biomedicines

Review
Functions of Differentially Regulated miRNAs in Breast Cancer
Progression: Potential Markers for Early Detection and
Candidates for Therapy
Kumar Subramanian † and Raghu Sinha *

Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA
* Correspondence: rsinha@pennstatehealth.psu.edu
† Current address: Department of Surgery, Georgetown University School of Medicine, Washington, DC 20007, USA;

ks1888@georgetown.edu.

Abstract: Breast cancer remains a major global health concern, emphasizing the need for reliable
biomarkers to enhance early detection and therapeutic interventions. MicroRNAs (miRNAs) are
evolutionarily conserved small non-coding RNA (~22 nt in length) molecules, which are aberrantly
expressed in cancer and seem to influence tumor behavior and progression. Specific miRNA dys-
regulation has been associated with breast cancer initiation, proliferation, invasion, and metastasis.
Understanding the functional roles of these miRNAs provides valuable insights into the intricate
molecular mechanisms underlying breast cancer progression. The diagnostic potential of miRNAs
as non-invasive biomarkers for early breast cancer detection is a burgeoning area of research. This
review aims to elucidate the functions of differentially regulated miRNAs in breast cancer pro-
gression and assess their potential as markers for early detection, stage-specific biomarkers, and
therapeutic targets. Furthermore, the ability of specific miRNAs to serve as prognostic indicators
and predictors of treatment response highlights their potential clinical utility in guiding personalized
therapeutic interventions.

Keywords: breast cancer; microRNA; biomarkers

Citation: Subramanian, K.; Sinha, R.

Functions of Differentially Regulated
miRNAs in Breast Cancer Progression:
1. Introduction
Potential Markers for Early Detection
and Candidates for Therapy. Breast cancer is a disease caused by genetic aberrations and deleterious environmental
Biomedicines 2024, 12, 691. exposure [1]. Breast cancer is a complex and enigmatic disease caused by a series of
https://doi.org/10.3390/ alterations in genes that control cell growth and proliferation. Breast cancer is the most
biomedicines12030691 common cancer in women and is a heterogeneous disease with diverse molecular subtypes
and clinical presentations. Despite advances in treatment, it is the leading cause of cancer
Academic Editor: Randolph C. Elble
mortality in women worldwide. Globally, it accounts for 16 percent of cancer deaths, and
Received: 13 December 2023 has over 20 distinct subtypes that differ genetically, morphologically, and clinically [2,3].
Revised: 15 February 2024 About 90% of these deaths are due to metastases. Currently, breast cancer is the fourth
Accepted: 21 February 2024 leading cause of cancer-related death, with 2.3 million new cases in 2022 worldwide [4].
Published: 20 March 2024 The metastatic spread of breast cancer, typically to the bone, lung, liver, and brain, accounts
for most cancer-related deaths [5]. There are few treatment strategies for metastatic breast
cancer, which is incurable with a median survival rate of 2–3 years [6]. Breast cancer
cases are increasing alarmingly, underscoring the importance of treating the disease in
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
many ways. Several trials have been conducted for the successful development of drugs
This article is an open access article
for breast cancers over the past few decades. As a result of inadequate early detection,
distributed under the terms and breast cancer patients have a high mortality rate and recurrence rate. Thus, to improve
conditions of the Creative Commons disease outcomes and prolong patient survival, it is vital to find novel early prognostic and
Attribution (CC BY) license (https:// diagnostic biomarkers and effective therapeutic methods [7].
creativecommons.org/licenses/by/ Recent research studies have shed light on the progression of breast cancer, its patho-
4.0/). genesis, and the molecular pathways involved in proliferation. As evidenced by recent

Biomedicines 2024, 12, 691. https://doi.org/10.3390/biomedicines12030691 https://www.mdpi.com/journal/biomedicines

Biomedicines 2024, 12, 691 2 of 23

research, molecular marker-based targeted therapies using miRNAs may improve the
prognosis and diagnosis of a wide range of diseases, including breast cancer [8]. A growing
body of evidence suggests that miRNAs play a critical role in tumorigenesis and breast
cancer development. These molecules are altered in different tumorigenic processes of
breast cancer. miRNAs are small, noncoding RNA molecules that regulate gene expression
through interfering with transcription. miRNAs play a significant role in regulating a
variety of cellular processes, such as angiogenesis, apoptosis, and the cell cycle. Due to
their ability to modulate multiple targets within these pathways, they play a significant
role in maintaining cellular homeostasis. It is often observed that dysregulation of miRNAs
in these processes contributes to various diseases, including breast cancer, which makes
them potential therapeutic targets or diagnostic markers for cancers [9]. It is important to
understand the complex network of miRNA-mediated regulation to gain insight into the
molecular mechanisms that govern these vital processes within cells.
miRNA was discovered by Ambros and co-workers in Caenorhabditis elegans (Nema-
tode), during their genetic study to investigate defects in the temporal control of C. elegans
development [10]. Recent studies have demonstrated that miRNAs play critical roles in the
development of breast cancer, differentiation, proliferation, and other physiological pro-
cesses [11]. A growing body of evidence suggests that miRNAs might have very important
clinical implications.
It is well established that miRNAs are critical regulators of mRNA expression and
cell activity, both in normal and abnormal biological processes, including breast cancer.
As miRNA dysregulation occurs in various types of cancers including breast cancer and
leads to tumor initiation, drug resistance, and metastasis, the therapeutic strategies aimed
at modulating miRNA expression levels and identifying their targets are promising strate-
gies [9,11]. miRNAs are secreted by a variety of cells and transported to a variety of bodily
fluids in remarkably stable forms (i.e., peripheral blood, saliva, cerebrospinal fluid, ascites,
urine, and breast milk) through extracellular vesicles, and interestingly, the levels of miR-
NAs circulating in cancer patients differ from those of healthy donors [12–16]. Therefore, a
quantitative analysis of such potential circulatory miRNAs would be an ideal approach
for detecting breast cancer via liquid biopsy in the early stages. This review summarizes
the major functions of miRNAs in breast cancer progression and discusses the clinical
applications of differentially regulated miRNAs, especially circulating miRNAs in early
diagnosis and as targets for therapy.

2. miRNA Biogenesis and Maturation

Pri-miRNAs are processed step-by-step in the cytoplasm and nucleus during the
biogenesis of miRNAs. In humans, miRNA gene transcription takes place within the
nucleus following the cleavage of the ~80 nucleotide stem-loop pre-microRNA precursor
performed by the microprocessor complex consisting of Drosha, an RNase III-type nuclease,
a double strand RNA-binding protein co-factor, and the DiGeorge syndrome critical region
8 gene (DGCR8) (Figure 1). The complex cleaves the pri-miRNA to generate a shorter
hairpin-shaped precursor called precursor miRNA (pre-miRNA). The pri-miRNAs are
processed into 60–70 nucleotide hairpin structures (pre-miRNAs) and are exported from the
nucleus to the cytoplasm, supported by the nucleocytoplasmic shuttle protein Exportin-5
in a Ran-GTP-dependent manner. The enzyme Dicer further breaks down the pre-miRNA
into a double-stranded RNA duplex in the cytoplasm. The mature miRNA is one of the two
strands of the duplex that is chosen and loaded into the RNA-induced silencing complex
(RISC) [17,18].
By base-pairing with complementary sequences in the 3’ untranslated region (UTR)
of the target mRNA, the mature miRNA directs the RISC complex to its target mRNAs.
As a result of this interaction, the target gene may be downregulated due to translational
repression or mRNA degradation. Multiple steps and molecular players are involved in
miRNA biogenesis [17]. Breast cancer has been associated with dysregulation of miRNA
biogenesis or its function.
 of the target mRNA, the mature miRNA directs the RISC complex to its target mRNAs.
As a result of this interaction, the target gene may be downregulated due to translational
repression or mRNA degradation. Multiple steps and molecular players are involved in
miRNA biogenesis [17]. Breast cancer has been associated with dysregulation of miRNA
Biomedicines 2024, 12, 691 3 of 23
biogenesis or its function.

Biosynthesis of
Figure1.1.Biosynthesis
Figure of miRNA:
miRNA: Pre-miRNAs
Pre-miRNAsare arefurther
furthercleaved
cleavedinto an an
into asymmetric
asymmetric duplex
duplex us-
using
ing thethe actionofofDicer
action Dicer and
and accessory
accessoryproteins. Transactivation-responsive
proteins. Transactivation-responsive RNA-binding protein protein
RNA-binding
(TRBP) and
(TRBP) and PACT
PACTininhumans
humansremove the loop
remove sequence
the loop by forming
sequence a short-lived
by forming asymmetric
a short-lived duplex
asymmetric du-
intermediate
plex (miRNA:
intermediate miRNA),
(miRNA: with a duplex
miRNA), with aofduplex
about 22 of nucleotides in length. This
about 22 nucleotides in pre-cursor
length. This pre-
is cleaved
cursor to generate
is cleaved ~21–25-nucleotide
to generate mature miRNAs.
~21–25-nucleotide The matureThe
mature miRNAs. miRNA is loaded
mature miRNA into
is the
loaded in-
miRNA-induced silencing complex (miRISC), which binds to target mRNA, resulting
to the miRNA-induced silencing complex (miRISC), which binds to target mRNA, resulting in ei- in either
ther the degradation
the degradation of mRNA of or
mRNA or the ofblockage
the blockage translation ofwithout
translation
mRNA without mRNA
degradation degradation
(adapted
(adapted from [17,18]).
from [17,18]).

3. miRNA Targets and Their Role in Breast Cancer

3. miRNA Targets and Their Role in Breast Cancer
Discovering the regulatory network regulated by miRNAs requires identification
Discovering the
of miRNA-mRNA regulatory
target network
interactions. Theregulated
development by miRNAs
of miRNA requires identification of
target prediction
miRNA-mRNA target
algorithms is based interactions.
on several The Statistical
approaches. development of miRNA
inference based on target
machineprediction
learning algo-
and algorithms derived from characteristics of the mRNA sequence and/or based on the
miRNA-mRNA interaction are the two major categories. Pairings of miRNA and mRNA
seed sequences can be analyzed and evaluated. Instead of making “de novo” predictions
Biomedicines 2024, 12, 691 4 of 23

based on sequence features, the aim of machine learning is to classify miRNA targets
that reference miRNA-mRNA duplexes with known biological significance [19]. miRNA
sequences are complementary to 3′ -UTR sequences of mRNA targets. For miRNA to bind
to target mRNAs, the seed sequence of the miRNA 5′ region is essential. Specific seed
region characteristics, as well as those in proximity, have been linked to specific effects on
miRNA-induced gene repression [19].
Watson–Crick Pairing between miRNA and mRNA is needed for most target pre-
diction algorithms. miRNA prediction tools include miRNAFinder, miRscan, miRbase,
miRTarBase, and SSC profiler [20,21]. Most of them are focused on miRNA conservation
characteristics across ecosystems. The prediction of miRNA in a wide range of species from
the animal and plant kingdoms have proven successful with this technique.
The miRNAs play an important role in the regulation of gene expression, and the
dysregulation of these molecules has been implicated in various stages of breast cancer
development. Breast cancer miRNAs are classified based on their expression patterns,
functional roles, and clinical implications [22,23]. miRNAs can be classified into two main
categories established on their effects on tumorigenesis:
1. Oncogenic miRNAs (OncomiRs): these miRNAs are often upregulated in breast cancer
and promote tumorigenesis by inhibiting tumor-suppressor genes or regulatory pathways.
2. Tumor-Suppressive miRNAs: conversely, these miRNAs are downregulated in breast
cancer and typically act to inhibit oncogenes or other pro-tumorigenic processes.
To maintain normal cellular function, it is essential to maintain a balance between
oncogenic and tumor-suppressive miRNAs as reviewed earlier [24]. In breast cancer,
dysregulation of miRNA expression contributes to tumorigenesis (Table 1). Therapeutic
potential exists in manipulating miRNA expression. OncomiRs can be inhibited or replaced
with miRNAs (for tumor-suppressive miRNAs).

Table 1. List of miRNAs and the function of their targets in breast cancer.

MicroRNA Target Function References

Oncogenic miRNAs in breast cancer
Promotes cell migration, invasion,
miRNA-10b HOXD10 [25]
and metastasis
Promotes lymph node metastasis, enhanced cell
COL4A3, LAMA3, proliferation, colony formation, migration, and
miRNA-17/92 cluster [26,27]
TIMP2/3, ADORA1 invasion in Triple Negative Breast
Cancer (TNBC)
PDCD4, PTEN, TPM1,
miRNA-21 Promotes invasion, metastasis, and migration [28–31]
TIMP3
Nanog, Oct-3/4, BimL, F1H1, HIF-1,
miRNA-24 Hypoxia-inducible miRNA [32]
Snail, VEGFA
Pyruvate kinase and Promotes metastasis by reprogrammed
miRNA-122 [33]
citrate synthase glucose metabolism
LATS2, CDK2, Promotes cell proliferation and S-G2/M cell
miRNA-135b [34]
p-YAP cycle progression
miRNA-155 SOCS1, TP53INP1, FOXO3 Promotes cell growth, proliferation, and survival [35–37]
Promotes epithelial-to-mesenchymal transition
miRNA-181a Bim [38]
(EMT), migration, and invasion
Promotes apoptosis resistance and
miRNA-191-5p SOX4, caspase-3, caspase-7, p53 [39]
doxorubicin resistance
Biomedicines 2024, 12, 691 5 of 23

Table 1. Cont.

MicroRNA Target Function References

miRNA-200b Ezrin/Radixin/Moesin (ERM) Promotes metastasis and invasion [40]
Promotes breast cancer cell invasion, migration,
miRNA-206 NK-1 [41]
proliferation, and colony formation in vitro
miRNA-210 Pax-5 Modulating EMT and hypoxia [42]
HER2, HOTAIR, E2F1, Promotes metastasis and invasion by elevation
miRNA-331 [43]
DOHH in plasma of metastatic breast cancer patients
Promotes cell migration, invasion,
miRNA-373 CD44 [44]
and metastasis
miRNA-455-3p EI24 Promotes proliferation, invasion, and migration [45]
miRNA-498 BRCA1 Promotes TNBC cell proliferation [46]
Promotes cell migration, invasion,
miRNA-520c CD44 [44]
and metastasis
Tumor-suppressor miRNAs in breast cancer
Promotes NK cell antitumoral activity and
miRNA-17-92 Mekk2, cyclin D1 reduces metastasis, regulates G1 to S [47,48]
phase transition
Inhibits cell invasion and metastasis, decreases
miRNA-7 SETDB1 [49]
the BCSC population, and partially reverses EMT
let-7d Cyclin D1 Induces stem cells radiation sensitization [50]
Inhibits self-renewal of breast tumor-initiating
miRNA-30 Ubc9, ITGB3 [51]
cells (BT-ICs), trigger apoptosis
miRNA-33b HMGA2, SALL4, Twist1 Regulates cell stemness and metastasis [52]
miRNA-34a IMP3 Regulates TNBC stem cell property [53]
ERBB2, EPO, EPOR, ENPEP, CK2-α, Inhibits cell proliferation and differentiation,
miRNA-125b [54,55]
CCNJ, MEGF9 migration and invasion
miRNA-137 FSTL1 Suppresses TNBC stemness [56]
miRNA-143 ERK5, MAP3K7, Cyclin D1 Anti-proliferative [57]
Inhibits cell proliferation, migration and
miRNA-148a WNT1, MMP13 [58,59]
invasion
Regulates breast cancer cell growth and mtDNA
miRNA-200a TFAM [60]
copy number
Forfeiture of self-renewing capacity associated
miRNA-203 ∆Np63α with epithelial stem cells, suppresses [61]
proliferation and colony formation
Suppresses proliferation and invasion and
miRNA-205 HMGB3, Notch-2 [62,63]
inhibits EMT and stem cell properties
miRNA-206 Cyclin D2, Cx43 Reduces migration, invasion, and metastasis [64,65]
Re-sensitizes TNBC stem cells to tumor necrosis
miRNA-223 HAX-1 [66]
factor-related apoptosis
Cyclin E1, p-NPAT,
miRNA-483-3p Anti-proliferative and G1-S cell cycle arrest [67]
NPAT, CDK2
miRNA-497 Cyclin E1 Anti-proliferative and reduces migration [68]
miRNA-519d-3p LIMK1 Suppresses growth and motility [69]

Studying OncomiRs and tumor-suppressive miRNAs in breast cancer is of paramount

importance due to several reasons. Interactions between OncomiRs and tumor-suppressive
Biomedicines 2024, 12, 691 6 of 23

miRNAs provide insight into the molecular mechanisms underlying breast cancer pro-
gression. The level of dysregulation of these miRNAs in tumor tissue or bodily fluids
correlates with the cancer stage, aggressiveness, and clinical outcome. It is possible to
tailor personalized treatment strategies for patients by identifying specific OncomiRs and
tumor-suppressive miRNAs associated with breast cancer subtypes or treatment responses.

4. Significance of miRNAs in Breast Cancer Development

miRNAs function by binding to the 3′ UTR of target mRNAs, leading to translational
repression or mRNA degradation. Breast cancer is characterized by dysregulation of miR-
NAs and their targets, which contribute to various aspects of cancer initiation, progression,
and metastasis. Below is an overview of some key miRNAs and their known roles in the
regulation of their targets in breast cancer.

4.1. Breast Cancer Initiation and Progression

As a multistep process, cancer initiates and progresses with the gradual transformation
of human cells into highly malignant forms through progressive genetic changes [70]. It
has been recognized that malignant transformation occurs through successive mutations
in specific genes, leading to the activation of oncogenes and the inactivation of tumor-
suppressor genes. The activities of these genes may represent the final common pathway
using which many carcinogens act [71]. There are three main types of genes that play a
role in tumor initiation: proto-oncogenes, tumor-suppressor genes, and genes involved in
DNA repair. Changes in the genes, like mutations, amplifications, or deletions, may lead to
decoupling of biological mechanism involved in the regulation of normal cell growth and
differentiation [72].
Accumulating evidence suggests that cancer stem cells (CSCs) or tumor-initiating cells
(TICs) with stem cell-like properties can propagate human tumors with heterogeneous
tumor populations in immunodeficient mice, such as human-breast-tumor-initiating cells
(BTICs) or breast cancer stem cells (BCSCs) that can regenerate breast tumors in an in vivo
model. Additionally, BTICs self-renew and asymmetrically divide into differentiated cancer
cells, and these are trusted to be accountable for cancer stem-like cells that drive breast
tumor formation, recurrence, metastasis, and drug resistance [66,73]. Liu et al. proved that
BCSCs are involved in the spontaneous metastases of human breast cancer in mouse breast
cancer orthotopic models [74]. Several miRNAs have been implicated in the regulation
of CSC properties. The dysregulation of miRNA might contribute to the self-renewal of
BCSCs and cancer progression.
The pivotal roles of miRNA-200 are well characterized in breast cancer initiation.
For example, miRNA-200 family members are significantly downregulated in CD44+ ,
CD24−low -lineage human primary BCSCs when compared to non-tumorigenic cancer cells.
Moreover, miRNA-200b regulates BCSC growth by directly targeting Suz12, a subunit of
a polycomb repressor complex (PRC2) and regulates EMT by repressing the E-cadherin
gene. Higher expression of the polycomb protein EZH2, which is important in the stem
cell self-renewal capability of embryonic and adult stem cells, has been associated with
breast cancer progression [75]. Shimono et al. reported that the expression of the tumor-
suppressor miRNA-200c decreased the self-renewal ability of BCSCs in vitro and tumor
formation ability in vivo [76].
Yu et al. showed that let-7 and miRNA-30e were downregulated in BCSCs and SK-3rd
mammospheres compared to non-tumorigenic cells or more differentiated cells, respectively.
Enforced expression of miRNA-30e inhibited mammosphere formation and tumorigenesis
of SK-3rd cells in vitro and in vivo, respectively, by targeting ITGB3 and UBC9 [51]. The
miRNA-128 was found to be downregulated in BCSCs (in CD44+ , CD24−/low , and mam-
mospheres) compared to non-cancerous cells [77]. Furthermore, miRNA-128 is significantly
low in chemoresistant BTICs enriched from breast cancer cells (SK-3rd and MCF-7) and
primary breast tumors, and that its target Bmi-1 and ABCC5 are overexpressed in these
cancers. The ectopic expression of miRNA-128 sensitizes BTICs to the proapoptotic and
Biomedicines 2024, 12, 691 7 of 23

DNA-damaging effects of doxorubicin, showing its therapeutic potential [77]. In another

finding, miRNA-30c regulates invasion of breast cancer by targeting the cytoskeleton net-
work genes encoding Twinfilin 1 (TWF1) and Vimentin (VIM), both of which regulate
EMT [78].
The miRNA-34c expression level and function were reduced in the BTICs of MCF-7
and SK-3rd cells, and these cell lines were enriched for BTICs [79]. When ectopic expres-
sion of miRNA-34c decreased the self-renewal of BTICs, inhibited EMT, and suppressed
migration of the tumor cells through inhibition of the target gene Notch4, miRNA-495 was
significantly upregulated in CD44+ /CD24−/low BCSCs, reflecting its potential importance
in maintaining common BCSC properties, and its ectopic expression promoted colony for-
mation in vitro and tumorigenesis in mice. In addition, the overexpression of miRNA-495
is associated with the lowered expression of E-cadherin, which enhances the stem-like phe-
notype in BCSCs [80]. miRNA-181 family members and miRNA-155 oncogenic miRNAs
promote self-renewal, sphere formation, colony formation, or tumor development in breast
cancer cells [81].

4.2. miRNA in Metastatic Breast Cancer

Metastatic breast cancer is a complex, multistep malignant process through which
tumor cells migrate from their primary tumor (site of origin) to colonize distant tissues
(e.g., liver, brain, bones, or lungs) and is often responsible for 90% of cancer-related mortal-
ity [82,83]. EMT is one of the important processes by which cancer metastasis starts, and
EMT induces morphological changes in epithelial cells by which epithelial cells transform
into mesenchymal cells. During this process, cancer cells lose their cell–cell communication
and become mobile and invasive, spreading into distant organs and tissues [84]. Sup-
pression of E-cadherin expression in epithelial cancer cells is a hallmark of EMT. Various
miRNAs have been linked to the control of EMT in cancer, and several genes, includ-
ing ZEB [85], Twist [86], Snail [87], and Slug [88] are known to restrict the expression
of E-cadherin.
One of the most important miRNAs that control metastasis is the miRNA-200 family
(miRNA-200a/200b/200c/141/429), and this prevents cell migration and invasion by
targeting ZEB in several cancer types, including breast cancer [89], and miRNA-200/ZEB
plays a central role in the EMT/MET processes. In the meantime, inhibition of miRNA-
200 reduces the E-cadherin level while supporting VIM expression, thereby increasing
cell motility [89]. Many studies also demonstrated that TGF-β-induced EMT might be
inhibited only with the ectopic expression of the miRNA-200 family [90]. Xie et al. reported
that miRNA-193a-WT1 interaction plays an important role in breast cancer metastasis
and indicates that restoring miRNA-193a expression is a therapeutic strategy in breast
cancer [91].
Ma and co-workers discovered that upregulation of miRNA-10b promotes invasion
and metastasis by indirectly activating the pro-metastatic gene RHOC through repressing
HOXD10 [25]. In addition, miRNA-373 regulates the CD44 gene, which promotes tumor
invasion and metastasis [44]. miRNA-9, a MYC/MYCN-induced miRNA, has been demon-
strated to directly target E-cadherin to promote breast cancer metastasis via activation of
β-catenin signaling, through increasing tumor invasiveness and angiogenesis [92].
Recently, several studies have reported that Dicer, an endonuclease that processes
miRNAs, is also associated with EMT and metastatic progression. Dicer inhibition promotes
metastasis, and restoring its expression suppresses metastasis. Certain miRNAs also target
Dicer for controlling metastasis. For example, miRNA-103/107 induced EMT by targeting
Dicer expression [93]. In addition, the miRNA-221/222 cluster has been shown to induce
EMT in breast cancer cells by targeting Dicer, and estrogen receptor 1 (ESR1) [94]. In
breast cancer, miRNA-107 acts as an endogenous suppressor of let-7, and it interacts with
mature let-7 to inhibit its function, which plays a major role in determining metastatic
progression [95]. According to Huang et al., both in vitro and in vivo cancer cell migration
and invasion were promoted by miRNA-373 and miRNA-520c [44]. Wu et al. reported
Biomedicines 2024, 12, 691 8 of 23

that in breast cancer cells, miRNA-29a regulates the critical roles of EMT and metastasis by
targeting SUV420H2 [96].
The overexpression of miRNA-17/92 is also involved in metastatic breast cancer [27].
miRNA-454-3p plays an important role in breast cancer’s early metastatic events, promotes
the stemness of breast cancer cells, and promotes early distant relapse in both in vitro and
in vivo conditions. The higher expression of miRNA-454-3p was found to be significantly
associated with both a poor prognosis and early recurrence in breast cancer through Wnt/β-
catenin signaling activation [97]. Higher expression of miRNA-373 was found in breast
cancer samples from tumors exhibiting lymph node metastasis [44]. Dobson et al. identified
a novel target of miRNA-30c, the nephroblastoma overexpressed gene (NOV), which is an
inhibitor of the invasiveness of metastatic TNBC (MDA-MB-231) cells [98]. The miRNA-34a
plays a key role in the proliferation, invasion, and metastasis of breast cancer cells [53].
Moreover, miRNA-373, miRNA-520c, miRNA-210, and miRNA-29b were also shown to
influence the invasion and migration of breast cancer [44]. In addition, oncogenic miRNA-
224 expression is significantly upregulated in highly invasive MDA-MB-231 cells and
correlated with increased metastasis [99]. Another important oncogenic miRNA, miRNA-
155, is frequently overexpressed in invasive breast cancer tissues and is directly targeted
to RhoA and contributes to breast cancer metastasis. Inhibition of miRNA-155 suppressed
TGF-β-induced EMT and tight junction dissolution, along with cell migration and invasion.
Further, the ectopic expression of miRNA-155 reduced RhoA protein and disrupted tight
junction formation [100]. miRNA-21 is an important oncogene and has a role in breast
cancer tumorigenesis. It regulates invasion and tumor metastasis by targeting multiple
tumor and metastasis suppressor genes. Also, inhibiting miRNA-21 reduced the invasion
and lung metastasis of MDA-MB-231 cells [30]. All these studies demonstrated that some
of the miRNAs have a metastatic role in breast cancer cells.

5. miRNA Expression in Breast Cancer Subtypes

Breast cancer comprises a diverse pathophysiology, with several molecular subtypes
exhibiting varying clinical features, therapeutic responses, and prognoses. The immuno-
histochemical expression of hormone receptors such as the estrogen receptor (ER), proges-
terone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is a widely
accepted and commonly used classification system for breast cancer [101–103]. There
are ER+ /PR+ , HER+ , or triple negative (ER− , PR− , HER2− , i.e., does not express these
three receptors) clinical definitions for breast tumors. Further, breast cancer can be sub-
typed including Luminal A (ER+ PRhigh HER2− Ki-67low ), Luminal B (ER+ PR+ HER2+
or HER2− Ki-67high ), HER2-enriched (ER− PR− HER2+ ), and triple negative or basal-like
(ER− PR− HER2− ) [104]. Dysregulation of miRNAs has been associated with breast cancer
development at various stages. In addition to these markers, miRNAs have emerged as
additional biomarkers that contribute to the molecular and functional diversity of breast
cancers [22,23]. miRNAs in breast cancer are classified based on their expression patterns,
functional roles, and clinical implications. Table 2 lists some miRNA biomarkers associated
with breast cancer molecular subtypes. This classification allows us to stratify breast cancers
according to their molecular subtype, which guides the diagnosis and treatment decisions
and predicts prognosis. As far as the treatment of breast cancer is concerned, two main
targeted therapies are commonly used: hormone therapy for hormone receptor-positive
tumors [105] and anti-HER2 therapy for HER2+ tumors [106]. It is anticipated that future
studies will identify miRNA biomarkers and their associations with specific subtypes of
cancer. In breast cancer, miRNA profiling can improve molecular subtyping accuracy and
guide better personalized treatment approaches.
Biomedicines 2024, 12, 691 9 of 23

Table 2. Characteristics of miRNAs in subtypes of breast cancer.

Subtype miRNA Signature Findings Analysis Type References

miRNA-99a/let-7c/ High expression in Luminal A Breast cancer tissues-cluster
Luminal-A [107]
miRNA-125b compared to Luminal B tissue analysis of small RNAseq
Plasma samples—analyzed
Luminal-A miRNA-221 High expression [108]
using qRT-PCR
miRNA-16, miRNA-21, Higher expression than healthy Serum circulating miRNAs
Luminal-A [109]
miRNA-155, miRNA-195 controls analyzed using qRT-PCR
miRNA-29a,
Reliably differentiate between Microarray analysis and
Luminal-A miRNA-181a and [110]
cancers and controls confirmed using qRT-PCR
miRNA-652
Serum samples and analyzed
Luminal-A miRNA-206 Higher expression [111]
using qRT-PCR
Early-stage breast
cancer specimens—
Luminal-B miRNA-342 Higher expression [112]
microarray and
qRT-PCR analysis
Breast cancer tissue: global focus
miRNA-182-5p and Significantly higher than in
Luminal-B miRNA PCR Panel and analyzed [113]
miRNA-200b-3p normal breast tissues
using qRT-PCR
miRNA-21,
Overexpression in Luminal B Tumor tissues of patients with
miRNA-221,
Luminal-B breast cancer without Luminal B breast cancer and [114]
miRNA-200a and
overexpression of HER2/neu analyzed using qRT-PCR
miRNA-196a
Upregulated in the analyses of FFPE blocks: miRNA microarray
Luminal-B miRNA-210 [115]
all 40 patients’ samples and confirmed using qRT-PCR
Higher expression in
Luminal-B/(HER2+ ) subtypes Breast cancer tissues and
Luminal-B miRNA-222 [116]
than in Luminal A and analyzed using qRT-PCR
TNBC subtypes
Significantly increased
HER2- expression in breast cancer FFPE breast cancer tissues,
miRNA-125b [117]
enriched tissues compared with that in luciferase activity and qRT-PCR
the non-cancerous tissues
High levels of circulating
Serum specimens: Solexa deep
HER2- miRNA-375 and miRNA-375 and low levels of
sequencing and confirmed [118]
enriched miRNA-122 miRNA-122 were associated
using qRT-PCR
with HER2 status
miRNA-548ar-5p,
Significantly differential
HER2- miRNA-584-3p, Serum samples: Multiplexed
expression in the [119]
enriched miRNA-615-3p, and gene expression analysis
HER2-enriched subtype
miRNA-1283
let-7f, let-7g, miRNA-107,
Primary breast cancer biopsies:
HER2- miRNA-10b, Inversely correlated with
microarray and confirmed [120]
enriched miRNA-126, miRNA-154 and HER2 overexpression
using qRT-PCR
miRNA-195
Highly expressed and accurately Early-stage breast cancer
HER2- miRNA-520d,
predicted HER2 status in specimens- [112]
enriched miRNA-376b
early-stage breast tumors microarray and qPCR analysis
Significantly higher expression
TNBC or Breast cancer tissue and
miRNA-210 compared to ER+ /HER2− [121]
basal-like analyzed using qRT-PCR
breast cancers
Biomedicines 2024, 12, 691 10 of 23

Table 2. Cont.

Subtype miRNA Signature Findings Analysis Type References

TNBC or FFPE tissues and confirmed
miRNA-214 Higher expression [122]
basal-like using qRT-PCR
TNBC or
miRNA-493 High expression Tissue microarrays [123]
basal-like
TNBC or
miRNA-135b High expression TaqMan low-density array [124]
basal-like
Higher expression compared to
TNBC or Luminal A and Luminal Biopsies of tumor tissue; and
miRNA-20a, miRNA-221 [125]
basal-like B/HER2− breast confirmed using qRT-PCR
cancer subtypes
Overexpressed in high grade FFPE- tissue: miRCURY LNA
TNBC or
miRNA-17 family and TNBC associated with microarray and confirmed [126]
basal-like
aggressive behavior using qRT-PCR
Elevated in TNBC but reduced
in ER-positive breast cancer and Breast cancer cell lines:
TNBC or associated with poor outcome. high-throughput mRNA
miRNA-17-92 [27]
basal-like The miRNA-17–92 expression sequencing and confirmed
enhanced cell growth and using qRT-PCR
invasion of TNBC cells

6. miRNAs as Potential Diagnostic Biomarkers for Breast Cancer

In recent years, investigating biomarkers for early detection has rapidly expanded
for better diagnoses and prognoses and the prediction of treatment responses in breast
cancer. Using significant features, including stability, tissue specificity, ease of detection,
and manipulation, will attract the clinician’s attention to achieve personalized breast
cancer treatment [127]. miRNA are emerging, attractive, and promising biomarkers owing
to their detectability and stability in the bloodstream. Usually, a significant increase
in miRNA level is observed in cancer compared to controls; therefore, diagnostic and
monitoring applications for miRNAs must be examined. Determining the expression ratios
of genes and miRNAs has been a useful technique to improve diagnostic potential. The
circulating miRNAs are widely known to be relatively stable, accessible, less invasive,
easy to test, promising, and stage-specific biomarkers for the non-invasive diagnosis of
breast cancer [128]. miRNAs are exceptionally stable in the bloodstream against the action
of endogenous RNases, suggesting that miRNAs may serve as an effective blood-based
biomarker for the detection and diagnosis of cancer [129]. miRNA expression has been
observed to be higher in tumors than in normal tissues, leading us to hypothesize that
global miRNA expression reflects the level of differentiation of cells. Tumor tissues release
miRNAs into the bloodstream and other body fluids. miRNA levels may reflect abnormal
conditions in breast cancer [130]. Therefore, miRNA status in the body fluids could be
a novel, specific, and very sensitive blood-based diagnostic tool for the early detection
of breast cancer. Cancer-specific miRNAs are shedding light on the molecular basis of
cancer by being able to identify cancer-specific expression of miRNAs in the blood, which
plays a vital role in the early-stage diagnosis of breast cancer. Currently, blood-based
biomarkers are widely used for the early diagnosis of breast cancer and in response to
treatment [130,131].
Using microarrays and other conventional methods, it is possible to detect differences
in miRNA expression patterns between normal and cancerous tissue. Studies have shown
that the profiles of miRNAs can be correlated with different types and grades of tumors,
and that these profiles may be different from those of the normal breast tissue and cells [132].
Furthermore, exosome-derived miRNAs may influence the evolution of tumors by serving
as messengers between tumor cells and non-tumor cells like immune and stromal cells.
Biomedicines 2024, 12, 691 11 of 23

This phenomenon was extensively covered in a recent review on the emerging role of
exosomes in breast cancer progression [133].
The detection of circulating miRNA can be a useful method for the non-invasive
detection of breast cancer biomarkers. Because circulating miRNAs are very stable in clinical
sources such as sputum, plasma, serum, urine, and saliva, they have been extensively
used to classify breast cancer subtypes more accurately than circulatory cell-free DNA or
RNA [134].
The miRNA-21 gene is an important regulator of breast carcinogenesis and is the most
sensitive (87.6%) and specific (87.3%) biomarker for breast cancer diagnoses at early stages
compared to other biomarkers like CEA and CA153 [135]. Canatan et al. reported that
miRNA-21, miRNA-125, and are reliable candidates for circulating miRNA biomarkers for
the detection of breast cancer [136]. On the other hand, the serum miRNA expression profile
analysis using highly sensitive microarrays revealed five miRNA signatures (miRNA-1246,
miRNA-1307-3p, miRNA-4634, miRNA-6861-5p, and miRNA-6876-5p) that can be used
to detect early-stage breast cancer [137]. miRNA-195 and let-7a had significantly higher
levels in the circulating blood of the breast cancer cohort than in the healthy controls.
However, circulating levels of miRNA-195 and let-7a decreased in cancer patients following
curative tumor resection [138]. Similarly, preoperative serum miRNA-20a and miRNA-21
expression levels were significantly higher in patients with breast cancer and benign disease
than in healthy women. Serum miRNA-214 levels, on the other hand, could distinguish
between benign and malignant tumors and healthy controls. In addition, in postoperative
serum samples, miRNA-214 levels significantly decreased as compared to the preoperative
sample [139].
Breast cancer patients with lymph node metastasis have high levels of miRNA-10b and
miRNA-373 circulating in their blood, and their expression is associated with promoting
the migration and invasion of breast cancer cells. Furthermore, such miRNAs may serve
as viable biomarkers for detecting lymph node metastases in individuals with breast
cancer [140]. Some studies revealed that groups of circulating miRNAs such as miRNA-299-
5p, miRNA-411, miRNA-215, and miRNA-452 were differentially expressed in metastatic
patients and increased the expression of miRNA-20a, miRNA-214, and miRNA-210 in
lymph node-positive patient subgroups [139,141,142].
A few studies revealed significant high expression levels of circulatory miRNA-10b,
miRNA-34a, miRNA-155, and miRNA-122 in breast cancer patients, which are associated
with primary metastatic breast cancer [143]. Similarly, there were significantly higher
expression levels of serum miRNA-21, miRNA-29a, miRNA-130b-5p, miRNA-145, miRNA-
151a-5p, miRNA-206, miRNA-222-3p, and miRNA-451 in the breast cancer group than in
the control group [144,145]. Cuk et al. also identified dysregulated miRNAs (miRNA-148b,
miRNA-376c, miRNA-409-3p, and miRNA-801) in the plasma of 127 sporadic breast cancer
patients and 80 healthy controls using RT-qPCR [146]. Other studies revealed some of the
candidate biomarkers in plasma, such as miRNA-16, miRNA-21, miRNA-451, miRNA-409-
3p, and miRNA-652, in the plasma of breast cancer patients [147]. But interestingly, these
miRNAs were downregulated after surgery.

7. miRNA-Based Therapeutic Approaches to Breast Cancer Treatment

In the field of medicine, miRNAs have gained increasing interest in recent years due
to their potential as therapeutic targets. Despite their promise, miRNA-based therapeutics
face significant challenges, including issues related to delivery, off-target effects, and the
need for rigorous clinical trials. miRNAs are currently being researched to unlock their full
therapeutic potential.
For integrated cancer care, miRNA-based gene therapy provides an appealing anti-
tumor approach. miRNA expression imbalance in cancer is related to tumorigenesis, so
miRNA-based therapies are based on restoring miRNA function or inhibiting overexpressed
miRNAs as reviewed earlier [148]. Recent experiments have shown that the phenotype in
 pathways can be restored by correcting miRNA changes with miRNA mimics or an-
tagomiRs [149].
There are two different strategies and challenges to the clinical application of miR-
NAs. 2024,
Biomedicines One
12, strategy
691 is designed to inhibit oncogenic miRNAs using miRNA antagonists, 12 of 23
such as locked-nucleic acids (LNA), or antagomiRs, and is aimed at gaining function. To
inhibit miRNA activity, small-molecule inhibitors specific to certain miRNAs are also be-
cancer cells can be reversed and the gene regulation network and signaling pathways can
ing developed. The second strategy,
be restored miRNA
by correcting miRNA replacement, includes
changes with miRNA mimicstheor reintroduction
antagomiRs [149]. of
tumor-suppressor miRNA mimics
There are twoto restore
different the loss
strategies and of function
challenges [149–152].
to the clinical application of miRNAs.
One strategy is designed to inhibit oncogenic miRNAs using miRNA antagonists, such
as locked-nucleic acids (LNA), or antagomiRs, and is aimed at gaining function. To
7.1. miRNA Inhibitioninhibit
TherapymiRNA activity, small-molecule inhibitors specific to certain miRNAs are also
being developed.
By repressing oncogenic The second
miRNAs, we strategy, miRNA
can inhibit replacement, includes
tumorigenesis, the reintroduction
which is a promis-of
tumor-suppressor miRNA mimics to restore the loss of function [149–152].
ing cancer treatment strategy. Oncogenic miRNAs are often overexpressed in breast can-
cer and need to be suppressed to restore
7.1. miRNA Inhibition Therapythe normal expression of their target tumor-
suppressor genes. In theByrecent repressing oncogenic
past, miRNAs,
multiple we can inhibit
strategies havetumorigenesis, which isto
been developed a promising
inhibit
cancer treatment strategy. Oncogenic miRNAs are often overexpressed in breast cancer and
oncogenic miRNAs [153,154].
need to be suppressed to restore the normal expression of their target tumor-suppressor
The decision to genes.
use miRNA-based
In the recent past,therapies is because
multiple strategies have miRNA expression
been developed inoncogenic
to inhibit tumor
cells is altered and the tumor
miRNAs phenotype can be altered with miRNA expression modula-
[153,154].
The decision to use miRNA-based therapies is because miRNA expression in tumor
tion. miRNA inhibition therapy consists of the following agents: antisense anti-miRNA
cells is altered and the tumor phenotype can be altered with miRNA expression modula-
oligonucleotides (AMOs),
tion. miRNA LNA, antagomiRs,
inhibition miRNA
therapy consists sponges,agents:
of the following andantisense
miRNA small-
anti-miRNA
molecule inhibitors (SMIRs) [155]. The fundamentals of these approaches are that en-
oligonucleotides (AMOs), LNA, antagomiRs, miRNA sponges, and miRNA small-molecule
inhibitors (SMIRs) [155]. The fundamentals of these approaches are that endogenous miR-
dogenous miRNAs are isolated in an unrecognizable configuration, resulting in the inac-
NAs are isolated in an unrecognizable configuration, resulting in the inactivation and
tivation and removalremoval
of mature
of maturemiRNAs fromthethe
miRNAs from RISCRISC [151,152].
[151,152]. Figure
Figure 2 shows the2various
shows the
miRNA
various miRNA inhibition
inhibitiontherapies discussed
therapies discussed here.
here.

Figure 2. Variety of miRNA

Figureinhibition therapies
2. Variety of miRNA in breast
inhibition cancer
therapies discussed
in breast in thisinreview.
cancer discussed this review.

7.1.1. Anti-miRNA Oligonucleotides (AMOs)

AMOs are single stranded chemically modified anti-sense oligonucleotides with a
length of 17 to 22 nt and complement the miRNA target of interest. The AMOs target
and bind specifically to miRNAs, preventing them from interacting with the mRNAs
they are targeting. Inhibiting the function of miRNAs can modulate gene expression and
Biomedicines 2024, 12, 691 13 of 23

7.1.1. Anti-miRNA Oligonucleotides (AMOs)

AMOs are single stranded chemically modified anti-sense oligonucleotides with a
length of 17 to 22 nt and complement the miRNA target of interest. The AMOs target
and bind specifically to miRNAs, preventing them from interacting with the mRNAs
they are targeting. Inhibiting the function of miRNAs can modulate gene expression
and alleviate dysregulated pathways associated with diseases. A significant method for
uncovering miRNA biology has been the antisense inhibition of miRNA action. This
antisense oligonucleotide was designed to bind to and inhibit the interaction of that miRNA
with its unique mRNA targets and the complementary mature miRNA, thus enabling
normal translation. Unmodified AMOs, by comparison, are unable to inhibit in vitro
miRNA function. Okumura et al. stated that modified AMOs targeting miRNA-21 (CL-
miR21) interstrand cross-linked duplexes (CLDs) suppressed breast cancer cell proliferation
with greater efficiency compared to other types of AMOs. Furthermore, the miRNA-
21-controlled expression of tumor-suppressor genes was probably upregulated [156]. In
the regulation of miRNAs, CLD-modified AMOs are substantially successful and could
be a promising strategy for treating breast cancer. Clinical trials showed some progress
for experimental AMO-based drugs directly targeting miRNA-122 in chronic hepatitis C
patients (Santaris Pharma-developed Miravirsen and Regulus® -developed RG-101) and
might be worth considering for therapy of breast cancer [157].

7.1.2. Locked Nucleic Acid (LNA)

LNA is a modified nucleotide with a methylene bridge connecting the 2′ -oxygen and
4′ -carbonof the ribose sugar ring. As a result, the oligonucleotide becomes more stable and
binds better to complementary RNA sequences. LNA-modified AMOs specifically target
and inhibit miRNAs. LNA is an example of a modified AMO that binds to miRNA, can
modulate miRNA as antisense-based gene silencing, and has recently emerged as a possible
miRNA-targeting therapeutic alternative [158]. These are very similar to RNAs in that the
methylene bridge in the ribose ring binds the 2′ -oxygen atom and the 4′ -carbon atom (2′ -O
4′ -C methylene). This bridge forms a bi-cyclic structure that locks the conformation of
the ribose and is integral to its complementary RNA sequences due to the high stability
and low toxicity of the biological systems and the affinity of the LNA. In the regulation
of RNAs, LNA oligonucleotides are stable, have greater aqueous solubility, and may not
induce an immune response, indicating their utility in gene therapy for antisense-based
gene silencing [28,158,159]. An in vivo study demonstrated that LNA-modified antisense
oligonucleotides were capable of silencing upregulated miRNA-205-5p in breast cancer,
leading to a significant reduction in tumor growth and metastatic spread in mouse models,
thus suggesting the possible future use of this approach in therapy [160].

7.1.3. AntagomiRs
AntagomiRs, also known as anti-miRs, are another class of synthetic oligonucleotides
that inhibit the activity of miRNAs, like AMOs. Chemically modified antagomiRs com-
plement the targeted miRNAs with cholesterol-conjugated single-stranded 23-nt RNA
molecules. Their backbone consists of single-stranded oligoribonucleotides with 2′ -O-
methyl (2′ -O-Me) and partially modified phosphorothioate linkers [161]. The modifications
were made to improve the RNA’s stability and protect it from degradation. The strategy of
antagomiRs seems promising for suppressing miRNAs in therapeutic strategies.
The use of antisense miRNAs (antagomiRs) to knockdown miRNAs is one of the most
common approaches. Ma et al. demonstrated that systemic treatment of tumor-bearing
mice with miRNA-10b antagomiRs suppressed the metastasis of breast cancer, both in vitro
and in vivo. This achieved significant reduction in the levels of miRNA-10b and increased
levels of a functionally important miRNA-10b target, Hoxd10 [25]. In another study, the
effect of miRNA-10b antagomiR in a 4T1 mouse model of mammary tumor metastasis was
analyzed. AntagomiR-10b systemic delivery had a potent and highly specific metastasis-
suppressing effect on these malignant breast cancer cells without affecting their ability to
Biomedicines 2024, 12, 691 14 of 23

develop as primary tumors; in fact, antagomiR-10b prevented the spread of cancer cells
from the primary tumor but did not influence the late stages of the metastatic phase after
tumor cells had already disseminated [92]. AntagomiR-21 can affect breast cancer cells by
inducing apoptosis and reducing cell proliferation [29].

7.1.4. miRNA Sponges

A miRNA sponge is an artificial RNA molecule which binds to miRNAs and prevents
them from interacting with their target mRNAs. “miRNA sponges” or “miRNA decoys”
offer many synthetic miRNA binding sites which are compete with natural miRNA targets
for miRNA binding [162]. The vectors of expression constitute the source of the miRNA
sponge transcription, thus reducing the effects of miRNA and increasing the expression
of the native targets of miRNA [162]. Because these compounds strongly inhibit target
miRNAs and have a high specificity for miRNA sponges, they have been used more
frequently in miRNA loss-of-function studies [163]. In one study, a sponge plasmid against
miRNA-10b was transiently transfected into MDA-MB-231 and MCF-7, high and low
metastatic cell lines. This resulted in the miRNA-10b sponge effectively inhibiting the
growth and proliferation of these cell lines [164]. Furthermore, miRNA-21 sponges have
been effectively applied to the MDA-MB-231 and MCF-7 breast cancer cell lines [165], while
miRNA-9 sponges have been applied to the 4T1 metastatic breast cancer cell line, resulting
in a nearly 50% reduction in metastatic activity [92]. Clinically, miRNA sponges and decoys
have been developed for more stable suppression and targeted delivery of miRNAs.

7.1.5. miRNA Small-Molecule Inhibitors (SMIRs)

A small-molecule inhibitor of miRNA is a compound designed to modulate the ac-
tivity of miRNAs. This differs from traditional oligonucleotide-based approaches, which
include AMOS and miRNA sponges [166,167]. Due to the decreased time of development,
acceptance, and cost, inhibition therapy using SMIRs is an encouraging one. Instead of
inhibiting target recognition from miRNA-21, the small molecule’s mode of action is pri-
marily through the transcriptional regulation of miRNA-21. The small molecule enoxacin,
an antibacterial fluoroquinolone, has been documented to bind to the miRNA biosynthesis
protein TRBP and to increase tumor-suppressor miRNA output [168]. Enoxacin was also
shown to inhibit MDA-MB-231 and MCF-7 cell growth [168]. Linifanib, a multi-tyrosine
kinase inhibitor, could substantially inhibit miRNA-10b and reverse its oncogenic function
in both in vitro and in vivo breast and liver cancers [169]. A novel strategy for restoring
dysregulated miRNAs in cancer is thus defined using small-molecule modulators of miR-
NAs [153]. Overall, targeting miRNAs for cancer care with SMIRs is an evidence-based
approach with high potential and ability for success.

7.2. miRNA Replacement Therapy

Synthetic miRNA mimics are introduced into cells to restore or enhance the function
of a specific miRNA that is deficient or downregulated in breast cancer. By mimicking
the function of endogenous miRNAs, this therapeutic approach regulates gene expression
and cellular pathways. Synthetic miRNA mimics must be delivered efficiently for target
cells for miRNA replacement therapy to be effective. There are a variety of delivery
systems that can be used to ensure effective uptake by target tissues, including viral vectors,
lipid nanoparticles, and other nanocarriers [150–152,170]. A synthetic miRNA mimic is
incorporated into the RISC and acts similarly to endogenous miRNAs once inside a cell.
Once they bind to target mRNAs, they inhibit the translation or induce the degradation of
mRNA, thus regulating gene expression (Figure 3). As a potential treatment strategy for
breast cancer, miRNA replacement therapy is being actively explored.
 gration, and invasion in vitro in highly metastatic breast cancer cells [52]. MRX34 was in-
volved in the first phase I clinical trial for miRNA replacement therapy and was intend-
ed to restore miRNA-34 expression in patients with different solid tumors including
Biomedicines 2024, 12, 691 TNBC [173]. The use of miRNA-mimetic agents in patients is a promising15new of 23 way of
treating clinical breast cancer.

Figure 3.3.Potential
Figure Potentialapproaches
approachesto miRNA replacement
to miRNA therapy
replacement for breast
therapy cancer using
for breast cancera variety
using aofvariety of
miRNA delivery
miRNA deliverysystems.
systems.

The effects of these therapies may include the suppression of cancer cell growth, modu-
8. Conclusions and Future Directions
lation of immune responses, and restoration of tissue homeostasis. Tumor cell proliferation
can beTo conclude,
inhibited, being able
or apoptosis can to
be understand
induced usinghow miRNA differentially regulated
restoration therapy miRNAs influ-
by restoring
ence breast
exogenous cancer progression
tumor-suppressor miRNAsprovides
that arevaluable insightininto
downregulated tumortheir
cellspotential diagnostic
[153]. Differ-
ent studies
and have shown
therapeutic applications. of in vitro and
the efficacyUnraveling thein vivo miRNA
intricate webrestoration
of miRNAtherapies. For offers
functions
example, Liang
valuable et al.into
insights introduced miRNA
their utility asreplacement in radioresistant
both diagnostic tools andbreast cancer cells.
therapeutic targets for
For the first time, they showed that miRNA-302a’s enforced expression
breast cancer. By identifying stage- and subtype-specific miRNA signatures, effectively sensitizes early-
radioresistant tumor cells to irradiation by directly downregulating both AKT1 and RAD52
detection strategies can be improved and personalized therapeutic interventions can be
expression [171]. These results suggested that decreased miRNA-302 expression confers
implemented. Moreover, it is important to determine which miRNA or group of miR-
radioresistance, and the miRNA-302 baseline expression restoration sensitizes breast cancer
NAs
cells toisradiotherapy.
most dysregulated in breast cancer, specifically, at different stages of the disease
SuchWhen signatures
miRNAcould
mimicsserve
were as robust
used biomarkers,
to force the expressionfacilitating moreand
of miRNA-365 accurate and timely
miRNA-22,
breast cancer cell proliferation was inhibited and sensitivity to paclitaxel and fluorouracil
was boosted, respectively. By targeting GALNT4, miRNA-365 works on overcoming
chemoresistance [172]. In comparison, Jiang et al. found that in breast cancer cells and
tissues, miRNA-148a was downregulated, and its overexpression mimics the reduced
migration and invasion of breast cancer cells [58]. Also, in breast cancer cells, miRNA-
33b expression was downregulated, and it had a negative correlation with the lymph
node metastatic status of breast cancer patients. Ectopic overexpression of miRNA-33b
inhibited lung metastasis in vivo and compromised stem cell characteristics, migration,
and invasion in vitro in highly metastatic breast cancer cells [52]. MRX34 was involved
in the first phase I clinical trial for miRNA replacement therapy and was intended to
restore miRNA-34 expression in patients with different solid tumors including TNBC [173].
The use of miRNA-mimetic agents in patients is a promising new way of treating clinical
breast cancer.
Biomedicines 2024, 12, 691 16 of 23

8. Conclusions and Future Directions

To conclude, being able to understand how differentially regulated miRNAs influence
breast cancer progression provides valuable insight into their potential diagnostic and
therapeutic applications. Unraveling the intricate web of miRNA functions offers valuable
insights into their utility as both diagnostic tools and therapeutic targets for breast cancer.
By identifying stage- and subtype-specific miRNA signatures, early-detection strategies can
be improved and personalized therapeutic interventions can be implemented. Moreover,
it is important to determine which miRNA or group of miRNAs is most dysregulated
in breast cancer, specifically, at different stages of the disease. Such signatures could
serve as robust biomarkers, facilitating more accurate and timely diagnoses, which is
crucial for improving patient outcomes. This will assist in identifying and prioritizing the
most promising treatment targets, with an emphasis on developing early-detection and
-treatment strategies for breast cancer. Better understanding of miRNA-guided networks is
essential for improving breast cancer diagnoses and treatment. Ongoing research in this
field holds the promise of translating miRNA-based discoveries into clinically relevant
applications, fostering advancements in breast cancer management.

Author Contributions: Conceptualization, K.S. and R.S.; investigation, K.S. and R.S.; resources, K.S.;
writing—original draft preparation, K.S.; writing—review and editing, K.S. and R.S.; visualization,
K.S.; supervision, R.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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