Drug Discovery Today
Volume 00, Number 00
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Interplay between miRNAs and

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Mycobacterium tuberculosis:
diagnostic and therapeutic
implications
Amit Kumar Singh1, Mrinmoy Ghosh2, Vimal Kumar1, Sumit Aggarwal3 and
Shripad A. Patil4
1
Experimental Animal Facility, ICMR-National JALMA Institute For Leprosy & Other Mycobacterial Diseases, M. Miyazaki Marg, Tajganj, Agra, Uttar Pradesh, India
2
KIIT-Technology Business Incubator (KIIT-TBI), Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar-751024
3
Division of Epidemiology & Communicable Diseases, Indian Council of Medical Research, Ansari Nagar, New Delhi, India
4
Immunology Division, ICMR-National JALMA Institute For Leprosy & Other Mycobacterial Diseases, M. Miyazaki Marg, Tajganj, Agra, India

Increasing evidence suggests that mycobacteria change the host miRNA profile to their advantage. The
active participation of miRNAs in controlling immune responses in TB has raised the possibility of
utilizing miRNA-based therapy itself or canonically with a standard drug regimen for shortening the
duration of treatment. The development of delivery systems for optimal delivery of oligonucleotides,
including small interfering (si)RNA/miRNAs-based therapeutics has shown potential as a new
therapeutic intervention. However, studies related to the exploitation of miRNAs as both biomarkers and
as therapeutics in TB are scarce; thus, more in vitro and in vivo studies are required to fully determine the
role of miRNAs as potential diagnostic biomarkers and to improve the pharmacological profile of this
class of therapeutics.

Introduction treatment. As genomic data become increasingly used to guide

Mycobacterium tuberculosis infection is becoming a global health
problem with growing resistance to available drugs. In 2018, an
estimated 10 million people had TB and 1.5 million lost their lives.
Despite being declared a global health emergency by the WHO in
1993, TB remains the top leading infectious disease cause of death
worldwide. Although TB incidence is declining by 2% per year
globally, this needs to be accelerated to 4–5% per year to reach the
2035 targets set under the End-TB strategy [1]. TB pathogenesis is
determined by the cellular responses mounted by the host, deter-
mining whether an infected individual will remain asymptomatic
(latent TB infection) or develop clinical signs and symptoms
(active TB disease). The probability of M. tuberculosis-infected
individuals developing the disease at some point in the life ranges
from 5% to 10%, suggesting the role of host genetic factors in the
regulation of disease development and progression [2]. Further-
more, the spread of drug-resistant M. tuberculosis strains has re-
ignited interest in identifying new avenues for TB diagnosis and
Corresponding author: Singh, A.K. (amits.hq@icmr.gov.in)
tailored patient care, molecular diagnostics and therapeutics could
pave the path for achieving the targets of the End-TB strategy.
miRNAs are noncoding small, single-stranded functional RNA
molecules that post-transcriptionally regulate target gene expres-
sion by binding the 30 -untranslated regions (3’-UTRs) of target
mRNAs transcripts, leading to either degradation or inhibition of
translation of the respective mRNA [3,4]. miRNAs occur in plants
and animals and are 22 nucleotides in length; they account for 1–5%
of all expressed human genes but can control up to 30% of protein-
coding genes [5]. The biogenesis of miRNAs is well characterized [5].
A single miRNA is often capable of regulating the expression of
multiple mRNAs. An mRNA transcript can be targeted by many
different miRNAs because of numerous miRNA-binding sites [6].
Recent investigations have revealed the regulation of miRNAs as a
result of infection by bacteria, viruses, and so on, as well as the role of
miRNAs in cytokine expression, activation of apoptosis and autop-
hagy, major histocompatibility complex class II expression (MHC
class II), and phagolysosome maturation [7]. miRNAs either inhibit
or promote the innate function of macrophages, dendritic cells

1359-6446/ã 2021 Elsevier Ltd. All rights reserved.

https://doi.org/10.1016/j.drudis.2021.01.021 www.drugdiscoverytoday.com 1
Please cite this article in press as: Singh, A.K. et al. Interplay between miRNAs and Mycobacterium tuberculosis: diagnostic and therapeutic implications, Drug Discov Today (2021),
https://doi.org/10.1016/j.drudis.2021.01.021
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(DCs), and natural killer (NK) cells, and adaptive immune responses
such as T cell differentiation and function [8,9].
miRNAs have been revealed in recent years to have a close link
to pathogenesis mechanisms in M. tuberculosis infection. Profile of
gene expression in macrophages and NK cells derived from TB-
infected and healthy controls demonstrated variations that might
be controlled by miRNAs [10,11]. Various other studies have
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as new tools for diagnosis and drug development against myco-
bacterial infections. Correcting post-infection altered miRNAs
using miRNA substitutes and antagonists is of particular interest
for future research.
Here, we discuss recent advances in understanding the altered
expression of miRNAs during M. tuberculosis infection and its effect
on immune responses. We also discuss their potential as diagnostic

revealed changes in the production of tiny miRNAs in host cells
following infection with M. tuberculosis, suggesting the vital role of
miRNA in guiding the immune response [12]. Recent findings
highlighted the essential role of miRNAs in regulating macrophage
roles in M. tuberculosis-induced autophagy and apoptosis (Fig. 1)
[13–15]. A detailed understanding of the relationship between
miRNAs and TB infection is a future research area in this field.
Accumulating evidence suggests that miRNAs have the potential
biomarkers in TB. miRNAs have been utilized to treat several
disorders, including cancer and, thus, we also review opportunities
for miRNA-based host-directed therapies (HDT) for the treatment
of TB.
The role of miRNAs in TB
Recent studies have established the involvement of miRNAs in
mycobacterial infection, thus opening a new field of research

Drug Discovery Today

FIGURE 1
Schematic presentation of miRNA regulation of autophagy and apoptosis. miR-31 is potential inhibitor of WNT signaling-mediated autophagy by targeting
protein phosphatase 2A (PP2A). miR-30/33/33*, miR-125a-3p, and miR-144-3p suppress autophagic responses during Mycobacterium tuberculosis (Mtb) infection
by targeting UV radiation resistance-associated gene (UVRAG) and autophagy-related (ATG)/lysosomal-associated membrane protein 1 (LAMP-1). miR-155
interferes with both autophagy and apoptosis during infection by inhibiting mammalian target of rapamycin (mTOR) and forkhead box O3,1 (FOXO3,1)
pathways, respectively. It also negatively regulates suppressor of cytokine signaling 1 (SOCS1) and suppresses tumor necrosis factor alpha (TNF-a) or reactive
oxygen species (ROS)-triggered apoptosis. miR-21 inhibits apoptosis by targeting Bcl2. miR-209-5p and miR-20a-5p also regulate apoptosis. miR-125 is a
negative regulator of Toll-like receptor (TLR) signaling in M. tuberculosis-infected macrophages. miR-146a directly targets TNF receptor-associated factor 6
(TRAF6) and suppresses nitric oxide production, thereby promoting mycobacterial survival. Moreover, miR-146a acts as a negative regulator of TLR signaling by
targeting TRAF6 and interleukin 1 receptor-associated kinase 1 (IRAK1,4). miR-21 regulates the immune response by targeting programmed cell death 4
(PDCD4). miR-223 interferes with nuclear factor (NF)-kB signaling and inhibits proinflammatory cytokine secretion.

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Please cite this article in press as: Singh, A.K. et al. Interplay between miRNAs and Mycobacterium tuberculosis: diagnostic and therapeutic implications, Drug Discov Today (2021),
https://doi.org/10.1016/j.drudis.2021.01.021
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DRUDIS-2912; No of Pages 11
TABLE 1
https://doi.org/10.1016/j.drudis.2021.01.021
Please cite this article in press as: Singh, A.K. et al. Interplay between miRNAs and Mycobacterium tuberculosis: diagnostic and therapeutic implications, Drug Discov Today (2021),

miRNAs and its regulatory effects on genes involved in immunity against Mycobacterium spp.
miRNA Expression level Target Function Species Intracellular survival Cell/tissue a Refs
of mycobacteriab
miR-144-3p Upregulated ATG4a Inhibits autophagy Mycobacterium bovis BCG " RAW264.7 cells [28]
miR-20a Upregulated ATG7andATG16L1 Inhibits autophagy M. bovis BCG " RAW264.7 cells [84]
miR-23a-5p Upregulated TLR2/MyD88/NF-kB Inhibits autophagy Mycobacterium tuberculosis " RAW264.7 cells and BMDMs [28]
miR-26a Downregulated KLF 4 Inhibits autophagy M. tuberculosis " RAW264.7 cells [31]
miR-17-5p Downregulated Mcl-1/STAT3 Inhibits autophagy M. tuberculosis " RAW264.7 cells [27]
miR-33 Upregulated ATG5, LAMP1 Inhibits autophagy M. tuberculosis " Human THP-1 and HEK293 [23]
cells
miR-125a-3p Upregulated UVRAG Inhibits autophagy M. tuberculosis " RAW264.7 cells and J774A.1 [85]
macrophages
miR-17-5p Downregulated Mcl-1 Inhibits autophagy M. tuberculosis " RAW264.7 cells [65]
miR-17-5p Upregulated ULK1 Inhibits autophagy M. bovis BCG " RAW264.7 cells [86]
miR-144 Upregulated ATG4a Inhibits autophagy M. bovis BCG " RAW264.7 cells [87]
miR-155 Upregulated Rheb Enhances autophagy M. tuberculosis # RAW264.7 cells, BMDMs [88]
miR-142-3p Upregulated N-Wasp Inhibits phagocytosis M. tuberculosis " Mouse J774A.1 cells, hMDMs [89]
miR-21 Upregulated Bcl-2 Inhibits apoptosis M. tuberculosis " RAW264.7 cells,THP-1 [90]
miR-20a-5p Downregulated JNK-2 Promotes apoptosis M. tuberculosis # hMDMs [91]
miR-223 Upregulated FOXO3 Inhibits apoptosis M. tuberculosis " hMDMs, THP-1 [92]
miR-155 Upregulated FOXO3 Inhibits apoptosis M. tuberculosis " Human PBMCs, THP-1 [93]
let-7e Upregulated Caspase3 Inhibits apoptosis Mycobacterium avium " hMDMs [94]
miR-29a Upregulated Caspase 7 Inhibits apoptosis M. avium " hMDMs [94]
miR-140 Upregulated TRAF6 Decreases IL-6, TNF-a, IL-1b, and IFN-g M. tuberculosis " Human PBMC, THP-1, U937 [95]
secretion cells
miR-27a Downregulated TAB-2/p38/JNK Increases IL-10 secretion M. avium subsp. paratuberculosis " RAW264.7 cells, BMDMs [96]
miR-27a Downregulated IRAK4 Increases IFN-g, IL-b, IL-6, and TNF-a M. tuberculosis # Human THP-1 [97]
miR-206 -1 Upregulated TIMP3 Increases inflammatory cytokines and M. tuberculosis " M. tuberculosis– Human THP-1 [98]
MMP-9 induced inflammatory
response
Let-7f Downregulated A20 (TNFAIP3) Decreases TNF-ɑ and IL-1b M. tuberculosis ESAT-6 protein " RAW264.7 cells, BMDMs, [65]
hMDMs
miR-146a Upregulated IRAK1, TRAF6, and PTGS2 Negative feedback loop in NF-kB M. bovis BCG # RAW264.7 cells, BMDMs [99]
signaling
miR-99b Upregulated TNF and TNFR-4 Inhibits TNF-a and its receptor M. tuberculosis " Mouse DCs [17]
synthesis
miR-203 Upregulated MyD88 Inhibits proinflammatory cytokine M. bovis BCG " RAW264.7 cells [100]
production
miR-142-3 Downregulated IRAK-1 Promotes proinflammatory cytokines M. bovis BCG " RAW264.7 cells [101]
miR-21 Upregulated IL-12p35 Inhibits IL-12 production M. bovis BCG " Mouse BMDCs and BMDMs [30]
miR-155 Upregulated Unknown Inhibits COX-2 and IL-6 M. tuberculosis " RAW264.7 cells, BMDMs [33]
www.drugdiscoverytoday.com 3

miR-144 Upregulated Unknown INF-g and TNF-ɑ M. tuberculosis " Human whole blood [102]
miR-155 Upregulated C/EBPb Inhibits NO production Mycobacterium marinum " RAW264.7 cells, human THP- [103]
1 cells
miR-146a Upregulated NF-kB, MAPK Inhibits NO production M. bovis BCG " RAW264.7 cells [19]
miR-155 Upregulated SHIP1/protein kinase B (Akt) pathway Inhibits apoptosis M. tuberculosis " BMDMs [34]
miR-21 Upregulated CYP27B1 and IL-1B Antimicrobial peptides cathelicidin Mycobacterium leprae " Human monocytes [104]
(CAMP) and defensin beta 4A (DEFB4A)

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a
Abbreviations: BMDC, bone marrow-derived dendritic cells; hMDMs, human monocyte-derived macrophages; RAW264.7 cells, mouse RAW264.7 macrophages; THP-1, human THP-1 monocyte cell line.
b
" increased; # decreased.

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(Table 1). The maturation and differentiation of B cells, antibody
generation, and T cell development and function in the adaptive
immune response are controlled by miRNAs [16,17]. Myeloid
proliferation and differentiation have been reported to be regulat-
ed by the miR-155, miR-223, and miR-17-92 cluster. miR-223
negatively regulates the proliferation and differentiation of neu-
trophils, whereas miR-155 and miR-223 augment myeloid prolif-
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eration and differentiation [18]. Li and colleagues investigated the
function of miR-146a during mycobacterial infection. They found
that it promotes mycobacterial survival by suppressing nitric oxide
(NO) production via tumor necrosis factor (TNF) receptor-associ-
ated factor 6 (TRAF6) at the post-transcriptional level [19]. M. bovis
Bacillus Calmette–Guérin (BCG) infection in mice induced inter-
feron (IFN)-g production via downregulation of miR-29 expression
in IFN-g-producing NK cells, activated primary CD4+ T cells, and
CD8+ T cells. Studies in transgenic GS29 mice (mir-29 downregu-
lated) corroborated these observations and established that miR-
29 downregulates immune responses against mycobacteria by
targeting IFN-g [20]. The upregulation of miR-223 has been ob-
served in blood and lung parenchyma of patients with TB and also
in mouse model of TB. MiR-223 deletion caused increased suscep-
tibility to TB infection in M. tuberculosis-resistant mice. The study
concluded that miR-223 controlled TB susceptibility via infiltra-
tion of myeloid cells to the lungs by directly targeting chemoat-
tractant chemokine (C-X-C motif) ligand 2 (CXCL2) chemokine
(C-C motif) ligand 3 (CCL3) and interleukin (IL)-6 [21].
Several studies have reported a correlation between mycobacte-
rial virulence and differences in miRNA expression patterns. Zheng
et al. identified14 miRNAs as differentially expressed following
THP-1 macrophage infection with M. tuberculosis Beijing and non-
Beijing clinical strains. The Beijing strain repressed various miR-
NAs, including miR-150 and miR-21, which have been identified
as major regulators of mature T and B cell count in patients with
active TB [22].
Apoptosis and autophagy have a crucial role in host defense
against M. tuberculosis. Mycobacteria subvert host defense
responses to survive within the host for a long period of time.
Ouimet et al. showed that M. tuberculosis infection in murine
alveolar macrophages inhibited autophagy via the upregulation
of miR-33 and miR-33* expression, which target key autophagy
effectors, such as ATG5, ATG12, LC3B, and LAMP1. The same
study revealed that Mir-33 reprogrammed host lipid metabolism
and promoted cellular lipid accumulation within macrophages
[23]. The role of miR-33 in lipid accumulation and immune
polarization has been substantiated by various other researchers
[24,25]. A recent study demonstrated that the upregulation of MiR-
144-5p inhibited autophagy by targeting the 30 -untranslated re-
gion of DNA damage-regulated autophagy modulator 2 (DRAM2),
thereby inhibiting antimicrobial responses against M. tuberculosis
[26]. Another study demonstrated that suppressed miR-17 expres-
sion in M. tuberculosis-infected macrophages enhanced the target
proteins Mcl-1 and Signal transducer and activator of transcription
3 (STAT3), a transcriptional regulator of Mcl-1, which in turn
interacts with BECLIN-1 to inhibit autophagy [27]. Guo et al. noted
that miR-144-3p overexpression in Mycobacterium bovis BCG-chal-
lenged macrophages attenuated the induction of autophagy
via targeting autophagy-related gene 4a (ATG4a). miR-144-3p
overexpression downregulated ATG4a protein levels in infected
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February 2021
macrophages, resulting in inhibition of autophagosome forma-
tion, thereby contributing to enhanced mycobacterial survival
[28]. miR-144-3p-dependent autophagy pathway regulation has
been noted in various diseases and could be a potential target for
therapeutic intervention [29].
Cytokines and chemokines have a crucial role in controlling TB
disease progression and are dependent on miRNA-regulated path-
ways for their function. M. bovis BCG induced MiR-21 disturbed IL-
12 production by suppression of IL-12p35in human macrophages
and DCs, and inhibited antimicrobial T cell responses against M.
tuberculosis [30]. Interestingly, a recent study observed that the
downregulation of miR-26a during M. tuberculosis infection led to
the upregulation of Krüppel-like factor 4 (KLF4). KLF4 upregula-
tion resulted in decreased trafficking of the mycobacteria to lyso-
somes, thereby enhancing mycobacterial survival. The study
revealed a crucial function of miR-26a in innate immunity and
contributes to understanding of how M. tuberculosis leverages miR-
26a and KLF4 differential regulation to regulate autophagy and
trafficking of M. tuberculosis to lysosomes for survival in its intra-
cellular niche [31].
miR-155 has been identified as the most expressed miRNA upon
M. tuberculosis infection in macrophages and has a pleiotropic role
in several biological processes associated with response to infec-
tion and immunity [32]. M. tuberculosis-induced MiR-155 repressed
the expression of BTB and CNC homology 1 (Bach1), a transcrip-
tional repressor of hemoxygenase-1 (HO-1), IL-6, cyclooxygenase-
2 (Cox-2), and SH2-containing inositol 50 -phosphatase 1 (SHIP1),
an inhibitor of the serine/ threonine kinase AKT, thereby facilitat-
ing M. tuberculosis survival within macrophages [33]. A separate
study by Rothchild and collaborators substantiated previous find-
ings and demonstrated that miR-155 regulates the SHIP1 pathway
in both macrophages and T cells. However, the miRNA-induced
downstream process had the opposite effect on M. tuberculosis
survival depending upon the cell type. On the one hand, miR-
155 augmented macrophage survival and propagation of bacteria,
whereas, on the other hand, it promoted the survival and function
of M. tuberculosis-specific T cells required to control infection. An in
vivo study in a miR-155–/– mouse model of TB corroborated in vitro
observations. The mice exhibited earlier control over mycobacte-
rial replication during the initial phase when macrophage func-
tion is crucial. During the chronic stage of infection after the onset
of adaptive immunity, higher mycobacterial load with increased
pulmonary damage was observed in miR-155–/– mice because of an
inadequate T cell response [34]. A similar observation was revealed
by Iwai and coworkers in miR-155-deficient C57BL/6 mice, indi-
cating the crucial role of miR-155 in the regulation of host
responses during TB infection [35]. The distinct miRNA expression
profiles in TB suggest the regulatory function of miRNAs in im-
munity to disease and their role in articulating macrophages
orchestrating innate and adaptive immune responses and evasion
from immune destruction.
miRNA as biomarkers for TB
Improved and rapid methods are urgently required to identify TB
and further distinguish between active and latent TB. miRNA-
based diagnostic biomarkers have been extensively studied in
many diseases, including cancer, and researchers are currently
exploring their roles as possible diagnostic biomarkers for TB
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[36]. miRNAs have shown high stability in serum and are hard to
degrade by RNases, strengthening their potential use as biomarkers
for early diagnosis of infection [37]. Moreover, identified miRNA
candidates must be discernible in surrogate tissues and sufficient
to establish disease stages, treatment efficacy, or susceptibility
despite inherent variability.
Several studies have identified abnormally expressed miRNA
miR-424 was differentially upregulated in peripheral blood mono-
nuclear cells (PBMCs) in patients with TB, whereas significant
downregulation of miR-223, miR-144*, and miR-421 was observed
in pleural fluid mononuclear cells [39]. Fascinatingly, miR-146a
was suppressed in both peripheral and pleural fluids mononuclear
cells of patients and was identified as a negative regulator of host
immunity [40]. Several researchers are investigating urine-based

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patterns and have proposed miRNAs for use as prognostic or
predictive biomarkers (Table 2). Zhang and colleagues identified
serum miRNA signatures for the diagnosis of active TB and latent
TB infection. The authors proposed miR-196b and miR-376c miR-
NAs as potential diagnostic markers for TB; however, further
studies are required to confirm the finding in a larger subset of
patients with TB. Differential miRNA expression between active
and latent TB infection suggests that miRNA expression modulates
target gene expression and, therefore, has a crucial role in disease
pathogenesis [38]. Another study by Spinelli et al. identified varia-
tion in miRNA expression in mononuclear cells obtained from
both peripheral blood and pleural fluids of patients and suggested
compartmental-specific miRNA-dependent gene regulation.
diagnostic procedures for the confirmation of various diseases,
including TB. A recent study identified miR-625-3p, mannose-
binding lectin-2 (MBL2), and inter-a-trypsin inhibitor H4 as a
three-biomarker combination in urine samples for TB diagnosis.
Protein–miRNA interaction network analysis identified a positive
correlation between MBL2 protein and miR-625-3p expression and
envisaged mannose-binding lectin 2 protein as a direct target of
miR-625-3p [41]. Another study identified increased expression of
host genome-encoded (miR-146a-5p and miR-125b-5p) and M.
tuberculosis genome-encoded (M. tuberculosis-miR5) miRNAs in
active TB patients serum samples. One of the major limitations
of the study was the limited sample size and, thus, the
results require verification in a larger cohort of patients with

TABLE 2
Differential expression of miRNAs in TB and their potential as biomarkersa,b
Cohorts examined Sample Candidate biomarkers Platform/assay Refs
TB versus healthy controls Serum miR-484 ("), miR-425 ("), and miR-96 Bioinformatics analysis, qRT-PCR [105]
(") (candidate approach: three miRNAs)
Exosomes isolated miR-20a ("), miR-20b ("), miR-26a Microarray, qRT-PCR [107]
using plasma ("), miR-106a ("), miR-191 ("), and
miR-486 (")
Latent TB versus active TB versus Exosomes isolated LTBI: miR-let-7e-5p ("), miR-let-7d- HTS, qRT-PCR [106]
healthy contacts using serum 5p ("), miR-450a-5p ("), and miR-
140-5p (")
TB: miR-1246 ("), miR-2110 ("), miR-
370-3P ("), miR-28-3p ("), and miR-
193b-5p (")
Tuberculous meningitis versus viral PBMCs miR-126-3p (#), miR-130a-3p (#), Microarray, qRT-PCR [108]
meningitis versus healthy controls miR-151a-3p (#), and miR-199a-5p
(#)
Patients with active TB versus PBMCs miR-892b (#), miR-199b-5p ("), and Bioinformatics analysis using gene [47]
healthy controls miR-582-5p (") and miRNA expression data sets,
qRT-PCR
Serum miR-423-5p ("), miR-17-5p ("), and HTS, qRT-PCR [109]
miR-20b-5p (")
Healthy controls versus patients Serum Human: miR-146a-5p (") and miR- HTS, qRT-PCR [42]
with pulmonary TB or 125b-5p ("), M. tuberculosis: miR-5
extrapulmonary TB (")
Patients with pulmonary TB versus Urine miR-625-3p ("), Mannose-binding Urine proteomic profiles analysis, [41]
healthy controls lectin 2 ("), and inter-a-trypsin qRT-PCR
inhibitor H4 (")
Plasma miR-29a ("), miR-99b ("), miR-21 (#), miRNA PCR panels (Exiqon), qRT- [43]
miR-146a (#), and miR-652 (#) PCR
Serum miR-378 ("), miR-483-5p ("), miR-22 HTS, qRT-PCR [12]
("), miR-29c ("), miR-101 (#), and
miR-320b (#)
Patients with childhood TB versus PBMCs miR-150 (#), miR-146a (#), miR-125b Microarray, qRT-PCR [49]
healthy contact (#), miR-31 (#), miR-10a (#), miR-1 (#),
miR-155 (#), and miR-29 (")
Cows with bovine TB versus healthy Milk miR-146a qRT-PCR (candidate approach using [110]
cows four miRNAs)
Healthy household contacts of TB Serum miR-148b-3p (#), miR-21-5p ("), and qRT-PCR (candidate approach using [46]
index cases miR-484 (") 608 c-miRNAs)
a
Abbreviations: HTS, high-throughput sequencing; LTBI, latent TB infection; qRT-PCR, quantitative real-time PCR.
b
" increased; # decreased.

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extrapulmonary TB and pulmonary TB, including individuals with
other lung pathologies [42].
Treatment with anti-TB drugs affects the miRNA expression
profile and is associated with response to treatment. A study in
a Chinese population revealed that the expression levels of miRs
-29a, -99b, -26a, and -146a changed following successful treat-
ment, although not all miRNAs returned to baseline by treatment
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completion [43]. The study suggests the potential of miRNA-based
biomarker signatures to measure treatment response; however,
these observations need further examination in a larger cohort.
Another study in an Indian cohort identified miR-16 and miR-155
as surrogate biomarkers for studying TB infection and response to
treatment. The miR-16 level was significantly upregulated in
patients with TB compared with uninfected controls, whereas
the miR-155 expression level was reduced considerably. The levels
of miR-16 and miR-155 after successful therapy returned to those
observed in healthy subjects. miR-155 was downregulated in
serum from patients with multidrug-resistant (MDR) TB compared
with control groups but higher than in TB-naı̈ve patients [44]. The
discordance between miRNA expression between these two studies
could result from the ethnic variation of the study populations,
infection with different mycobacterial strains or variation in
patients’ ages at diagnosis [45]. Nevertheless, the studies demon-
strate the potential of miRNA signatures to predict response to
treatment and early recognition of treatment failure and possible
drug resistance.
Duffy et al. analyzed Uganda and South Africa cohorts of house-
hold contacts (HHCs) of TB index cases. They recorded a serum
circulating miRNA-based signature for increased risk of progres-
sing to clinical disease among household contacts. The study
included a panel of 47 highly expressed and technically replicable
c-miRNAs; of the 608 candidate miRNAs, a miR-148b-3p, miR-21-
5p, and miR-484c miRNA-based signature could discriminate be-
tween HHCs who remained healthy and those who developed the
disease within 6 months [46]. A recent study using integrated
bioinformatics analysis and a candidate validation-based ap-
proach identified significant deregulation of miR-892b, miR-
199b-5p, and miR-582-5p in PBMCs of patients with active TB
[47]. A study in Chinese pediatric patients with pulmonary TB
compared PMBCs from patients with pulmonary TB and healthy
controls and identified the downregulation of miRNA-31 expres-
sion in patients’ samples. Further analysis identified an inverse
correlation between miR-31 expression and Th1 cytokine levels,
which are considered to be correlated with protection against TB
infection [48]. Another study in Chinese pediatric patients with TB
identified alterations in 29 miRNA by microarray analysis. Bioin-
formatics analysis identified 14 miRNAs as crucial regulators of
disease progression in pediatric patients with TB, and observed
downregulation of miR-1, miR-155, miR-31, miR-146a, miR-10a,
miR-125b, and miR-150, whereas miR-29 was upregulated com-
pared with uninfected children [49]. Interestingly, miR-31 was
identified in both the studies and emerged as a potential biomarker
for diagnosing childhood TB; however, the findings need to be
validated in a larger population.
Drug resistance is one of the major obstacles in the control of
TB. Recent studies highlighted the role of miRNAs in the develop-
ment of drug resistance in M. tuberculosis strains. Ran et al. revealed
similar expression of six miRNAs between drug-sensitive and
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drug-resistant TB strains, whereas 108 miRNAs were exclusively
expressed only in the MDR M. tuberculosis strain [50]. However, the
role of miRNA in regulating gene expression within M. tuberculosis
is still not clear, and more research is required to determine the
mechanism. A separate miRNA profiling study identified down-
regulation of miR-320a expression level in patients with drug-
resistant TB compared with those with drug-susceptible TB; how-
ever, further studies are required to elucidate the role of miR-320a
in the development of drug resistance [51]. Wang et al. proposed a
biomarker-based potential MDR-TB diagnostic model using CD44,
KNG1, miR-424-5p, miR-4433b-5p, and miR-199b-5p [52]. The
integrative miRNA and proteomic-based biosignatures might
not only help to increase the diagnostic accuracy, but might also
uncover key roles for miRNA in the development of drug resis-
tance.
Thus, numerous studies have identified various miRNAs as
biomarkers to differentiate healthy controls, latent TB, and active
TB; nevertheless, there is no clear-cut evidence for the association
of those miRNAs in the physiology and pathogenesis of the disease
or disease-specific expressions. A recent review by Pedersen et al.
examined all published miRNA–TB biomarker studies to identify a
reproducible miRNA signature of TB disease [53]. Of interest, only
eight miRNAs were differentially expressed between patients with
TB and healthy controls in two or more studies. There was no
consensus over which miRNA signature is more informative for
the identification of TB disease. The variation in results might
result from the source of samples used for miRNA isolation,
differences in sample preparation, and methodology used for
quantifying miRNA expression level. Further research is expected
to identify the most appropriate sample source and methods to
quantify differential expression correlated with TB and its closely
related diseases and contribute to biomarker signature develop-
ment for easier and earlier diagnosis of TB.
RNA-based therapeutics
The therapeutic application of nucleic acid was first approved in
1998 and has since progressed from antisense nucleotides (ASOs)
to RNAi [small interfering (si)RNA]-based and miRNA-focused
therapies. As the expression of miRNAs is altered in many diseases
and has a significant role in disease pathogenesis, it has opened a
new avenue for the use of miRNA as therapeutic targets.
Antisense oligonucleotides
ASOs are single-stranded, short, synthetic nucleic acid sequences
designed to bind selectively to selected nucleic acid sequences
because of its complementarity. The binding trigger modulation of
RNA function, either by the degradation of RNA transcript because
of RNAse H or by altering splicing factor recruitment in protein
translation, results in RNA metabolism aberration or protein
translation inhibition [54]. By contrast, siRNA, another class of
nucleic acid, is a short, synthetic, and double-stranded sequence,
which utilizes the RNA-induced silencing complex pathway (RISC)
RNA decay pathway for its degradation. Fomivirsen1 was the first
US Food and Drug Administration (FDA)-approved ASO drug for
the treatment of retinitis caused by cytomegalovirus infection.
Milasen1 is the latest personalized ASO drug approved for a 6-
year-old child with neuronal ceroid lipofuscinosis, a disorder of
the central nervous system [55]. Similar to siRNAs, miRNAs
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February 2021
downregulate gene expression post transcriptionally and can be
synthesized chemically within the lab. ASOs can regulate the
overexpression of miRNAs during disease conditions and have
been demonstrated safe in various diseases, including TB [55].
The application of ASOs as therapeutics, approved drugs, their
clinical trials, and associated toxicity have been discussed in detail
elsewhere and are beyond the scope of the present review [56].
Within the content of this review, will discuss the application and
limitation of miRNAs as therapeutic targets for TB treatment,
delivery systems, and crucial challenges for its translational suc-
cess.
miRNAs as therapeutics
Research in the field of HDT development has opened new ave-
nues in the fight against TB. HDTs targeting autophagy pathways,
immunometabolism, and granuloma structure lead to realign-
ment of the immune response against M. tuberculosis. Recent
studies established the potential of miRNAs as adjunctive HDTs
to improve therapeutic outcomes in TB. The first successful pre-
clinical study in Hepatitis C virus (HCV)-infected chimpanzee
using anti-miR-122 ‘antagomir’ oligonucleotides initiated the
era of miRNA-based therapeutics [57]. Reports showed that some
miRNAs can serve as valuable therapeutic targets for a large num-
ber of diseases, including cardiovascular diseases [58], cancer [59],
and HCV [60]. There are two distinct possibilities to exploit
miRNAs for therapeutic interventions. Exogenous miRNA mimics
can be applied to restore optimal miRNA expression for endoge-
nously expressed miRNA [61], and miRNA inhibitors or antago-
mirs can reduce mature miRNA target activity [62].
M. tuberculosis utilizes the suppression of apoptosis and autop-
hagy as a strategy for its survival within the intracellular niche. In
recent years, several researchers have explored the miRNAs in-
volved in apoptosis and autophagy as targets to treat TB. In mice,
anti-miR-27a reduced M. tuberculosis infection by enhanced autop-
hagosome formation [14]. The results showed that alternate intra-
peritoneal injection for 4 weeks post infection suppressed miR-27a
expression in lung tissues and spleens of the mice, decreasing
mycobacterial load and improving lung pathology. Ouimet et al.
showed that targeting miR-33 and miR-33* by genetic or pharma-
cological means promoted autophagy of M. tuberculosis-infected
macrophages via AMPK-dependent activation of the transcription
factors FOXO3 and TFEB [23]. Etna et al. noticed that antagonizing
M. tuberculosis-induced miR-155 expression in infected DCs re-
stored autophagy, resulting in decreased mycobacterial survival
and persistence. Among their results, the authors highlighted that
the ATG3 protein content post treatment was restored to untreat-
ed and untransfected control cell levels [13].
There is also evidence that miR-143 and miR-365 increased the
growth of M. tuberculosis in bone marrow-derived macrophages
(BMDMs) and monocyte-derived macrophages (MDMs) alterna-
tively activated by IL-4/IL-13. miR-143. miR-365 increased M.
tuberculosis growth via enhanced heme oxygenase-1 expression
and anti-inflammatory IL-10 [63]. Zhang et al. noted that treat-
ment of M. tuberculosis-infected macrophages with MiR-20b-5p
antagomir enhanced mycobacterial survival in macrophages.
miR-20b-5p downregulation attenuated apoptosis via the Mcl-1
pathway in infected macrophages, resulting in enhanced myco-
bacterial survival [47]. Zhou et al. developed a CD-11b antibody
Please cite this article in press as: Singh, A.K. et al. Interplay between miRNAs and Mycobacterium tuberculosis: diagnostic and therapeutic implications, Drug Discov Today (2021),
https://doi.org/10.1016/j.drudis.2021.01.021
REVIEWS
and a poly(ethylene imine) (PEI)-modified nanoparticle (NP)-
based nanoplatform and conjugated miRNA-124-3p onto the
surface of the NPs. In this study, Mycobacterium marinum-infected
microglia transfection with miR-124-3p promoted apoptosis
through upregulation of Caspase 3 or downregulation of Bcl-2
and Bcl-xl, and decreased mycobacterium proliferation [64]. An-
other study showed that MiR-99b antagomir transfection in M.
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GENE TO SCREEN
tuberculosis-infected DCs aided enhanced bacterial clearance by
augmenting TNF-a and tumor necrosis factor ligand superfamily-
4 (TNFSF-4) [17]. Another study directly scored for bacterial
survival after M. tuberculosis H37Rv infection of the murine
macrophage cell line RAW264.7 as well as BMDMs after transfec-
tion of cells with either let-7f mimic or let-7f inhibitor. Over-
expression of miR-let-7f decreased M. tuberculosis survival and
augmented the production of inflammatory cytokines, such as
TNF- and IL-1b [65]. Results from Lou et al. indicated that
intravenous administration of miR-20b mimics could ameliorate
the inflammatory response in a mouse model of TB by targeting
the NLRP3/caspase-1/IL-1b pathway and have potential for fur-
ther evaluation as TB therapeutics [66]. These studies clearly show
that mycobacteria subvert cytokine-regulated immune responses
for survival via miRNA-regulated pathway and, thus, are attrac-
tive therapeutics target for manipulating the host response to M.
tuberculosis infection.
Although miRNA-based therapeutic options are still in their
infancy and most miRNA-based drugs are currently in clinical
trials, recent studies in other diseases and TB provide a valid basis
for future miR-based HDTs. Nevertheless, several challenges need
to be overcome, including devising methods for efficient drug
design and delivery of miRNA mimics/antagomiRs to protect them
from circulating RNAse enzymes. Targeted efficient release of
miRNAs toward the site of infection is also required to overcome
the probability of off-target effects that would otherwise limit the
efficacy and safety of this approach [67].
miRNA delivery techniques
Susceptibility to degradation by serum nucleases and renal clear-
ance restricts the delivery of naked miRNAs in vivo. The intrinsic
negative charge of oligonucleotides blocks their interaction with
anionic components of the membrane, subsequently resulting in
poor cellular internalization. Technologies to deliver miRNAs in
vivo include viral vectors, such as lentiviral vectors, or nonviral
vector methods, such as lipid conjugates, liposomes, dendrimers,
small exosome-like vesicles, and synthetic and natural polymers,
have been studied with varying success (Fig. 2) [68]. A study
showed that conjugation of miR antagomir with high-affinity
ligands for the hepatocyte-specific asialoglycoprotein receptor
(ASGPR) resulted in significant viral load reduction in patients
with chronic HCV [69]. The latest experimental data and potential
of dendrimers and dendrimer-based nanoconstruction delivery
systems as promising candidates for the delivery of short siRNAs
and miRNAs at the cell and organism levels are summarized in a
recent review [70]. Lipid-based delivery is another popular ap-
proach, and research has demonstrated the delivery of miR-29b
and miR-133b with a mixture of 1,2-di-O-octadecyl-3-trimethy-
lammonium propane lipid (DOTMA), cholesterol, and polyethyl-
ene glycol (PEG) [71,72], or pre-miR-107 encapsulation with solid
lipid NPs [73]. The use of a neutral lipid delivery system reduces the
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 DRUDIS-2912; No of Pages 11

REVIEWS Drug Discovery Today
Volume 00, Number 00
February 2021

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Drug Discovery Today

FIGURE 2
Schematic of viral and nonviral miRNA delivery vectors. miRNA delivery systems are divided into viral carrier-based and nonviral carrier-based delivery vectors.
Nonviral vectors are divided into three categories: inorganic material-based delivery systems; cell-derived membrane vesicles; and organic material-based
delivery systems, which includes polymeric nanoparticles (NPs), lipid-based carriers, and dendrimer-based vectors.

adverse-effect profile with minimal adverse effects and a uniform
distribution in different tissues, including the lung [74].
Exosomes are similar to liposomes in terms of their lipopolysac-
charide, periplasm, and phospholipid content, and have low
toxicity and immunogenicity because of their biogenesis. Alexan-
der et al. revealed that intraperitoneal injection of miR-146a-con-
taining exosomes into C57BL/6 mice inhibited the inflammatory
response to lipopolysaccharide endotoxin stimulus. The authors
also noted that treatment with miR-146-containing exosomes
delivered the miRNA to the spleen, liver, and bone marrow,
thereby contributing to the amelioration of inflammatory
responses [75]. Song et al. supported these earlier observations
and found that treatment of macrophages with miR-146-contain-
ing exosomes resulted in decreased inflammatory cytokine secre-
tion and increased survival in septic mice [76]. Zhang et al.
demonstrated that intratracheal instillation of serum-derived exo-
somes loaded with miRNA targeted alveolar macrophages and
could ameliorate inflammatory lung responses [77]. The emerging
prospects of using exosomes as miRNA delivery vehicles are dis-
cussed in detail elsewhere and are beyond the scope of the present

8 www.drugdiscoverytoday.com
Please cite this article in press as: Singh, A.K. et al. Interplay between miRNAs and Mycobacterium tuberculosis: diagnostic and therapeutic implications, Drug Discov Today (2021),
https://doi.org/10.1016/j.drudis.2021.01.021
DRUDIS-2912; No of Pages 11
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February 2021
review [78]. However, isolation of exosomes of high purity signifi-
cantly complicates pharmaceutical development, and future stud-
ies are required to confirm their clinical safety.
NP-based delivery of miRNAs offers excellent scope for the clinical
application of miRNA-based therapeutics for various diseases. The
ability of NPs to protect encapsulated materials from inactivation or
degradation besides targeted delivery supports the rationale for NP-
based approaches for miRNA delivery. Numerous preclinical studies
have reported successful application of poly(lactic-co-glycolic acid)
(PLGA), PEI, poly(e-caprolactone) (PCL), and polyurethane-based
NP delivery systems for miRNA delivery [79–81]. miR-19b-3p miRNA
mimics encapsulated in PLGA microparticles caused a significant
reduction of secretory leucoprotease inhibitor (SLPI), with an anti-
inflammatory effect, within macrophages in vitro [77]. Liu et al.
observed that treatment of a mouse model of gastric cancer with
miR-200c and docetaxel-encapsulated PCL NPs resulted in higher
accumulation of drug-elevated miR-200c, resulting in suppressed
tumor growth [81]. To date, no studies have compared different
miRNA formulations in TB infection models; however, considering
the outcomes of miRNA NP-based therapeutic entities, miRNA-
based therapeutics might emerge as a therapy option for TB
treatment in the near future. The successful delivery of PLA micro-
particles containing anti-TB drugs by inhalation in a mouse model of
TB potentiates their prospective application for the delivery of
miRNA-based therapeutics to treat TB [82].
Concluding remarks and prospects
There is significant scientific evidence demonstrating the central
role of miRNAs as regulators of immunity against M. tuberculosis.
Blood, sputum, and pleural fluid-based studies have revealed that
differential expression of miRNA can indicate disease progression
and could differentiate between TB-infected and healthy individ-
uals, and between active and latent TB infections. Such results
offer insights into the possible use of miRNAs as biomarkers for
diagnosing TB. Research indicates the therapeutic implications of
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GENE TO SCREEN
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Author contributions
A.K.S. devised the manuscript, collected the data, and wrote the
manuscript. V.K. and S.A. helped to compile the tables. M.G.
assisted in writing the manuscript and drawing figures. S.P. pro-
vided critical inputs during writing and reviewed the manuscript
before the final submission. All authors read and approved the
manuscript before submission.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgment
The work was supported by an intramural grant from the Indian
Council of Medical Research, New Delhi.
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