Cellular and Exosomal MicroRNAs: Emerging Clinical Relevance as Targets for Breast Cancer Diagnosis and Prognosis

Breast cancer accounts for the highest cancer cases globally, with 12% of occurrences progressing to metastatic breast cancer with a low survival rate and limited effective early intervention strategies augmented by late diagnosis. Moreover, a low concentration of prognostic and predictive markers hinders disease monitoring. Circulating and exosomal microRNAs (miRNAs) have recently shown a considerable interplay in breast cancer, standing out as effective diagnostic and prognostic markers. The primary functions are as gene regulatory agents at the genetic and epigenetic levels. An array of dysregulated miRNAs stimulates cancer‐promoting mechanisms, activating oncogenes and controlling tumor‐suppressing genes and mechanisms. Exosomes are vastly studied extracellular vesicles, carrying, and transporting cargo, including noncoding RNAs with premier roles in oncogenesis. Translocation of miRNAs from the circulation to exosomes, with RNA‐binding proteins in stress‐induced conditions, has shown significant cooperation in function to promote breast cancer. This review examines cellular and exosomal miRNA biogenesis and loading, the clinical implications of their dysregulation, their function in diagnosis, prognosis, and prediction of breast cancer, and in regulating cancer signaling pathways. The influence of cellular and exosomal miRNAs presents clinical significance on breast cancer diagnosis, subtyping, staging, prediction, and disease monitoring during treatment, hence a potent marker for breast cancer.


Introduction
Breast cancer is a complex disease that accounts for 13% [1] of all cancers globally.It is the most common type of cancer in women, according to the World Health Organization (WHO) 2020.The disparity of breast cancer pathology offers unprecedented challenges in disease mechanisms, phenotypically, immunologically, and pathologically.Diagnostic, prognostic, and predictive phases of the disease require biomarkers to determine an individual's clinical outcomes during and after therapy.Several biomarkers have been recognized for breast cancer detection and diagnosis, while its heterogeneity [2] presents multiple challenges in monitoring disease progression, treatment outcomes, development of metastasis, and drug resistance.Therefore, early breast cancer diagnosis and management are paramount before disease progression.This has proved to increase the survival rate by 40%-60%, [3] with better prognosis and clinical outcomes.Innovative strategies for biomarker identification to categorizing breast cancer from initial stages to disease monitoring during therapy, progression, disease recurrence, disease status, breast cancer subtype, and quality of survival of patients are dire.Therefore, specific, and highly sensitive biomarker detection is key to early breast cancer diagnosis.
Presently, diagnosis targets have centered on solid tumors [4] and genetic testing to detect the Breast Cancer gene (BRCA) mutation (BRCA1 and 2) for the genetic type of breast cancer [5] using DNA analysis methods such as enzymatic detection of mutation, next-generation sequencing, and fluorescent-based electrophoretic assays.Conversely, BRCA gene genetic testing has invasive modes of sample collection, a prolonged period of sample preparation and detection, and limited use to monitor the disease from onset to metastasis.Thus, the use of liquid biopsy to detect molecular and circulating biomarkers, including circulating tumor DNA, circulating tumor cells, circulating miRNA, [6] oncogenic proteins such as metallomembraneous proteins (MMP), Ki-67, human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor (PR), absence of the three receptors, HER2, ER, PR triple-negative breast cancer (TNBC), and epithelial cell adhesion molecule (EpCAM) [7] provide potential as diagnosis targets.The clinical role of most of these biomarkers has been used for predicting breast cancer disease status, monitoring drug responses, and disease recurrence statuses.However, the variability of breast cancer has hindered targeting these markers in treatment.Therefore, multitargeted diagnosis and therapy can suppress tumorigenesis while activating tumor inhibitory factors.
Remarkably, the recent discovery of extracellular vesicles, such as ectosomes and exosomes, has presented diverse opportunities for cancer research.Exosomes are natural nanoparticles ≈30-150 nm in size, [8,9] described in 1983 by Stahl and colleagues.They participate in the internalization of elements secreted by cells, cellular communication, and cargo transportation [10,12] Endowed by various loads, including 9769 proteins, 3408 mR-NAs, 2838 miRNAs, and 1116 lipids, [9,13,14] according to ExoCarta Database, [15] they are effective biomarkers for diagnosis, treatment targets, and drug delivery particles for cancer. [16]With the proteins on the surface, nucleic acids, and other transport proteins found intracellularly, [8,17] ; they exhibit multiplexity properties for detecting and targeting diseases, an integral approach to disease diagnosis and treatment.Moreover, exosomes derived from immune cells have the potential to deliver immune suppression markers, such as immune checkpoint inhibitors and miRNAs. [18]ecently, targeting exosomal surface markers has succeeded as diagnostic and predictive markers, [19,20] as therapeutic targets, [21] for breast cancer, and as engineering platforms for drug delivery. [22]This exploration of exosomal components presents novelty on the function of the internalized miRNA in regulating gene expression [23][24][25] and controlling signaling pathways associated with cancer, promoting cellular proliferative and differentiation properties of cancer cells, and promoting angiogenesis and tumorigenesis. [21,23]miRNAs are small non-coding RNA molecules generally dysregulated with post-transcriptional functions, [21,26,28] whose significance controls breast cancer outcomes in disease and treatment [29,31] Exosomes containing miR-NAs have been identified as stable and minimally invasive biomarkers for several human diseases. [32]Hence, they have been targeted to understand their role in tumorigenesis in several diseases, including prostate cancer, [16] non-small lung cancer, cervical cancer, [33] and breast cancer, [34,35] Therefore, exosomal miRNAs are potential target markers for diagnosis and disease prognosis.They are associated with the advantages of dysregulation on different phases of breast cancer development, defining phenotypic features of breast cancer and regulating cellular mechanisms of proliferation, differentiation and metastatic development, and drug resistance. [36]For example, upregulation of miR-21 is associated with early breast cancer development, while others have demonstrated that, it suppresses angiogenesis and metastasis. [37]Therefore, evaluating exosomal-derived miRNA will reveal both the functions of exosomes and the expression profile of exo-miRNAs that contribute to breast cancer progression.
In this review, we comprehensively explain the emerging clinical significance the breast cancer-derived cellular and exosomal miRNAs and their profiles, the biogenesis of miRNA, and its packaging into exosomes by RNA-binding proteins, including cross-talk of these miRNAs with signaling pathways that contribute to breast cancer progression.We provide literature-based clinical evidence of aberrant expressions of cellular and exosomal miRNA profiles as predictive, prognostic, predictive markers, and therapeutic targets for breast cancer.

Exosome Structure and Its Cargo
Exosomes are natural [9] circulating nano-vesicles that participate in physio-pathological and non-pathological processes to transfer cargo [38] from cells of origin to recipient cells and organs.Morphologically, exosomes are round or cup-shaped with a lipid bilayer and are lumen-containing. [13]Exosomes participate in cellular communications and transport elements from cell to cell.These cargoes include proteins, lipids, and nucleic acids (RNAs-mRNA, miRNA, mtRNA, lncRNAs), [9,12,13] as illustrated in Figure 1.Exosomes, exhibit properties of stability, [14,39,40] and non-immunogenicity, [11,13] biocompatibility, and low toxicity, [11,41] compared to other nanoparticles such as liposomes, polymeric, and inorganic nanoparticles.Advantageously, exosomes mirror the properties of the parent cells. [11]ence, they are used as diagnostic markers and particles to deliver therapeutic payloads.For example, exosomes derived from cancer cells or tumor microenvironments carry oncogenic proteins, aberrated tumor regulatory miRNAs, and mutated mRNA transcripts that promote tumorigenesis, angiogenesis, and metastasis. [42]This is aided by the exosomal size and biocompatibility, enabling evasion from cellular phagocytosis, invagination into cell membranes, and extravasation through blood vessels. [43]Therefore, exosomes can spread metastatic proteins, penetrate vast microenvironments, including the blood-brain barrier, [44,45] and disseminate cancer to distant organs.Therefore, profiling of exosomal release cargo will endow disease-promoting functions of EVs in altered cellular responses [46] and microenvironments, further presenting them as diagnostic markers for complex diseases such as cancer.

Biogenesis of miRNAs
miRNAs are conserved, non-coding RNAs of ≈21-25 nucleotides [47][48][49][50] encoded by nucleic DNA.miRNA biogenesis has two pathways, the conical and non-conical pathways.Both are significant, and they all produce single-stranded mature miRNA.Briefly, miRNA biogenesis is a post-transcriptional mechanism that originates from the intronic region of messenger RNA.Synthesis is initiated in the nucleus [51] as long primary miRNA (pri-miRNA) transcripts by RNA polymerase II.Pri-miRNA is ≈100 kilobases long with an internal stem-loop structure containing a double-stranded stem region and an apical loop. [52]Pri-miRNAs are then modified and cut by a couple of RNase III (Drosha [52][53][54] and RNA-binding protein DiGeorge Syndrome Critical region-8 [DGCR8]), [28] respectively.Drosha processes the pri-miRNA into a ≈70 nucleotide hairpin looped structure with a lagging end, called the precursor miRNA (pre-miRNA), which is then exported from the nucleus by exportin five (XPO5) complex and Ras-related nuclear protein-5′-triphosphatase (Ras-GTPase), [14,28] Ras proteins are GTPases, oncogenic switching proteins, [55] which control cell proliferative and differentiation signaling pathways.
The pre-miRNA in the cytoplasm is then modified by an RNAse III endonuclease, dicer, [28] which cuts the pre-miRNA into short duplex RNAs and loads an RNA-induced silencing complex (RISC).The RISC is a multiplex protein, [28] with Argonauts 2 (Ago 2), which acts on the short duplex of miRNA to remove one of the strands.The strand removal is directional, such that the strand removed from the '5′ becomes a miRNA-5p, and if from the '3′, becomes a miR-3′OH.The strand attaches to the Ago-2 protein, becoming the leading or guide strand while the other, the passenger strand, is degraded.The leading or passenger strand selection depends on the purine: pyrimidine ratio.Ultimately, the leading strand and the Ago-2 complex act on the '3′ untranslated regions ('3′ UTR) of mRNA, inhibiting the stability of the mRNA, mRNA decay, the sequential prevention of protein synthesis, protein degradation, falling off-of ribosomes, [56,57] and degradation from the cell, [28] as shown in Figure 2a.Evidence shows that miRNA can interact with other regions, such as the '5′ UTR, coding sequences, and gene promoters, regulating transcription or activating translation mechanisms. [58]These interactions and potential outcomes are prompted by the subcellular location of miRNAs, miRNA abundance, target region on the UTR, and the ability of the miRNA to interact with mRNA. [58] has also been revealed that miRNA biogenesis can be cellindependent.Pre-miRNAs can be synthesized to mature miRNA within the exosomes and microvesicles. [59]Melo et al., 2014 revealed that miRNAs can mature from pre-miRNAs, Dicer, and Ago2, present in cancer cell-derived exosomes.Moreover, silencing of Exportin (XPO5) inhibited the translocation of miR-NAs from the exosomes into the cells, [59] supporting the cellindependent synthesis of miRNAs in exosomes.This implied that miRNAs can be loaded into exosomes through stressinduced mechanisms or/and processing in the exosomes by the presence of miRNA biogenesis components.

miRNAs are Sorted and Loaded into Exosomes with RNA-Binding Proteins
The sorting and shuttling of miRNAs into exosomes is an important aspect of understanding vesicular roles in cancer, but it is highly unclear.miRNAs are associated with a random response to physiological and pathological stimuli, [60,61] such as stress-induced conditions.For example, Lee et al., 2016, showed incorporation of altered miR-221 and miR-320a by cooperative functioning of the lung epithelial cells and macrophages during acute lung injury in hyperoxia-induced oxidative stress. [62]hey also established macrophage infiltration-induced inflammation of epithelial lung cells, due to acid exposure, predisposed the accumulation of elevated miR-17 and miR-221 containing micro-vesicles. [63]Conclusively, stress-induced microenvironments trigger miRNA translocation and packaging into extracellular vesicles. [63]This generates exosomal miRNA-regulated cascades in subsequent cellular interactions, influenced by the physiological and pathological state of the cells, [60] and functions, as schematically illustrated in Figure 2b.

Heterogenous Ribonucleoproteins A2B1 (hnRNPA2B1) and Caveolin-1
Heterogeneous ribonucleoproteins-A2B1 (hnRNPA2B1) recognize and cleave the sequence motifs, AGG/UAG [69] of RNA, controlling the localization and loading of the miRNA into exosomes. [64]hnRNPA2B1 undergoes SUMOylating, a posttranscriptional modification process with small ubiquitin-like proteins known as SUMOs, [70] which control the sorting of miRNA into exosomes by binding to the sequence motifs AGG/UAG of miRNA. [64]Evidence indicated that hnRNPA2B1 binds strongly to miR-19, miR-93, [69] and miR-198 [64] facilitating their loading and localization in exosomes.Moreover, hn-RNPA2B1 actively regulates the loading of various miRNAs into exosomes when it binds to caveolin-1, [68] a membranous protein with tumorigenic properties [71] Caveolin-1 is associated with endocytosis, distribution of lipid and cholesterol in the caveolin lipid rafts, [71,72] cell migration, proliferation, cellular signaling, and alteration of metabolic pathways that promote malignancy via anchorage-independent growth, [73,74] and promoting cancer drug resistance. [75]Induced oxidative stress on the macrophages, incorporated hnRNPA2B1 to bind to caveolin-1 facilitating miRNA translocation. [68]ndependently, caveolin-1 thrives in metabolically stressful conditions such as hypoxia, in cancer microenvironments. [75]t can also facilitate the loading of miRNA into EVs by guiding the RNA-induced silencing complexes (RISC) impacting the loading of other miRNAs dependent on the pathological properties, the status of the disease, and the administered therapies. [76]or example, during oxidative stress, Lee et al., 2019 confirmed that a CAV-1 tyrosine 14 (Y14) domain undergoes phosphorylation, inducing an interaction of CAV-1 and hnRNPA2B1. [68]This caused an O-GlycNAcylation and released hnRNPA2B1, with altered binding to miRNAs, leading to the CAV-1/hnRNPA2B1 complex-specific packaging and secreting of miR-17 and miR-93.Therefore, hnRNPA2B1 and caveolin-1 can independently or combined, shuttle miRNA into extracellular vesicles, such as exosomes.

Argonaute-2 (Ago-2)
Argonaute-2 (Ago-2), is a type of RNA induced silencing complex (RISC) [48] that binds to the guide strand of mature miRNA.This causes dissociation of the lagging strand, and degradation of mRNA (messenger RNA) and falling off of ribosomes. [28]Therefore, Ago-2 promotes miRNA stability during biogenesis and in circulation, as Ago-2-miRNA (Ago-2-miRNA) complexes. [77]cKenzie et al., 2016 discovered that activated Mitogen-activated protein kinase (MEK) reverse mutations in the KRAS cascade, indicating the active secretion of Ago-2 and sorting into exosomes. [65]Essentially, the KRAS-MEK pathway is regulated by kinases and RAS proteins, and mutations in components of the cascade lead to malignant transformation, abnormal cellular growth, and drug resistance in various cancer diseases such as breast cancer and prostate cancer. [78]Moreover, the regulation of the Ago-2 by localization and phosphorylation by KRAS-MEK was confirmed to control the sorting of let-7a, miR-320a, miR-100, [65,79] and miR-10b [79] into exosomes.

Y-Box 1 Proteins
Y-box 1 proteins are RNA-binding proteins and oncogenic proteins that provoke chromosomal instability. [80]They convert human mammary epithelial cells into breast cancer cells [55] promoting cell growth and proliferation, mediating drug resistance in HER2+, and potential prognostic markers in breast cancer. [80]-box binding proteins have been implicated in selectively sorting miRNAs.Shurtleff et al., 2016 identified an array of miRNAs packaged into exosomes of HEK293T cells using an in vitro packaging assay finding a predominant interaction between Y-box 1 protein and miR-223, that sorted and packed it into exosomes, [66] confirming Y-box 1 protein role in loading miRNAs into exosomes.

Cellular and Exosomal miRNA in Breast Cancer
miRNAs regulate and promote tumorigenesis by altering the behavior of recipient tumor and stromal cells. [21]It is known that several miRNAs can collaboratively regulate a single oncogene, and a single type of cancer can be associated with the dysregulation of multiple miRNAs. [16]Therefore, a myriad of regulatory mechanisms in gene expression is promoted by miRNAs, [84] defining breast cancer staging, subtype, clinical outcomes, and therapy responses.miRNAs, as promising candidates for breast cancer diagnosis, have cancer-specific expression arrays [85] depending on their dysregulation of cancer signaling pathways.They perform biological functions, including regulating gene expression of mRNA [86] by targeting the untranslated regions (3′-UTR) of the mRNA, cell proliferation and differentiation, cell reprogramming, tumorigenesis, apoptosis, metastasis, and immune responses in cancer, [14,29,87] This has contributed to identifying miRNAs as potential biomarkers for cancer detection. [88]or example, altered expression of the miR-200 family promotes breast cancer becoming primary miRNAs to explain metastasis, [89,90] and drug resistance. [90]

Cellular and Exosomal miRNAs in Breast Cancer Diagnosis and Prediction
Research has also established multiplexity in the detection of an array of miRNAs as diagnostic and prognostic biomarkers providing cognizant health status of the patients, especially in surrogate and clinical outcomes.42a) in breast cancer tumors with a significant downregulation in aggressive molecular subtypes, ER+, HER2+, and TNBC. [92]his inferred breast cancer onset on upregulated miR-200 family expression while downregulation was indicative of aggressive breast cancer (Luminal A, HER2+, and TNBC).
Similarly, using tumor-derived EVs, Kim et al., 2021 simultaneously targeted a multi-miRNA panel of miR-9, miR-16, miR-21, and miR-429, as shown in Figure 3A.Using different combinations of these miRNA panels, they predicted the subtype of breast cancer, such as luminal A, luminal B, HER2+, and TNBC at a sensitivity of 0.90, 0.86, 0.88, and 0.84, respectively. [93]Determining individual expression and selected combination levels, each miRNA was associated with a specific breast cancer tumor subtype.A summary of miRNAs with potential in diagnostic and prognostic applications is summarized in Table 1, and examples of expressions are in Figure 3.

Cellular and Exosomal miRNAs Predict Breast Cancer Progress and Staging
Significantly, the downregulation of several miRNAs indicates disease metastasis to distant organs.A study conducted by Jurkovicova et al., 2017 indicated the downregulation of oncomirs miR-17, miR-18a, miR-19a, and miR-20a, miR-21, miR-27a, and miR-155 and predicted advanced stages of breast cancer to lymph node metastasis, [94] as shown in Figure 3B.A decrease in miR-17 and miR-19a from early to advanced stages of breast cancer clinically procured the potential for their use in diagnosis and disease monitoring.This demonstrated that the downregulation of these miRNAs contributed to breast cancer staging and disease prognosis, [94] therefore, their expression profiles are relevant in  2+) and TNBC (ER-, PR-, and HER-2-).Reproduced with permission. [93]Copyright 2021, Wiley Publishers.B) Comparative individual expression level of miRNAs between tumor grade (G) and lymph node metastasis (LNM) in invasive breast cancer patients.Reproduced with permission. [94]Copyright 2017, Impact Journals.C) Volcano plot showing the differential expression levels of 45 exosomal miRNAs in 435 breast cancer samples of neoadjuvant therapy in the GeparSixto Trial, 211 HER2+ and 224 TNBC patients, all against 20 healthy women and a comparison between TNBC and HER2 positive BC.The green dots indicate downregulated miRNAs, and the red dots indicate upregulated miRNAs, while the grey dots show the threshold value corresponding to the p-value of 0.05.Reproduced with permission. [95]Copyright 2018, BioMed Central.multiplex detection for diagnostic and prognostic applications in breast cancer.Likewise, inducing overexpression of these miR-NAs as a treatment strategy for breast cancer [94] presents a potential area of investigation to evaluate gene expression patterns.Moreover, sensitive methods for expression miRNA quantitation during therapy can be applied to monitor breast cancer during treatment.

Cellular and Exosomal miRNA Function and Relevance in Breast Cancer Therapy
Consequently, specific signature miRNAs present their potential role in predicting patient outcomes during therapy.For example, a study on the expression of exosomal miRNA in the circulation of TNBC and HER2+ patients in the GeparSixto clinical trial [96] detected significant expression patterns in both subtypes.They revealed the risk factors, clinicopathologic, and complete predictive response to therapy, indicating that, during the neoadjuvant treatment administration with paclitaxel, non-pegylated liposomal doxorubicin, and carboplatin, HER2+ patients secreted more exosomes than the TNBC with significant upregulation of miR-27a/b, miR-365, miR-16, miR-328, and miR-660 in HER2+.Seemingly, miR-335, miR-376c, miR-382, and miR-433 were upregulated in TNBC, [95] as shown in Figure 3C.For example, miR-27a/b has been shown to possess oncogenic properties of proliferation, migration, and proliferation of breast cancer cells by targeting the SFRP1 (secreted Frizzled-related protein-1) gene in the Wnt pathway, [97] epithelial-mesenchymal transition, [98] and dysregulation of epithelial growth factors and tumor necrosis factoralpha by HER2/Neu [99] respectively.
Remarkably, some miRNAs possess dual roles in promoting oncogenesis.For instance, miR-335 possesses pro-oncogenicity and anti-oncogenicity functionalities in breast cancer.Hynes et al., 2014 demonstrated a dysregulated miR-335 expression upregulates BRCA1 mRNA activators, ER-, insulin-like growth factor 1 receptor (IGF1R), specific protein 1(SP1), and inhibitor of differentiation 4 (ID4).Contrary, miR-335 overexpression led to a tumor suppressive mechanism of reduced cell viability and increased cell apoptosis. [100]Hajibabaei et al., 2023 established that aberrated hypermethylation of miR-335 and miR-145 downregulated their expression and overexpressed, the immune checkpoint PD-L1, and induced apoptosis, arrested cell death, and reduced proliferation. [101]This showed the tumor suppressor function of miR-335 and miR-145 in breast cancer.Martin et al., 2017, revealed the synergistic mechanism of miR-335-5p and miR-335-3p inhibiting the expression of estrogen receptor-alpha (ER-) while promoting tamoxifen resistance hence posing both oncogenic properties and drug resistance capabilities, despite its functional role as a tumor suppressor. [30]Kodahl et al., 2014 profiled the miRNA expression in health against ER+ individuals, proving that miR-365 was downregulated in ER+ breast cancer compared to healthy individuals.However, they also demonstrated that it possesses oncomir properties due to its overexpression in ER-, HER2+ breast cancer. [29]Zhao et al., 2020 indicated that miR-375 suppressed breast cancer cell stemness by targeting and inhibiting (Janus kinase/Signal transducer and activator of transcription 3) JAK2/STAT3.They also showed that miR-375 reduced Adriamycin resistance. [102]This justified Ward et al., 2013 who showed miR-375 re-expression reversed tamoxifen resistance while reversing the EMT. [103]However, Moorthy et al., 2023 recently showed that miR-375 targeted and inhibited the transcriptional factor, homeobox protein A5 (HOXA5), while promoting cell proliferation, invasion, and cell migration. [104]This indicated the duality of miR-375 as a tumor suppressor miRNA and an oncomir of breast cancer.All these studies reveal duality in functions of miRNAs as oncomirs and as tumor suppressors hence more accurate studies are needed to understand pathological functions in breast cancer disease, to deliver discernable therapy targets.
Conversely, metastasis is the most common cause of death in breast cancer, [105] coupled with drug resistance after metastatic spread.There is a deficiency in clinically applicable prognostic markers to detect and monitor metastatic spread.For example, ER+ breast cancer bone metastasis [94] has shown the development of drug resistance with inadequate adjuvant therapy responses. [96]Prognostic markers can then be used to monitor disease progression during treatment.A recent study by Wu et al., 2021, revealed synergy and cooperation of exosomal miR-19a and integrin binding sialoprotein (IBSP) as prognostic markers for the development of bone metastasis. [106]Upregulation of miR-19a inhibits phosphatase and tensin homologous (PTEN), a phosphatase known to inactivate the nuclear factor-kappa beta (NF-k) and Akt signaling pathways, [107,108] Moreover, to identify possible miRNA therapy targets, Chai et al., 2018 established that miR-498 targeted the 3-UTR region of PTEN and negatively regulated its expression while increasing the expression of p-Akt, [109] a precursor of the P13/Akt pathway.Hence, upregulation of miR-19a and miR-498 inhibited tumor-suppressing PTEN [107][108][109][110] while activating NF-k and Akt pathways, respectively indicating potential use as prognostic markers.
Other significant miRNAs in breast cancer, including miR-376c and miR-382, are predominantly overexpressed across the various subtypes of breast cancer 78 .miR-382-5p proved to inhibit the Ras-related and estrogen-regulated growth inhibitor (RERG/Ras/ERK), [111] promoting aggressiveness of breast cancer by promoting cell viability, clonogenicity, survival, migration, tumorigenesis, and metastasis. [95]The miR-382-5p targets and represses RERG, a regulator and suppressor of the oncogenic Ras/ERK pathway.The Ras/ERK pathway is hyperactivated in breast cancer cases and with aggressive and poor prognosis. [111]istinctively, the expression profiles of these miRNAs during treatment can be targeted for disease monitoring and for targeted therapy to control their regulatory signatures.Additionally, combined therapy for the miRNA-regulated genes and corresponding miRNAs, provides an alternative therapy target for breast cancer.

Cellular and Exosomal miRNA Profiles Relevance in Clinical Outcomes
Other research has demonstrated that combined exosomal miRNA detection provides individuals with predictive clinical outcomes, forecasting prognostic parameters of the disease, subtype, disease occurrence, and probability of recurrence.For example, an RNA analysis by Ozawa et al., 2020 to determine the miRNAs expressed in characteristic EVs from serum samples of luminal A and TNBC identified that they possess a wide range of dysregulated miRNAs in the subtypes of breast cancer.They quantified miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5b, showing a combination of the miRNAs provided a better understanding of the breast cancer subtype.For example, combining the quantification of upregulated miR-142-5p and miR-320a revealed prominence in Luminal A breast cancer patients with a 100% sensitivity and 93.80% specificity.Consequently, the downregulation of the combination of miR-142-5p and miR-320a was indicative of large tumor sizes. [50]A similar study examined the expression of exosomal miRNAs derived from tissue and discovered an upregulation of miR-338-3p, miR-340-5p, and miR-124-3p in recurrent breast cancer cases and downregulation of miR-29b-3p, miR-20b-5p, miR-17-5p, miR-180-3p, miR-18a-5p, miR-195-5p, miR-485-5p, miR-93-5p in non-recurrent cases. [112]They revealed that combined quantification of the expression of several miRNAs could reveal the "patient" prognostic status, unlike the profiling of a single miRNA.Therefore, an array of miRNAs can be used to predict patient therapy outcomes and monitor the progress of patients at the clinical stages.Consequently, combined targeted therapy to inhibit the expression of these miRNAs provides a potential therapeutic target for breast cancer.Table 2 represents examples of known dysregulated miRNAs in different subtypes of breast cancer.

miRNAs Regulate Cancer-Promoting Signaling Pathways
Some top dysregulated pathways in breast cancer signaling, including P13K-Akt, Wnt pathways, epithelial mesenchymal transition (EMT), and NF-k pathways are influential in disease prognosis and drug therapy. [113]The general functions of EVs transporting signaling proteins, receptors, and regulatory RNAs affect the expression of transcriptional factors and gene expression in signaling pathways.Exosomal miRNAs inhibit or activate signaling pathways by dysregulating specific or multiple transcription factors on the cascades promoting breast cancer cell mechanisms and metastasis development, [65,106,114,116] Evidently, many cellular pathways and processes in both healthy and abnormal conditions are regulated by miRNAs, targeting over one-third of all cancer-associated genes, indicating that most pathways have miRNA involvement. [117]he functions of cellular and exosomal miRNAs have been well summarized [118,119] in regulating cellular mechanisms, breast cancer hallmarks, [120] and drug resistance. [121]Cellular crosstalk influenced by exosomes carrying dysregulated miRNAs control breast cancer cascades.For example, the Wnt/-catenin pathway is a key pathway in regulating development and cellular stemness, [122] whose aberrant signaling has been associated with driving the EMT and metastasis. [123]Overexpression of miRNA-374a induced hyperactivation of Wnt/-catenin regulators, including WIF1, PTEN, and WNT5A. [123]Consequently, miR-31, a pro-oncogenic miRNA regulated by NF-k, has been shown to activate several cascades, including the Wnt/-catenin, by targeting and inhibiting the expression of Dickkopf-related protein 1 (Dkk1).Dkk1 protein is a Wnt/-antagonist [124] whose expression in breast cancer acts as a suppressor protein, inhibiting migration and invasion by obstructing -catenin expression and down-stream inhibition of the MMP-7. [125]The expression of miR-31 was shown by Lv et al., 2017 to target and restrain Dkk1 expression, promoting mammary epithelial proliferation and expansion of mammalian stem cells. [126]u et al., 2021 proved that cellular miR-19a and exosomal miR-19a targeted the phosphatase and tensin homolog (PTEN) "3"UTR region, causing the differentiation of osteocytes cells, promoting breast cancer metastasis to the bone. [106]TEN, a suppressor protein functioning in cell proliferation and migration [127][128][129] negatively regulates the mechanism of the NF-k and PI3K/Akt pathways.PTEN is targeted and regulated by the PI3k/Akt expression, such that phosphorylation of the subsequent proteins in the pathways induces conformational changes that alter the expression of the PTEN suppressor.Its suppression promotes metastasis by activating tumorenhancing cascades and regulatory miRNAs acting synergistically with PI3K/Akt/mTOR genes.Kia et al., 2019 implicated exosomes carrying miR-9 and miR-155, derived from a metastatic MDA-MB-231, induced cell proliferation and metastatic effects on low metastatic MCF-7 by targeting and suppressing the expression of PTEN and DUSP14 genes. [130]Yang et al., 2021 explored the role of miR-134-5p, normally downregulated in breast cancer.They showed that overexpressed cellular and exosomal miR-134-5p restrained breast cancer progression by targeting the Rho GTPase Activating protein (ARHGAP1), an oncogene that regulated the PI3K/Akt and suppressed cell proliferation, migration, invasion, and promoted breast cancer cell apoptosis. [131]oreover, the inflammatory pathway NF-k is activated at the onset of chemotherapy and radiation treatment. [132]It activates transcriptional factors that promote inflammation, tumor metastasis, and drug resistance.MiRNAs that regulate the NF-k induce the regulatory proteins and transcriptional factors that activate or inhibit NF-k upregulation.For example, Mao et al., 2021 evidenced that overexpression of cellular and exosomal miR-370-3p promoted lymphatic and tumor node metastasis by inhibiting the expression of Fibulin 5 (FBLN5) proteins and activating the NF-k. [114]This was supported by Zhao et al., 2021 who revealed that EVs-derived miR-370-3p induced metastatic transformation of fibroblastic cells to cancer-associated fibroblasts by targeting the downregulation of CYLD Lysine 63 Deubiquitinase (CYLD) and activating the NF-k pathways [133] promoting disease prognosis.This suggested that miR-370-3p may be used as a potential prognostic marker in breast cancer treatment.Additionally, cellular and exosomal miR-1910-3p promotes proliferation, metastasis, and autophagy in breast cancer in combination with cancer antigen-15-3 (CA15-3) by inhibiting MTMR3 (Myotubularin Related Protein 3) and activating the NF-k pathway, as proved by Wang et al., 2020. [134]Subsequently, Dai T. et al., 2020 showed that miR-423 is upregulated in breast cancer, which targeted and degraded TNIP2 (TNIP interacting protein), activating the NF-k pathway. [135]These illustrated a distinct prognostic role of these miRNAs in controlling the function of NF-k and promoting breast cancer.
The Ras pathway activates such molecules' signaling, which triggers cell survival and growth, apoptosis, cell maintenance, and motility.Overactivity of Ras-GTPase-activating protein leads to the aggressive breast cancer phenotype.Ras/ERK (extracellular-signal related kinase) pathway is known to be activated in metastatic breast cancer. [111]The Ras-GTPase superfamily member RERG (Ras-related and estrogen-regulated growth inhibitor) is a tumor suppressor that reduces breast cancer cell proliferation.Ho et al., 2016 showed that miR-385-5p directly targets the RERG to stop its activity as a tumor suppressor and support the activation of the Ras/ERK pathway.miR-385-5p is also clinically associated with poor prognosis of breast cancer. [111]ltimately, understanding the underlying effects of miRNA expressions can determine cancer-promoting cascades, providing target therapies for the regulatory molecules and the miR-NAs.Therefore, cellular and exosomal miRNAs can be used as diagnostic and prognostic markers and therapy targets for breast cancer.Figure 4 represents the functions of selected miRNAs in regulating the signaling pathways, and a summary of this is provided in Table 3.

Conclusion
Breast cancer remains a global challenge in early diagnosis, and management, and limited effective target therapies after metastasis development.Being a heterogenetic disease, genetic and epigenetic alterations, molecular phenotyping changes, altered protein expression, and body metabolism are some eminent aberrated mechanisms.This is also attributed to the distinctive characteristics of the divergent tumor microenvironment. [46]utations such as single nucleotide polymorphism on miRNA in the components that promote miRNA biogenesis or in the miRNA sequence contribute to alteration in the biogenesis and [ 95,98]   miR-335 ER- signaling They repress the genes that activate ER- expression and promote tamoxifen resistance [30]   miR-376c Repression of Ras/ERK pathway Its downregulation suppressed aggressive form of metastasis, but upregulation through the neuropilin protein suppresses tumor growth in most subtypes of breast cancer [95,155]   the function of the miRNAs, the targets, and gene expression levels.This can stimulate disease progression and ineffective drug responses. [136]Identifying these anomalies, a systemic approach to breast cancer diagnosis, prognosis, and treatment depends on the biomarker targets at the various levels of the disease cycle, trickling down to effective therapeutic interventions, such as multitargeting therapies.Distinctively, variations in circulating, cellular, and exosomal miRNA expressions are still indistinguishable, and little evidence has established comparative expressions.Some studies have revealed insufficient and unstable quantities of circulating miR-NAs for routine detection, in blood. [85]However EV-derived miR-NAs are stable and protected from degradation by circulating RNAses. [19]There is a lack of enough evidence to substantiate the differences in expression levels from these sources.Despite their sources and quantities, cancer-derived miRNAs from cells or extracellular vesicles play the same roles in their molecular dynamics promoting or restricting tumor growth, [137] cancer recurrence, [50,88] and fostering drug resistance, [116,138,139] The advantage of using exosome-derived miRNAs is the ease of collection of exosomes from a wide range of body fluids, including tears [140] and saliva, [141] which mitigates invasive methods for sample collection from cancer individuals.This also expedites sample recovery and quantitative and qualitative analysis in diagnosing the disease and obtaining results for the individ-uals.Additionally, of crucial need are effective tools to detect these exosomal miRNAs.Initially detected by northern blotting and RNA microarrays, several more sensitive and selective tools have emerged in the field of nanotechnology, [38,93,142] Currently, RT-PCR is the gold standard approach to detect miRNAs. [143]t the same time, next-generation sequencing and CRISPR-Cas have been used to identify single nucleotide polymorphisms (SNPs) that distinguish similar miRNA generations during biogenesis and influence disease. [144]his literature review focuses on cellular and exosome-derived miRNAs as biomarkers, and their diagnostic and prognostic value in breast cancer at the clinical level.It presents a comprehensive perspective to understanding breast disease, subtyping and staging breast cancer, breast cancer regulatory mechanisms and pathways, and predicting disease outcomes during treatment using miRNAs.Additionally, exosomes possess multitargeting properties in diagnosis and functions in prognosis and targeted treatment, including exosomal proteins and miRNAs which are translocated into exosomes in stress-induced conditions.Moreover, research has also indicated that miRNA biogenesis can occur independently in exosomes [59] by carrying processor complexes, such as the Drosha and Dicer, that process immature miRNAs.This makes EVs independent machinery for miRNA synthesis that promotes transcriptome alteration and induces tumorigenesis in normal cells hence possessing comprehensive profiles of cellular and exosomal miRNAs usable as potential biomarkers for breast cancer diagnosis and prognosis.Besides, diverse biomarker identities present a multifaceted approach to breast cancer disease diagnosis, disease state, and more therapeutic targets in breast cancer, in the era of miniaturization and precision medicine.
In summary, studies on cellular and exosomal miRNAs and exosomes in general as biomarkers for breast cancer have great potential for effective diagnostics, prognosis, and targeted therapy.Understanding the complex interplay between exosomes, exosomal cargo including miRNAs, cancer-promoting cascades, transcriptional factors, and breast cancer progression can help develop effective interventions and personalized therapeutic strategies for breast cancer patients.

Figure 1 .
Figure 1.Diagrammatic representation of exosome structure showing the major components of the exosomes, including surface protein markers such as Tetraspanins proteins (CD63, CD81), immune regulatory receptors such as the major histocompatibility complex (MHC), immune inhibitory receptors such as the PD-L1, CTLA, Trim-3, lipids such as cholesterol, and surface integrin.Encapsulated in the exosomes are nucleic acids-DNA, small non-coding RNAs, long non-coding RNAs, heat shock proteins, membranous proteins, and cytoskeletal proteins.Created by Biorender.com.

Figure 2 .
Figure 2. a.A schematic diagram of conical miRNA biogenesis in the cell.Overview, miRNA synthesis is initiated in the cell nucleus on the intronic region and acted on by the RNA polymerase II complex (Drosha/DGRC8), into precursor miRNA, and transported out of the nucleus by exportin 5 and Ras GTPase.The dicer acts on the pre-miRNA into short duplex miRNAs which is then dissociated with Argonate-2 with a degradation of the passenger strand while the guide miRNA strand is loaded with the RISC to act on the untranslated region (UTR) and open read frames (ORFs) region of the target mRNA.b.Schematic of normal cell transformation to cancer cells that gain immortal replication, forming the tumor microenvironment (TME).A normal cell i) is transformed into a cancer cell inducing an immortal replication mechanism, to create a tumor microenvironment [TME] ii).iii.miRNAs and extracellular vesicles/exosomes are released from the TME into the circulation through the vasculature of the TME iv) or miRNAs are shuttled and loaded into the released exosomes by various RNA-binding proteins including caveolin-1, hnRPA2B1, Argonaute-2 through stress-induced mechanisms.Various regulatory roles of exo-miRNAs outlined include regulation and induction of metabolic stress, regulation of transcriptions, promote drug resistance, regulation the EMT, promoting angiogenesis and metastasis, induce immune suppression, and signaling pathways.Created by BioRender.com

Figure 3 .
Figure 3. Graphical representations showing the expression profiles of miRNAs and EV-derived miRNAs dependent on breast cancer subtypes and tumor grade and dissemination A) Differential tumor-derived extracellular vesicles-miRNA expression by fold change based on breast cancer subtypes, Luminal A, Luminal B, HER2, and TNBC against healthy controls.Each of the boxes represents the relative miRNA expression in Luminal A (estrogens receptors {ER+}, Progesterone receptor {PR+}, and human epidermal growth receptor {HER2+}), Luminal B (ER+, PR+, and HER-2+), HER-2 (ER-, PR-, and HER-2+) and TNBC (ER-, PR-, and HER-2-).Reproduced with permission.[93]Copyright 2021, Wiley Publishers.B) Comparative individual expression level of miRNAs between tumor grade (G) and lymph node metastasis (LNM) in invasive breast cancer patients.Reproduced with permission.[94]Copyright 2017, Impact Journals.C) Volcano plot showing the differential expression levels of 45 exosomal miRNAs in 435 breast cancer samples of neoadjuvant therapy in the GeparSixto Trial, 211 HER2+ and 224 TNBC patients, all against 20 healthy women and a comparison between TNBC and HER2 positive BC.The green dots indicate downregulated miRNAs, and the red dots indicate upregulated miRNAs, while the grey dots show the threshold value corresponding to the p-value of 0.05.Reproduced with permission.[95]Copyright 2018, BioMed Central.

Figure 4 .
Figure 4. miRNAs that regulate, promote, or inhibit breast cancer signaling pathways.Several signaling pathways are activated or inhibited during breast cancer development, depending on their function.The most commonly upregulated signaling pathways include Wnt/-catenin, NF-k, P13K/Akt, and the EMT.Examples of miRNAs known to activate these pathways are represented above the pathways, with the subsequent transcription factor they inhibit or activate, while examples of those known to inhibit these pathways are represented.The roles of these signaling pathways in breast cancer are also described.Created by BioRender.com

Table 1 .
Examples of known miRNAs, oncogenic or tumor suppressor properties or duality function, and their potential applications in breast cancer diagnosis, prognosis, and disease prediction.

Table 2 .
Significantly upregulated and downregulated miRNAs in various subtypes of breast cancer.

Table 3 .
miRNAs are known to regulate signaling pathways by activation or inhibition of tumor promoting transcriptional factors to promote breast cancer.As indicated, the selected miRNAs modulate the breast cancer promoting pathways including Epithelial Mesenchymal Transition (EMT) pathway, Notch pathways, Wnt/-catenin, P13K/Akt, NF-k, ER- while repressing suppressive pathways such as Ras/ERK.The Main mechanism involved includes promoting activation of tumor signaling and promoting transcriptional factors, attenuation of drug resistance, and suppression of inhibitory factors.