diabetes Diabetes is a complicated and persistent clinical condition requiring
continuous biomedical support and preventive measures which is just not
concerned about glucose management . Diabetes may be classified by three
conditions namely, Type 1 Diabetes (T1D) and Ttype 2 Diabetes (T2D) and
gestational diabetes . T1D out of the above is concerned with an
autoimmune abnormality wherein the β-cells producing Insulin in the
pancreas are destroyed . Relative insulin deficiency in T2D is
predominantly impaired by β-cell activity of the pancreas and resistance
to the action of insulin in specific organs . The occurrence of diabetes
in adults varying in the age group of 21 to 80 over the world was
predicted to shoot upto 10.6% (536.7 million) in 2021 and 12.3% (783.3
million) in 2045 .
Small endogenous miRNAs are predisposed to control cellular cycles,
proliferation, differentiation, and apoptosis by inhibiting the biogenic
molecules that control these processes, such as RNA transcripts, or by
degrading mRNA .
According to various research groups, miRNA-375 was found to be
upregulated, impacting the beta cells that secrete insulin along with
other miRNA molecules such as miRNA-29a, miRNA-29b, miRNA-200, and
miRNA-7 . A cross-sectional nested, case-control based research study
was carried out by Barutta et al. to look at the differential expression
of miRNAs in the blood samples of patients suffering from T1D.. A
considerable upregulation of the miRNAs miRNA-140-3p, miRNA-574-3p,
miRNA-139-5p, miRNA-106a, miRNA-17, miRNA-486-3p, miRNA16, miRNA-222,
and miRNA-885-5p was found . Children with new-onset T1D were found to
have blood samples significantly overexpressed miRNA-197. It was also
claimed that miRNA-197 can accurately predict residual beta-cell
functioning . miRNA-155, miRNA-92a, and miRNA-126 were all noticeably
downregulated in the blood samples of T1D patients . Additionally,
diabetes is frequently characterized by endothelial homeostasis, which
is maintained by miRNA-126. Additionally, miRNA-126 regulates
endothelial inflammation in individuals with micro/macrovascular
problems related to diabetes, establishing a connection between these
issues and reduced levels of miRNA-126 . Patients suffering from T2D had
shown greater levels of miRNA-29a in their blood. It was determined that
miRNA-29a overexpression inhibits glucose absorption by stimulating
insulin, eventually leading to insulin resistance. Another study
involving plasma samples from T2D patients to look into the
dysregulation of miRNA-34a found that miRNA-34a was highly elevated and
connected to senescence or death in pancreatic cells with a decline in
SIRT1 activity. miRNA-130b was shown to be severely reduced in the blood
samples that were collected from the affected individuals along with
high urine albumin-to-creatinine ratios (UACR) in a research that
included 327 T2D patients with different levels of UACR . Additionally,
it was shown that blood levels of miRNA-130b had a bad correlation with
HbA1c and HIF1- levels. Furthermore, the serum miRNA-130b level was
closely correlated with insulin resistance and blood glucose levels in
T2DM. Evidences suggest that miRNA-214-3p and miRNA-27-3p might serve as
potential T2D biomarkers comes from reports of their downregulation in
whole-blood samples from T2D patients as well as in a population with
T2D risk factors . miRNA-26a in pancreatic beta cells has successfully
reduced Type 2 Diabetes by increasing peripheral insulin sensitivity and
maintaining beta cell activity . An alteration to the daily routine,
furthermore following a holistic diet, biomedical or surgical
therapeutic procedures, or considering a combination of these methods
can lead to diabetes remission. Lifestyle changes that affect daily
activities linked to nutrition and exercise have health impacts that go
well beyond those that are specifically relevant to diabetes .3.3. Multiple Sclerosis MS is categorized as an auto-immunogenic inflammatory disease inducing
persistent demyelination in the central nervous system (CNS). According
to a study conducted between September 2019 and March 2020, it was
statistically estimated that 2.8 million individuals across the globe
suffer from MS approximately 35.9 per 100,000 population . In younger
individuals, MS is the predominant contributor to atraumatic
neurological impairment . The fundamental clinical features of MS
include several foci which are found to be dispersed across the white
matter which reverts back throughout the illness’s progression time and
again . The etiological factors that lead to the onset of MS include
T-cell-mediated immune responses that trigger cytokine release , alter
the barrier permeability between the brain and cerebrospinal fluid ,
axonal inflammation, gradual demyelination and a rise in the
pro-inflammatory miRNAs and its relevant hallmarks. Macrophages,
followed by CD8+ T-cells are significantly observed in the influx, along
with a lesser number of CD4+ T cells, B cells, and plasma cells. Also,
the lymphocytes that majorly contribute to the progression of MS are Th1
and Th17 . Diverse genetic, epigenetic, microbiological, and ecological
variables aid the pathogenesis of MS. miRNAs are quintessential
epigenetic phenomena that regulate abnormal cellular events during the
prognosis of the disease. The key purpose of miRNA is to monitor and
control the translation of genes, either by suppressing translation or
by cleaving the target mRNA. In MS, miRNA expression profiles are
altered in CNS lesions as well as in the immune system, which has
significant implications on gene expression in the array of cell types
and ultimately promotes the disease . In the study by Baulina et al., a
range of miRNAs having altered expression during the disease prognosis
from varied sources were outlined that including miRNA-155, miRNA-146a,
miRNA-181c, miRNA-326, miRNA-346, miRNA-17, miRNA-320a, miRNA-34a,
miRNA-340, miRNA-132, and their target sequences . Based on multiple
data identified, miRNA-155 is regarded as the most potent promoter of
inflammation and is crucial for the pathophysiology of the disease, by
virtue of myeloid cell polarization to a morphological and active
pro-inflammatory type. Upregulation of miRNA-155 has been detected in
the cellular blood samples of individuals affected by MS which may be a
sign of a severe disease progression. miRNA-155 modulates the MS risk
genes PIK3R1 and PIK3CA which encodes for proteins belonging to the
phosphoinositide 3-kinase (PI3K) family. Dysfunction of the PI3K family
leads to oncogenesis, neurological and immune disorders, and
demyelination in MS . Dysregulation of miRNA-3606-3p in patients of
systemic sclerosis is a prominent biomarker and can lead to the
reduction of TGFBR2 expression . This can lead to the severity of the
diseased condition. However, an upsurge in miRNAs like miRNA-126 and
miRNA-139-5p in systemic sclerosis have found a correlation to the
inflammatory cytokines and signaling molecules like IFN-B . With
reference to a study on miRNAs to ascertain their correlation with
gadolinium-enhancing (Gd+) lesions for evaluating their utility as MS
activity biomarkers, it was observed that peripheral blood mononuclear
cells (PBMCs) of MS patients had overexpression of miRNA-146a/b in
comparison to the control. They hypothesized that this elevation was
particular to the critical stage of MS and contributed to the
proliferation of Th cells, which are effective in the aggravating
mechanisms that occur in the CNS of MS patients. First, activated T
cells traverse through the blood-brain barrier from the periphery into
the CNS. Cytotoxic CD8+ T cells can directly cause axonal damage, while
CD4+ T cells aid in retaining the CNS inflamed. Furthermore, regulatory
T cells (T reg), that sustain immune tolerance by inhibiting effector T
cells, carry out this control through the secretion of miRNA-based
exosomes, a route for gene silencing . miRNA-20a-5p and miRNA-20b-5p
were shown to be key regulators of 1,000 targeted genes according to
database analysis of miRNA-mRNA interactions 46, indicating that they
may be key players in MS pathogenesis . By analyzing the association of
the expression of miRNAs and its potential target genes, the involvement
of miRNAs in the disease prognosis has been established. Nevertheless,
the exact functioning of miRNA in the pathogenesis of MS is not well
identified. New medications and therapeutic modalities that effects
miRNAs will undoubtedly emerge with the development and use of new
advances in research, providing the groundwork for the eventual
eradication of different immunogenic disorders.3.4. Systemic lupus erythematosus SLE is identified as a persistent inflammation associated with
auto-immunogenic ailment, caused by the progressive decline of
resistance to the self–antigens, stimulation of dysfunctional immune T
cells and B cells, synthesis of autoantibodies (auto-Abs), and altered
cytokine activity. Due to the advent of complex molecular associations
between epigenetic triggers, unbalanced hormone levels, genetic
susceptibility, epigenetic control, immune status, and other unknown
factors, SLE is strongly related to the downregulation of the
immunological responses both innate and adaptive . miRNAs are
significant regulators of ADs, where miRNA-146a and miRNA-155 are
demonstrated to be crucial in the pathogenesis of SLE. miRNA-146a was
found to be adversely correlated with the expression of the type I IFN
regulatory signaling system. The toll-like receptor (TLR)-myeloid
differentiation factor 88 (MyD88) pathway, which includes IRAK1 and
TRAF6, was proven to be a critical regulator of signaling pathways .
miRNA-155 is verified to upregulate the alteration of Treg phenotype in
MRL/lpr mice by modifying the CD62L expression . miRNA-17 is associated
with the production, division and activation of immune B cells, T helper
cells namely Th1, Th2, Th17 . In a study conducted by Kaga et al.,
miRNA-17 production was found to be more in the healthy control as
compared to that of the SLE patients and it was also observed that
miRNA-17 has an antagonistic relation with the interferon alpha mRNA
that may play a role in the etiology of the disease . miRNA-142-3p was
discovered to be considerably downregulated in SLE patients.
miRNA-142-3p, which is mostly produced in hematopoietic stem cells, has
been identified to be crucial for the immune response, particularly in
macrophages and Regulatory T cells . miRNA-20a downregulation was linked
to lupus nephritis and vascular thrombosis . miRNA-125a comprises the
inflammatory chemokine pathway. Recent studies have demonstrated that
miRNA-125a targets KLF13 in SLE, which results in increased production
of the inflammatory chemokine RANTES . miRNA-15a specifically has a
deleterious impact on the B-10 subpopulation, and miRNA-15a reduction
may help treat SLE . In a scientific study designed by Yan et al.,
miRNA-124-3p and miRNA-377-3p were highly expressed in PBMCs and serum
collected from patients affected by SLE in comparison to the healthy
controls . miRNA-21 is upregulated due to the hyperactivity of immune T
cells whereas miRNA-7 is related to the overproduction of B cells and
autoantibodies. miRNA-34a is responsible for the collapsing of the
immunological tolerance .3.5. Inflammatory bowel disorder The term ”IBD” corresponds to a group of relapsing, chronically
inflammatory gastrointestinal illnesses, notably Crohn’s Disease and
Ulcerative Colitis . Over the past decade, as lifestyles and dietary
practices became more westernized, IBDs expanded around the world .
According to experimental data, excessive consumption of certain
macronutrients in the modern food habit triggers an inflammatory
response in colon by preying on innate immune sensors and disrupting the
metabolism of gut microbes. Even though incidence is stabilizing in
western nations, the burden is still significant since prevalence
exceeds 0.3%. These findings underline the need for research into IBD
prevention and advances in healthcare systems to manage this difficult
and expensive condition . Immunometabolism regulates vulnerability to
intestinal inflammation, and the risk IBD genetically. This partially
influences metabolism and stress related signaling of innate immunity .
Some micro-RNAs, including miRNA-101, miRNA-515-5p, miRNA-623,
miRNA-325, miRNA-876-5p, miRNA-1224-5p, miRNA-1226-5p, and miRNA-1253,
have been found to invade bacterial membrane and subsequent regulate
gene transcription. This promotes bacterial proliferation and mobility,
which in turn affects the population and diversity of the gut
microorganisms . By reducing the over-expression of inflammatory
cytokine receptors like IL7R and IL17RA as well as signal proteins like
GP130, miRNA-31 reduced the inflammatory response in a mouse model of
Dextran Sodium Sulfate -induced colitisl, which Tian et al., 2019 found
to be hyper-expressed in tissues from patients suffering from IBD.
Another study using the DSS mouse model revealed that miRNA-155 binds to
SHIP-1 mRNA directly, resulting in a considerable drop in SHIP-1 levels,
which control cell membrane trafficking . The most often researched
miRNAs in relation to IBD appear to be miRNA-21, miRNA-155, and miRNA-31
. With links between miRNA-21 and IBD being reproduced in numerous
research and functional relevance being shown in mice models of IBD,
miRNA-21 is arguably considered to be prevalent in IBD. miRNA223
regulates innate immunity in intestinal inflammation . miRNA 214-3p and
miRNA 206 activate the NF-kB pathway and promote intestinal inflammation
. Some members of the regenerative gene (REG) family, I, including (REG
Iα, REG Iβ and REG IV) are expressed in Crohn’s disease and ulcerative
colitis and have a role in proliferative mucosal components in IBD.
Through the downregulation of miRNA-24, LPS caused REG IV expression in
human intestinal epithelial cells. Intestinal epithelial cells’
RAGE/TLR4 receptors controlled the LPS signal. Damaged intestinal cells
were replaced under IBD circumstances by intestinal cell growth. Since
REG IV is a growth factor for intestinal epithelial cells, it could help
to multiply intestinal cells to restore intestinal mucosa .
Epigenetic networks underlying AD progression
Epigenetic parameters have a great influence on cell signaling,
differentiation, gene expression and morphogenesis of cellular
development in an organism. Hence, they have an essential role to play
in the onset of a disorder and its related genetic background . There
are various epigenetic factors which are common in various disorders,
due to their long lasting effect on the nature and progression of the
genetic regulation in a diseased condition. The mechanisms like DNA
methylation and modification of histone proteins can contribute to the
pathogenesis of various ADs . These epigenetic parameters can correspond
to the initiation and prolongation of inflammation inADs. It has been
observed in various studies that miRNAs play a centric role as
epigenetic regulators and prevent the progress of inflammation . DNA
methylation is a process in which the methyl group is removed from the
methyl cytosine structures, which results in the loss of structural and
functional entities in various metabolic pathways. Ten-eleven
Translocation (TET) enzymes act as DNA methylases and take part in the
process of hypo or hyper methylation . Histone modifications act as
major epigenetic markers. They are involved in transcriptional
upregulation or downregulation, chromosomal compaction and DNA repair
techniques. One of the major histone modifying enzyme inhibitors, is
histone deacetylase inhibitor (HDACi) which is involved in the changes
that contribute to the growth and advancement of ADs .
The interplay and corresponding effects of the epigenetic regulation of
miRNA is an area of ongoing research. Epigenetic therapies is a new area
of clinical advancement that helps in the reversal of epigenetic
alteration or aberrations . Corresponding researches are being done in
the line of the development of epigenetic modifying drugs in conjugation
with adjuvant therapy. This can be used in T cell polarization and
control of the inflammatory processes.4.1. Rheumatoid ArthritisRA is a disease caused by loss of autoimmunity, that encompasses
synovial fluid hyperplasia and inflammatory joint degradation . The
inflammation in RA is mediated by several factors like ACPAs which
further result in structural damage of the bone joints . The dynamic
expression of the diseased condition in the patients is due to the
epigenetic regulation of the genes directly associated with the immune
system. The synovial fibroblasts consist of a unique methylation
pattern, which varies during the course of gene progression and
consequently affects its severity . L1 (LINE) is hypomethylated and
indirectly induces the surge of cytokine, growth factors, receptor and
inflammatory co factors . Local hyperinflammation spike is noticed in
the RA patients due to the hypomethylation of the CpG islands associated
with the IL6 promote gene. This leads to the overexpression of IL6 and
related proinflammatory cytokines . Recent research has verified that
epigenetically regulated gene networks in the peripheral blood
mononuclear cells consequently result in the severity regulation of RA .
The synovial cells modify themselves in such a way that they start to
show tolerance toward the apoptosis. This in turn triggers the
inflammatory cascades in the pathogenesis pathway of RA development.
This is majorly induced by the histone modification and hypermethylation
of the NFkB promoter gene . The histone modifications like acetylation,
methylation, citrullination, phosphorylation and ubiquitination
contribute to the changes in the transcriptional factors, making the
chromatin available for gene expression. The different histones that
take part in the RA pathogenesis and histone modification are - H3K9,
H3K14, H4K5, and H4K16 (for acetylation), H2BK5, H3K4, H3K36, and H3K79
(for methylation), H3S10 and H3S28, H4S1 (for phosphorylation) and
H2BK120 (for ubiquitination) . These developments can in turn be used as
biomarkers and contribute to the therapeutic application of this
disease.4.2. DiabetesAutoimmune dysfunction of
pancreatic β cells leads to the development of type I diabetes. Its
progression is monitored on a wider scale as it is widely affected by
the epigenetic regulation of the various related genes . Glucose and
insulin levels in the blood, affect the degree of methylation. They
regulate the homocysteine metabolism, which further modulates the DNA
methylation by DNMTs . These epigenetic factors contribute in the
glucose intolerance, due to the alteration in the methionine metabolism.
The immunogenic response is seen to be related to the DNA methylation of
several regulator genes. FOXP3 promoter region of the CD4+ is
hypermethylated in the case of latent autoimmune diabetes in adults
(LADA) patients . The INS gene promoter in diabetic patients is seen to
be hypomethylated, which has consequently culminated in the release of
inflammatory cytokines like TNF-β , IFNγ, IL6 and IL-1B . The histone
modifications are mainly regulated by histone-acetyltransferases (HATs)
and histone-deacetylases (HDACs), which regulate the process as
transcription coactivators. RNA modification and ncRNA regulation are
also noticed in the aged group of diabetic patients, which also
attributes in cellular senescence and post transcriptional modification
. Miao et al have documented the histone modification of H3K9Ac,
H4K16Ac, H3K4me3, H3K9me2,3, H3K27me3 genes in the diabetic pathway . As
these genes are closely located to the DQB1 and DRB1 genes. This leads
to a surge in the transcription process in the monocytic cell line,
thereby triggering the immunogenic pathway . One of the most significant
autoimmune markers of the progression type I diabetic condition is the
reduced expression of the H3 histone acetylation that develops a
consequent the expression of GAD autoantibodies .4.3. SysMS is a neurodegenerative, inflammatory disease that results in physical
and cognitive impairment. The pathogenesis primarily roots from the self
intolerance towards the neuronal antigens. Most patients develop
relapsing-remitting MS (RRMS) and others develop primary progressive MS
(PPMS). In case of PPMS, DNA methylation of the primary epigenetic
regulation that controls the progression of MS. Studies are being
conducted to identify and analyze the differentially methylated CpG
sites of CD4+ T lymphocytes genome . The progression and mortality of
the disease depends upon the interaction and interplay of the genetic
and environmental factors, combined . The T cell exhibits autoreactivity
that causes demyelination which consequently leads to an inflammatory
cascade in the central nervous system. Studies have shown that the
hypomethylation of promoter region peptidyl arginine deaminase (PAD) -II
contributes in the citrullination of myelin basic protein (MBP) . This
may lead to irreversible molecular changes that can cause insatiable and
chronic inflammation. ATXN1 gene which encodes for polyglutamine
protein ataxin-1, which in turn protects the system from the
demyelination event. Studies have shown that in the event of
hypomyelination of ATXN1 gene, the pathogenetic mechanism scales
up in the cellular and molecular regulation of MS . Thelper (Th) 17 and
T regulatory (Treg) molecular balance has an important role to play in
the progression and dynamics of MS, due to their epigenetic dynamic
nature. Histone modification of forkhead Box P3 (FOXP3)-
cell-type-specific regulatory regions (CSRs) and RAR related orphan
receptor C (RORC)-CSRs are polarized Th17 cells are regulated by
estrogen in pregnant women suffering with MS, during their third
trimester. A recent study shows the correlation of epigenetics and
disease progression via epigenomic and transcriptomic profiling, where
they have tried to compare demyelinated MS lesions and normal-appearing
white matter (NAWM). Human-iPSC-derived oligodendrocytes were
epigenetically edited to understand the region-dependent
hypermethylation of MBP which is responsible for the myelination and
axonal development . During the pathogenesis of MS, antibodies are
formed against different histone proteins like H2b, H1, H3, H4, MBP, and
DNA, that result in the hydrolysis of H2A histone. This cross reactivity
between the abzymes and histone proteins can result in the aggravation
of the disease condition . Pedre et al. reported the increased
expression of H3 histone acetylation in chronic MS patients, which has
also been seen as a side effect to in turn increase the transcription
pattern of the inhibitors of oligodendrocytes and histone
acetyltransferase (HAT) gene expression in MS patients . The acetylation
and deacetylation balance is hindered during the early phase of MS
progression, which can further modulate that severity of the disease .4.4. Systemic Lupus ErythematosusAutoantibodies and dysfunctional antigen presenting cells are the
classical characteristics of SLE. The pathogenetic pathway of SLE is
still a matter of debate, and has been seen to be largely affected by
the epigenetic and environmental regulation over the patient’s immune
system . The large network in the immune system contributes to lupus
pathogenesis. Regulatory factor X (RFX) 1 acts as an immune-suppressor
and aggrevates the DNA methylation of the promoter region of CD70 and
CD11a . This promotes autoimmune response . According to Hedrich et al,
CpG-DNA methylation patterns are highly conserved and close to the
promoter region of IL7F. It is observed that the SLE patients show low
degree of methylation in the T- lymphocytes, as drawn parallels with the
study on healthy individuals . In other recent findings, it has been
seen that there is an increment in the levels of 5-hydroxymethylxytosine
(5-hmC). This is due to the upregulation of ten eleven translocation
(TET)-2 and TET-3 factors, which are one of the prominent DNA methylases
and contribute in the enzymatic conversion of 5-methylcytosine (5-mc)
into 5-hmC . In several SLE patients, DNA hydroxymethylation pattern is
observed in the signaling pathway genes, which are then consequently
related to the immune response genes and factors like SOCS1, NRF2F6 and
IL15RA. The methylation product - 5-hmC and its aberrant regulation may
contribute in the therapeutic application of SLE . The Th17 cell
maturation takes place through the epigenetic modification if the
transcriptional factors associated with it, in the SLE patients. The
STAT3 pathway plays a pathbreaking role in the modification and
modulation in T cell maturation and progression of SLE pathogenesis .
Histone modifications in the SLE is reported in the form of
trimethylation of histone H3 in H3K27me3, which contributes to the
increase of H3K27me3 in the CD4+ T cells of the SLE patients. This
trimethylation is made possible with the help of enhancer of zeste 2
polycomb repressive complex 2 subunit (Ezh2). The cumulative effect of
these histone modifications may lead to the t cell lineage development
in the SLE patients, which may attribute to the pathogenesis of the
disease . Recent research and studies have shown and possibility of 3D
genome alteration with respect to the progression of disease. The study
included testing the viability of cells when subjected to histone
modification of H3K27ac, H3K4me1, H3K4me3, SP11 knockdown and
transcription factor motif enhancement. These studies indicate the
indirect effect of epigenetic regulation over the genome structure and
function .4.5. Inflammatory Bowel DiseaseIBD is an autoimmune, chronic, recurrent gastrointestinal disorder.
Several factors like environmental factors, gut microbiota, immune
dysregulation contribute to the advancement and cause of this disorder.
Pathogenesis of this disease can not be clearly elucidated as it can be
manifested into colorectal cancer, fistula development or stenosis
syndrome . The IBD has two twin forms- ulcerative colitis (UC) and
Crohn’s disease (CD). IBD related genes undergo methylation and histone
modification for reshaping the disease progression . Mucosal methylation
of HRAP2, FANCC, GBGT1, DOK2 and TNFSF4 in the progressive IBD, may
cause a significant progression and severity of the disease . There are
counter active findings which are noticed in the case of CD patients
where GBGT1, IGFBP4, FAM10A4 genes are hypermethylated and in case of UC
patients, IFITM1 is hypomethylated. This helps us to regulate and
differentiate between the two subtypes. However, there is also the case
of leukocyte methylation in the CD sub type of IBD . At the molecular
level, most methylations occurring in the case of this disease, has a
close proximity to GWAS risk genes, like CARD9, CDH1, ICAM3 . According
to a group of British scientists, the region of the gut that is more
prone to DNA methylation of the intestinal region. Major histone
modifications are found to be abundant in the following histone proteins
- H2BK5ac, H3K36me1, H3K4me3, macroH2A and Rme2sym, in the case of CD
patients, as compared to the healthy people. These modifications can be
consequently analyzed with the presence of natural killer (NK) . This
accounts for the epigenetic biomarkers for the progressive and
detectable approach of IBD .
Diagnostic Measures using miRNA as a Biomarker for AD
Globally, AD affect nearly 8% of the population, which include
approximately 80 disorders and exhibit several geo-epidemiological
variations; making it the fourth leading cause of mortality worldwide,
after cancer and heart disease . It has been found that women are 2.7
times more susceptible for developing AD as compared to men . Hence,
rapid and early diagnosis of AD is gaining prime importance in the light
of improving the quality of patients’ lives . Traditionally, the
diagnosis of AD has been limited to the detection of autoantibodies in
patients’ samples through western blotting, indirect immunofluorescence
and ELISA-based commercial assays . However, the pitfalls of such
techniques include expensive antibodies, longer incubation time, the
need for sophisticated instruments and complex procedures . Moreover,
the accuracy and sensitivity of these techniques is not upto the mark .
To overcome these limitations, a more sensitive and reliable technique
is needed for the diagnosis of AD.
Recently, miRNAs have proven to be effective non-invasive biomarkers for
the diagnosis and prognosis of a disease, to study its progression and
analyse the drug responses following treatment . This is because the
abnormal miRNA expression profile is strongly correlated with diseased
states and research shows that they are present in almost all biofluids
. Despite the advent of high-throughput technologies, the detection of
miRNA comes with a number of predicaments. Firstly, they are of short
length (~15-25 nt), making it difficult to develop a
highly specific probe. Secondly, distinct miRNAs harbour homologous
sequences which are prone to give false-positive results due to cross
hybridization . Conventional approaches detecting miRNA include
qRT-PCR,, northern blotting, enzymatic assays, oligonucleotide
microarrays, cloning and sequencing which come with several drawbacks
such as limited selectivity, low sensitivity, and ineffectiveness in
detecting extremely low miRNA concentrations in blood samples . All
these molecular techniques lack an integrated transducer element and are
amplification-based . In addition to being time-consuming and laborious,
these techniques do not take into account the susceptibility of miRNA to
degradation and hence give rise to bias, unreliable results .
Furthermore, these detection techniques do not encompass multiplexed
analysis, in vivo analysis, detecting circulating miRNAs and specificity
towards single-nucleotide, all of which are indispensable in clinical
settings .
With the breakthrough of nanotechnology in medicine, amplification-free
biosensors have drawn considerable attention from the academia as well
as the industry . A biosensor is an integrated analytical device
consisting of mainly three components: (i) a bio-recognition element
such as enzymes, antibodies, DNA, RNA, aptamers,etc. (ii) a transducer
element which detects a biological response and converts it into an
electrical signal and (iii) a signal- processing system that consists of
an amplifier, a processor and a display unit . This analytical device is
highly effective in quantitative or semi-quantitative detection of an
analyte . It incorporates the use of specific DNA probes which have
complementary sequence with target miRNA with highly adaptable
transducer sensing system . This enhances their multiplexing potential,
allowing them to provide rapid, label-free, extremely selective as well
as sensitive real-time detection of miRNA with respect to point-of-care
(POC) aims for clinical applications . On the basis of the type of
signal transducers used, biosensors for detecting miRNA are broadly
classified into optical and electrochemical biosensors . Optical
biosensors are highly effective in detecting biomarkers as they
transduce the absorbance or fluorescence signals of an optically-active
reporter linked to a nucleic acid probe when hybridized to the target
miRNA . This class of biosensors have a fairly simple and feasible
design, wherein the bio-recognition elements are immobilized on the
surface of a signal detection platform by either physical adsorption,
covalent or electrostatic bonding . The detection platforms are
fabricated with nanomaterials such as graphene oxide (GO), gold
nanoparticles (AuNP), and quantum dots (QD) . AuNPs are highly
compatible with nucleic acids as well as proteins, provide adequate
surface area to volume ratio, have excellent fluorophore quenching
ability and exhibit good LSPR absorption in the visible range . Surface
Plasmon resonance (SPR) is another robust and sophisticated optical
method for fast and direct miRNA detection . It measures the shifts in
refractive index when an analyte forms a complex on the surface of the
electrode . Surface plasmon resonance Imaging (SPRi) is a label free
hybridization-based method and can measure miRNA concentration for as
low as 2 pM in less than 30 minutes . Their limit of detection (LOD) is
low as far as complex biological samples are concerned . Several
strategies are employed to enhance its sensitivity such as the use of
nanoparticles like AuNPs coupled with DNA sandwich, GO–AuNPs hybrids,
hybridization chain reaction method, hairpin assembly,
streptavidin-biotin approach, etc . Surface-enhanced Raman scattering
(SERS) is employable for rapid and accurate detection of miRNA but is
not suitable for medical diagnostics due to low sensitivity . Another
biosensing strategy under the optical methods is fluorescence detection.
Examples include Ag nano-cluster DNA probes, GO with dye-labelled
probes, carbon nanoparticles, magnetic beads, etc . Polyaniline-gold
(PANI-Au) nanomaterial and ruthenium (Ru)-based electrochemiluminescence
immunosensors have also been designed for label free, highly sensitive
quantification of miRNA . Overall, SPR-based biosensors are commonly
employed optical methods for diagnosis of AD due to their high
selectivity, high efficacy, affordability, and reliability. However,
biosensors working on the principle of electrochemiluminescence
demonstrate highest sensitivity . Electrochemical biosensors are the
most predominantly used sensors for the diagnosis of AD . The transducer
is a solid electrode which is sensitive to changes in electrode
properties caused by the hybridization between the immobilized
nucleotide probe and the complementary sequence . In these, the
transduction element is often Au, indium tin oxide, glassy carbon, and
graphite, the sensitivity of which is enhanced by incorporation of a
variety of nanoparticles, nanowires and enzymes . In contrast to optical
biosensors, electrochemical biosensors have simpler electronic design,
are inexpensive, and serve as excellent platforms for point-of-care
tests because of their easy-to-use miniaturized portable integrated
systems . This allows them to provide fast, accurate and real-time
quantification of analyte. These are further classified into
amperometric, impedimetric and voltammetric methods . The amperometric
and voltametric techniques measure changes in the current when a target
miRNA is hybridized to its complementary sequence; with the only
difference being that the current is measured at a fixed potential value
in amperometry, while the current is measured in a certain potential
ramp in voltammetry . Cyclic voltammetry (CV), linear sweep voltammetry
(LSV), differential pulse voltammetry (DPV) , and square wave
voltammetry (SWV) are different techniques under voltammetry . These are
further categorized as label-free biosensors if the redox reaction is
generated due to an electroactive nucleic acid base (adenine or guanine)
signals or enzymes such as duplex-specific nuclease; or as label-based
biosensors if the electrochemically active reporter species are
nanoparticles (Eg. Au,Ag, OsO2, or Ruthenium NPs) or
hybrid nanoparticles (Eg. GO–AuNPs, MoS2 microcubes,
Au@NPFe2O3 nanocubes . Electrochemical impedance spectroscopy (EIS) is
another robust, label-free, and well-established technique for
quantification of miRNA . It is the most practical method to study
reaction mechanisms, biofunctionalization, nanostructure formation and
hybridization . EIS is more efficient and biocompatible as compared to
amperometric and voltammetric methods since it is less destructive and
more sensitive .Figure 4 – Illustrative depiction of biosensor
construction deploying miRNA signature molecules corresponding to
respective ADs using several biorecognition elements along with
assistive nanomaterials for eliciting robust signal amplification to
detect pathogenic miRNA indices.Both optical and electrochemical methods have their pros and cons in
detecting miRNA with some giving a much lower limit of detection and
hence higher sensitivity than others . However, selecting an optical or
an electrochemical biosensor would depend upon the experimental aim
which would be different for research and clinical purposes. In
conclusion, biosensors offer unique advantages over the use of
traditional assays which makes them highly suitable POC devices in the
clinical diagnosis of AD . The incorporation of nanoparticles in these
biosensors have significantly brought down the LOD to pM and fM range
which is instrumental in the rapid and early-stage diagnosis of AD.
Further innovation is needed to develop in vivo sensing platforms for AD
. Table 2 lists down the different optical and electrochemical
techniques used for detecting miRNAs in RA, Diabetes, MS, SLE and IBD,
along with their references.Table 2 - Different techniques, their range and mechanism
of detection of miRNAs in different autoimmune diseases