Breast cancer: miRNAs monitoring chemoresistance and systemic therapy

With a high mortality rate that accounts for millions of cancer-related deaths each year, breast cancer is the second most common malignancy in women. Chemotherapy has significant potential in the prevention and spreading of breast cancer; however, drug resistance often hinders therapy in breast cancer patients. The identification and the use of novel molecular biomarkers, which can predict response to chemotherapy, might lead to tailoring breast cancer treatment. In this context, accumulating research has reported microRNAs (miRNAs) as potential biomarkers for early cancer detection, and are conducive to designing a more specific treatment plan by helping analyze drug resistance and sensitivity in breast cancer treatment. In this review, miRNAs are discussed in two alternative ways-as tumor suppressors to be used in miRNA replacement therapy to reduce oncogenesis and as oncomirs to lessen the translation of the target miRNA. Different miRNAs like miR-638, miR-17, miR-20b, miR-342, miR-484, miR-21, miR-24, miR-27, miR-23 and miR-200 are involved in the regulation of chemoresistance through diverse genetic targets. For instance, tumor-suppressing miRNAs like miR-342, miR-16, miR-214, and miR-128 and tumor-promoting miRNAs like miR101 and miR-106-25 cluster regulate the cell cycle, apoptosis, epithelial to mesenchymal transition and other pathways to impart breast cancer drug resistance. Hence, in this review, we have discussed the significance of miRNA biomarkers that could assist in providing novel therapeutic targets to overcome potential chemotherapy resistance to systemic therapy and further facilitate the design of tailored therapy for enhanced efficacy against breast cancer.


Introduction
The most common malignancy worldwide is breast cancer (BC). According to the status update on the GLOBOCAN 2020 projections of cancer incidence and mortality, BC is the primary cause of cancer death in women and accounts for 1 in 4 cancer diagnoses among females (1). An estimated 684,996 people died from breast cancer in 2020, with low-resource areas accounting for a disproportionate share of these deaths. According to statistics, the prevalence of BC ranges from 2-6% in Western nations to 10-20% in Asian nations (2), indicating that BC is becoming a global health concern, even in nations with sizable young populations like India. Although breast cancer diagnoses have increased recently (3), the prognosis for the disease has significantly improved, with expected 5-year survival rates rising from 40% to approximately 90% over the last 50 years. With few exceptions, en-bloc radical resections in the form of Halstead mastectomy and axillary clearing were formerly thought to be essential for managing BC (4). Recent advancements in clinical trials have been brought about by a greater understanding of the molecular processes associated with the heterogeneity of breast tumors. This understanding has allowed for more conservative surgical procedures and the personalization of treatment plans to maximize sensitization to the tumor while minimizing unneeded morbidity to the patient. This includes the era of cancer diagnostics, which has recognized BC as a diverse disease and routinely subcategorized these cancers into four genetically distinct, integral subgroups -luminal A breast cancer (LABC), luminal B breast cancer (LBBC), human epidermal growth factor receptor-2 enriched breast cancer (HER2+) and triplenegative breast cancer (TNBC). These subgroups have different clinical behavior, prognosis, treatment approaches, and clinical outcomes in the known treatments (5).
For BC patients, chemotherapy is regarded as the most successful and crucial therapeutic approach. Anthracyclins, Tamoxifen, Taxane, 5-FU and trastuzumab are the major chemotherapeutic drugs which are administered to BC patients (6)(7)(8)(9)(10)(11)(12). Doxorubicin, daunorubicin, and epirubicin are some of the anthracycline antibiotics that are frequently used. Anthracyclines can be administered at all BC stages and have demonstrated a crucial function in treating BC (8). Tamoxifen is specifically used for the oestrogen receptor (ER) positive subtype of BC (9). Anthracyclines and taxanes are used as the predominant treatment for TNBC (10). The two most widely used taxanes arepaclitaxel and docetaxel, causing acute hypersensitivity responses (HSRs) in 5% to 10% of patients (11). The human epidermal growth factor receptor 2 (HER-2) is frequently used to categorize BC patients based on its overexpression (also known as HER-2 positive) or lack of expression (also known as HER-2 negative) (13). The likelihood of BC metastases and poor prognosis are strongly correlated with HER-2 overexpression (13). A targeted therapy for HER-2 is trastuzumab (TRS), a humanized monoclonal antibody (12). Despite our efforts to categorize tumors into prognostic categories, tumor behavior and prognosis remains unpredictable, which makes it challenging to develop strategies that would improve disease control while minimizing toxicities to patients. Although, a better understanding of the disease has led to advancements in treatment over the past few decades, but drug resistance remains a challenge and the underlying molecular causes are still largely undefined (14). Drug-resistant cancer cells multiply rapidly and grow more hostile, increasing the likelihood that the tumor may aggressively spread to other organs. Drug resistance can be categorized in two different ways. One is internal resistance or inherited resistance, which occurs when tumors are resistant to treatment even before receiving it, meaning that even early detection and treatment are ineffective. Another form of resistance is received resistance or acquired resistance which occurs following an initial positive response to the therapy (15). Here, the targets and processes associated are a focus of significant research, and the mechanisms of such drug resistance are largely still under investigation (16). For instance, Martz et al. (2014) demonstrated that stimulation of the Notch-1, mitogen-activated protein kinase (RAS-MAPK), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), PI3K/AKT and estrogen receptor (ER) signaling pathways resulted in resistance to a variety of drugs (17). It was observed that when Notch-1 is activated, BRAF (V600E) melanoma cells develop acquired resistance to MAPK inhibitors and breast cancer cells also exhibit resistance to tamoxifen (17). Hence, the research group used a Notch-1 inhibitor to restore sensitivity, indicating that Notch-1 knockdown could be a therapeutic strategy in melanomas and drugresistant breast malignancies (17). Likewise, it seems, resistance to chemotherapy is also related to the epidermal growth factor receptor (EGFR) pathway. Genetically modified murine model (GEMM), human cell lines, and a clinically applicable model of KRAS-mutant colorectal cancer (CRC) have all been used to study EGFR and PI3K/mTOR (18). According to the evidence, PI3K/ mTOR and EGFR inhibition boost drug sensitivity and are increasingly used in cancer therapy to combat drug resistance. Additionally, the use of systemic drugs as neoadjuvant enables the production of in-vivo data on tumor sensitivity, which has been shown to have predictive importance for disease survival and recurrence. These contemporary aspects of traditional breast cancer management shed light on the potential value of emerging biomarkers in advancing the current treatment model. There are currently few biomarkers that may reliably predict response and resistance to systemic and targeted therapy and attempts to use non-invasive approaches to collect such biomarkers have largely been ineffective (19). This highlights how important it is for researchers to find new biomarkers that can assess patient response to therapy, predict the prognosis of breast cancer patients, and offer clinicians cutting-edge oncogenesis-targeting therapeutic approaches. In the context of BC chemoresistance monitoring and systemic therapy, this study focuses on the function of microRNA (miRNA) as new clinical biomarkers.
miRNAs are small non-coding RNAs ranging from 19 to 25 nucleotides in size and are involved in a variety of biological activities, including cell cycle, apoptosis, survival, and gene control (20). miRNAs primarily bind to the 3′ or 5′ untranslated region (UTR) of their target mRNAs and, depending on the degree of binding, participate in controlling the translation of proteins or destruction of the mRNA itself (21). A single miRNA may target several mRNAs, while many miRNAs may target single mRNA with varying degrees of efficiency (22). Therefore, changes in miRNA expression levels and gene expression silencing by miRNAs have a significant impact on human health and the emergence of diseases such as cancer, diabetes, neurological disease, and cardiovascular disorders (23-25). In the context of cancer, miRNAs can function as both tumor suppressors and oncogenes/oncomirs (26). In contrast to their counterparts in normal tissue, many miRNAs are reported to be up-or down-regulated in cancer tissues. For instance, practically all cancer types have increased miR-21 expression (27). Numerous B-cell malignancies have been shown to express miR-155 at high levels (28). One of the first miRNAs to be found was let-7 which is essentially missing throughout embryonic stages or tissues, although it is highly expressed in the majority of differentiated tissues (29). Similar to the fall in let-7 expression during development, the decline in let-7 expression in malignancies is more pronounced in cancer cells that are more advanced, less differentiated and have mesenchymal features (29). The generation, biology and function of miRNAs in cancer have been discussed in detail in further sections.
This review focuses primarily on the latest findings about the involvement of miRNAs in breast tumor resistance to chemotherapeutic agents and in the development of systemic therapy. Targeting miRNAs-either reducing or raising their expression-seems to be an attractive approach for designing novel, more effective, and personalized treatments for BC. Boosting drug efficacy by examining the downstream targets/ pathways influenced by miRNA targeting and predicting patient response to various therapies can lead to better treatment outcomes for BC patients.

Breast cancer chemotherapy
Breast cancer bears 7% of the total number of cancers related deaths in 2020. To date, many strategies have been adapted to combat this disease. Complete surgical removal has usually enabled efficient breast cancer disease management (30). Regardless of the severity of the disease, William Halstead's radical mastectomy (which required significant removal of all the breast parenchyma, local lymph nodes, and pectoralis major muscle) used to be the cornerstone of breast cancer treatment (4). Cyclophosphamide, m e t h o t r e x a t e , a n d 5 -fl u o r o u r a c i l ( C M F ) , t h e fi r s t chemotherapeutic treatment prescribed by Bonadanno et al. in 1976 with the intention of curing breast cancer, significantly decreased breast cancer relapse (94.7% of 207 patients administered chemotherapy vs 76.0% of 179 patients constrained chemotherapy) (31). However, since 1950s, Bernard Fisher and the National Surgical Adjuvant Breast and Bowel Project (NSABP) have hypothesized that aggressive surgery for breast cancer has only limited scientific and biomolecular justification because it is frequently insufficient to achieve complete disease control (32). Fisher's theory that all breast cancer patients needed systemic therapy (especially with chemotherapy) has, however, been thoroughly refuted.
However, the inherent advantage of treating cancer patients with chemotherapy in the neoadjuvant setting has now been recognized as an oncological practice. Neoadjuvant chemotherapy (NAC) benefits comprised tumor downstaging, expanding patient suitability for breast conservation surgery (BCS), and producing in vivo data related to tumor resistance, which has been shown to hold predictive value for cancer recurrence and overall survival (OS) (33, 34). The Early Breast Cancer Triallist's Collaborative Group (EBCTCG) recently published data from a meta-analysis of randomized clinical trials showing that locoregional recurrence (LRR) rates are higher following neoadjuvant therapy (21.4% vs. 15.9%), despite the fact that disease-free survival (DFS) and overall survival (OS) results are parallel with those treated in the adjuvant setting (35). Additionally, there is growing proof that people who have a pathological complete response (pCR) with NAC have a higher chance of living longer than those who have a latent disease (34, 36). Nevertheless, the clinical usefulness of NAC has been integrated into best-practice guidelines for HER2+ and Triplenegative breast cancer (TNBC). HER2+ malignancies should be treated with NAC and trastuzumab, with the exception of T1a-T1b N0 disease (37). High-risk LN (lymph node)-patients and those with LN positivity should receive anthracycline-and taxane-based chemotherapy along with trastuzumab (37). Further, until TNBC is identified with cancer stages T1a-T1b N0, patients with TNBC should always be provided with an anthracycline and taxane-based treatment (37). American Society of Clinical Oncology (ASCO) also supports the inclusion of platinum-based chemotherapy in TNBC based on the results of a recent meta-analysis because of a higher likelihood to obtain pCR (52.1% versus 37.0%) (38). Pembrolizumab and NAC significantly increased the pCR rates in the KEYNOTE522 trial's preliminary findings (pembrolizumab and NAC: 64.8% versus placebo and NAC: 51.2%) (39).

Need for miRNA-based therapy
The idea of improving pCR rates, simplifying the de-escalation of adjuvant therapy post pCR, and minimising treatment-related toxicities for patients receiving these neoadjuvant medicines are the main directions for translational research efforts in the future (40). Therefore, numerous clinical trials have focused on practices that improve pCR rates (40). To further improve the pCR, the idea of manipulating treatment with miRNA-based therapies may be helpful in boosting pCR rates to NAC in breast cancer is now popular, and the same has been discussed in depth in this review.

Chemoresistance in breast cancer
Various molecular aspects are known to be involved in inducing chemoresistance in cancer cells (Figure 1). Some of them have also been summarized below: ♦ Resistant genes:

Twist
Twist is a key player in the invasion and metastasis of tumors because it regulates the epithelial-mesenchymal transition (EMT) (41). It has been reported that NF-kB up-regulation of twist-1 is a factor in the chemoresistance (42). Through the downregulation of estrogen receptor alpha (ERa) activity, twist overexpression can also contribute to hormone resistance in breast tumors (43). ♦ Efflux proteins: Another mechanism of resistance to chemotherapy is mediated by ATP-dependent efflux pumps, which decrease the intracellular concentration of drugs. By using the energy from ATP hydrolysis, or the MDR phenomenon, the ATP-dependent efflux transporters in cancer cells can actively transport a range of substrates outside the cell membrane (46 The major chemoresistance mechanisms of cancer cells.  (68) and glutathione S-transferases (GST) are some of the key enzymes that contribute to MDR in cancer cells. These agents have the potential to enhance the transformation and catabolism of anti-neoplastic drugs, shorten the term of effective concentration of chemotherapeutic drugs in tumor cells, decrease drug accumulation in target areas, and ultimately limit drug efficacy (69). For example, GST-p can be employed as a separate index to direct a clinical treatment against BC as its expression in breast cancer patients was associated with the histological grade, the number of lymphatic metastases, and the age of the patients (70).

MicroRNAs
MicroRNAs are a class of small noncoding RNAs (ncRNAs), which function in post-transcriptional regulation of gene expression and are powerful regulators of various cellular activities and have been linked to many diseases (20). RNA polymerase II (Pol II), which produces the main transcripts, participates in several stages of microRNA synthesis (pri-miRNA). The pri-miRNA are split up into precursor miRNAs (pre-miRNAs) by the RNase III Drosha (71). The pre-miRNAs are subsequently moved from the nucleus into the cytoplasm by Exportin-5 (Exp5), where they are further split by Dicer into a mature single-stranded miRNA. The miRNA is induced to either degrade or suppress the translation of mRNA targets when the mature miRNA is removed from the pre-miRNA hairpin and attached to the RNA-induced silencing complex (RISC) (72).  MiRNA expression and function. RNA polymerase II enzyme in the nucleus transcribes the miRNA-encoding genes, forming the "Pri-miRNA" hairpinshaped molecule. DROSHA and DGCR8 molecules work together to transform the "Pri-miRNA" molecule into the "Pre-miRNA" precursor molecule. Pre-miRNA then travels to the nuclear export receptor "Exportin 5" and reaches the cytoplasm. This precursor is cleaved by Dicer complex in the cytoplasm to create a double-stranded molecule called "miRNA duplex." One of these two strands is left active after this process, and it has the ability to suppress or even activate the target downstream genes at the transcriptional or translational level. and function. Besides miRNAs, other ncRNAs are long-chain noncoding RNAs (LncRNAs), piRNAs, and circle RNAs (circRNAs), which make up just 1% of the whole genome's RNA (73).
The molecular revolution enables us to design strategies to maximize patient outcomes, reduce toxicity, and control disease with less strenuous and more focused therapies. The development of chemotherapeutic response biomarkers is necessary in the future to speed up the removal of tumors and reduce the need for extended and excessive treatments. The utility of detecting miRNA expression (both in tumors and in the blood) is now being discussed in the scientific community. Doing so may help doctors prescribe medicines that are suitably targeted, address early relapse, or even enable miRNA-directed therapies. In general, miRNAs can be either tumor suppressors (tumor suppressor miRNA) or oncomirs, and they can affect the development of cancers in either way. Numerous miRNAs, along with their downstream targets, have been shown to be differentially expressed in breast cancer patients when compared to healthy controls (either circulating or in tumors) ( Table 1).

miRNAs in BC subtyping
The three main subtypes of breast cancer that exist are (1) Positive ER and PR (2); HER-2 positive; and (3) (ER, PR, and HER-2 negative) Triple negative. However, this subtyping is expanded to a more precise one using the microarray approach for identifying miRNA profiles, including (1):Luminal A (ER-positive with low grade) (2); Luminal B (ER-positive with high grade) (3); HER-2 positive (4); Basal-like (Almost equal to triple-negative condition).
There are several miRNAs that are used for the breast cancer subgrouping as shown in Table 2 (87). Currently, it is possible to use miRNA profiling for the subgrouping of breast tumors. This capability can therefore aid in the selection of cancer patients who will get adjuvant therapy. In addition, miRNA profiling can be successful in identifying new therapeutic targets by revealing the genetic underpinnings of distinct subgroups of breast cancer.

Role of miRNA in BC drug resistance
It is well known that miRNAs can regulate drug resistance to traditional chemotherapeutic medicines, endocrine hormone treatments, and radiotherapies in cancer cells (90)(91)(92)(93). It has been shown that miRNA expression can influence a breast cancer patient's ability to respond to or reject systemic treatment as shown in Table 3. The miRNAs along with other ncRNAs significantly reverse the BC cell drug resistance by suppressing signaling pathways such as Wnt/b-catenin, Hippo, AKT, TGF-b, or mTOR signaling pathways. A summary of molecules which can participate in target diversity in miRNA interactions leading to drug resistance using different chemotherapeutic drugs is mentioned in Table 3. According to various reports, there are scientific explanations and processes for chemotherapeutic resistance, including altered drug-target interactions, lower active drug doses, and increased tumor tissue survival (143). Numerous miRNA expression profiles have been linked to the prediction of    (150). The role of miRNAs in BC chemoresistance has been attributed to some of the following molecular mechanisms (Figure 3): ♦ miRNAs and cell cycle: Cell cycle deregulation is an established hallmark of cancer, and it has been linked to both drug resistance and poor prognosis when it is aberrantly activated. Various miRNAs have been reported to target genes linked to cell cycle regulation, resulting in either drug sensitivity or resistance such as miR-93, involved in G1/S phase arrest, was reported to be downregulated in paclitaxel-resistant BC samples compared to responder patients ( Figure 3A) (151). Direct  (142) targets of this miRNA were discovered to include CCND1 and the E2F transcription factor 1 (E2F1), which upon downregulation resulted in cell cycle arrest in G1 phase and increased apoptosis via inhibiting AKT phosphorylation (p-AKT), and BCL-2 expression, and increasing the expression levels of BCL-2-associated X, apoptosis regulator (BAX), which could increase the paclitaxel sensitivity.
♦ miRNAs and DNA repair machinery: As mentioned above, most chemotherapy drugs used today to treat breast cancer cause either direct or indirect DNA damage. To counteract DNA damage, however, CSCs activate DDR pathways, explaining why chemotherapy that destroys DNA could result in drug resistance ( Figure 3B). One such DDR pathway involves BRCA1 which is engaged in different cellular processes that maintain genomic stability like DNA damage repair, DNA damage-induced cell cycle checkpoint activation, chromatin remodelling, protein ubiquitination, transcriptional control, and cell death [43]. Drug sensitivity is thus impacted by its miRNA influenced control, for instance miR-182 inhibits BRCA1 expression to induce drug sensitization. Furthermore, it has been shown that overexpressing miR-182 makes BC cells more susceptible to PARPi (PADP-ribose polymerase 1 inhibitor). In contrast, miR-182 suppression raises BRCA1 levels and results in PARPi resistance (152).
♦ miRNAs and cell death: The interests of investigators are growing in drug-miRNA combination anticancer therapy since miRNAs can influence cell death ( Figure 3C). Examples include miR-125b, which confers paclitaxel resistance by inhibiting the expression of BAK1 (BAX and/or BCL-2 Antagonist/Killer 1), which causes the release of cytochrome C from mitochondria to the cytoplasm, where it binds Apoptotic peptidase Activating Factor 1 (APAF-1) and triggers caspase activation (76). Similar findings were also made from miR-149-5p, whose overexpression was shown to boost BAX expression (153), and from miR-663b, which imparts tamoxifen resistance by indirectly upregulating BAX (154).
♦ miRNAs, CSCs and epithelial to mesenchymal transition (EMT): Breast cancer stem cells (BCSCs) are a small population of cells with a high ability for tumorigenesis and are involved in therapy resistance (155). The modulation of BCSCs' phenotype is mediated by several molecular mechanisms, the most significant of which is EMT. This process takes place as cancer develops, and it involves a decrease in the expression of molecules associated with epithelial growth, such as E-cadherin, and a rise in molecules associated with mesenchymal development, such as N-cadherin, vimentin (VIM), and fibronectin (FN1) (156). Thus, the cells become more capable of invasion and migration (155) and can nest in various tissues where they can multiply and create new tumors through a process called metastasis (155). In this context, miRNAs play a significant role in controlling stemness and EMT by targeting a few genes implicated in these two pathways ( Figure 3D). Among those engaged in the control of EMT, the miR-200 family has received the greatest research attention. Five different miRNAs make up this family: miR-141, miR200a, miR200b, miR200c, and miR-429 (157), which can inhibit the expression of ZEB1 and ZEB2 (Zinc finger E-Box-binding homeobox genes) (157). As a result, it has been demonstrated that overexpressing miR-200 in many cancer cell lines can reverse EMT (158). Another factor contributing to stemness in BC is the Wnt/-catenin signaling pathway. It has been shown that several miRNAs, including miR-105 and miR-93-3p, regulate this pathway. The Wnt/-catenin signaling pathway suppressor Secreted Frizzled Related Protein 1 (SPFR1) is the target of those miRNAs. This led Li et al. to show that those miRNAs encourage cisplatin resistance (159).
Four circulating miRNA patterns linked to pCR were recently identified using profiling of circulating miRNA (ct miRNA detected in plasma) to categorize NAC responders (from non-responders) in Her2+ patients (160). These results demonstrate the potential of miRNA signatures as prognostic and predictive biomarkers that could individualize breast cancer treatments and enhance patient sampling techniques for current therapies, including traditional cytotoxic chemotherapies. The following is a discussion of a few of them: ♦ miR-638 -miR-638 was shown to be downregulated in cases with BC chemoresistance in a microarray analysis (161). A minimal patient-derived xenograft (MiniPDXTM) was also developed by the researchers to assess the chemosensitivity of various drugs. The results of this study demonstrated that in patients, who received 5-FU, miR-638 levels were relatively low in the 5-FU-resistant group compared to the 5-FU-sensitive group. So, according to the MiniPDX™ model, MDA-MB-231 BC cells overexpressing miR-638 were more susceptible to 5-FU treatment in vivo.
♦ miR-17/20 -The serine-threonine kinase Akt1 has been linked to the regulation of cellular homeostasis, proliferation, and growth as well as hyperactivation in human malignancies (162

miRNAs in neoadjuvant chemotherapies: predicting response
As already said, breast oncology research has advanced recently to realize that treating patients with chemotherapy in the neoadjuvant setting is both rational and beneficial (173,174). Although conventional clinicopathological traits have been shown to correlate with response to NAC (33), it is still difficult for oncologists to identify patients who are likely to experience such reactions since success rates are frequently unpredictable. The latest research has linked miRNA expression profiles with breast cancer patients' responses to NAC therapy.  Table 4 shows systematic trials examining the function of miRNAs in determining how patients would respond to neoadjuvant therapy and lists the miRNAs that are important in this setting (160,176,178,179,183,184,(186)(187)(188)(189)(190). Using miRNA expression profiles to assess response to adjuvant chemotherapy is substantially more difficult. It is quite challenging to measure whether medication improved oncological outcomes for patients who were most likely to succumb to recurrence, estimate the timing of miRNA sampling, and analyze treatment response rates in a crude way. Therefore, it is not surprising that most research evaluates miRNA expression patterns using metrics that indicate response to NAC rather than adjuvant chemotherapy (e.g., RECIST, Miller-Payne grade, Sataloff score, etc.).

MicroRNAs for therapeutic use in breast cancer
The use of miRNAs for the development of novel treatment approaches has been made easier by the current molecular technology. These entail the administration of carefully chosen miRNAs into the tumor microenvironment for therapeutic purposes or to improve the efficacy of currently available therapeutic modalities employed in standard clinical practice, such as systemic chemotherapy (143,191). miRNAs can act as tumor suppressors or oncomirs, so there are two possible methods for using them as therapeutics (1): miRNA replacement therapy, which involves inducing and overexpressing specific miRNA to reduce oncogenesis or increasing sensitivity to systemic treatment, or (2) oncomir inhibition, which involves lowering targeted miRNA expression characteristics (i.e., miRNA silencing) by incorporating inhibitory miRNA to lessen the translation of the target miRNA ( Figure 4).
♦ miRNA Replacement Therapy -By inhibiting oncogenes and the genes that regulate cell proliferation and death, tumor suppressor miRNAs can prevent the development of cancer (192). MiRNA replacement treatment includes reintroducing tumor-suppressing miRNA (or mimics) into the tumor microenvironment in order to inhibit tumor growth and restrain the spread of malignancy (193). They might be delivered into the cytoplasm of cancer cells through a variety of transporters, such as chemicals, electroporation, and modelling of the endogenous miRNA (192).  (198).
9.1 miRNA delivery strategies used for cancer therapy miRNAs can be introduced therapeutically into cancer cells through a variety of methods. These approaches are typically divided into two categories of local and systemic delivery, which are thoroughly discussed below and in Figure 5: Local delivery of miRNAs: Target gene suppression with less toxicity may result from the local delivery of miRNAs as opposed to the systemic delivery of miRNAs. According to Møller et al., 2013, the aforementioned strategy has been examined mainly for primary tumors including melanoma, breast, and cervical cancers (203). Recently, different local delivery techniques, such as the direct injection of miRNA vectors into the tumor site and the nanoparticles (NP) formulation with surface modifications, have been devised. For instance, glioblastoma multiform was treated using the intracranial miRNA delivery approach (203). In a study by Trang et al., 2010, let-7 was introduced into non-small-cell lung cancer using viral vectors, which inhibited the growth of KRASdependent tumors (204). The topical distribution approach is an additional technique for treating skin conditions. The target region is more accessible with fewer adverse effects when topical administration is used (205). Moreover, the local delivery system makes the use of modified miRNAs. For instance, astro-cyte elevated gene-1 (AEG-1) was the target of intratumoral miR-375 mimics in cholesterol-conjugated 2′-O-methyl modified form, which significantly suppressed tumor growth in in-vivo models of hepatoma xenograft (206).
However, as the local delivery system employs direct injection or local application of miRNAs with or without carriers, it cannot be recommended as a good strategy for treating late-stage metastatic disease. Therefore, developing a systemic delivery strategy is essential to provide efficient miRNA cancer therapy.
Systemic delivery of miRNAs: The systemic miRNA delivery technique represents a significant advancement in the effort to increase the effectiveness of cancer therapy and get over the drawback of miRNA delivery in vivo. Different systemic delivery strategies have been devised up until this point. A few of them are covered below:  ♦ Viral delivery of miRNAs -miRNAs can be transmitted by being encoded in several types of vectors, including viral and non-viral vectors. In this regard, viral delivery is an advantageous strategy. One of its benefits include low offtarget rate, resulting from given miRNAs being translated by tumor cells. Lentiviruses, adenoviruses, and adenoassociated viruses (AAVs) are among the viruses that have been identified as delivery vectors for miRNAs. As a result, targeting components were added to the viral capsid to strengthen the affinity between viral vectors and cancer-specific receptors, allowing for better transportation into tumors (209). However, due to the immunological reaction they cause and the difficulty of scaling up the production process in comparison to nonviral delivery systems, there are still some significant challenges to overcome. Additionally, the potential for a virus with replication competence may raise the risk of the pathogenic condition. For instance, some retroviruses can cause the start of a CNS illness as a result of their active reproduction (210).
♦ Non-viral delivery of miRNAs -The use of non-viral vectors is a beneficial strategy for miRNA delivery. In this approach, sitespecific delivery, system optimization, or polyethylene glycol (PEG) molecule augmentation could be employed to achieve targeted ligands or lengthen circulation times. Additionally, nanocarriers are produced in a secure and straightforward manner, and they are distinguished by their affordability, minimal immunogenicity, and adaptability. Non-viral delivery vectors can be divided into three primary categories: inorganic materials, lipid-based carriers, and polymeric carriers (211, 212).
However, non-viral-based approaches to miRNA delivery have their own shortcomings such as lower loading efficiency, lack of cargo protection, lower endosomal escape, nonspecific interaction with target cells and nucleic acids, etc (193).

Discussion
Considering that drug resistance continues to be a major obstacle in the clinical context, causing relapse and metastatic spread in many cancer types, novel treatment approaches are of the utmost importance. The discovery of miRNAs has provided a novel perspective on the molecular processes behind cancer, increasing the possibility of creating novel and more potent therapeutic approaches. This review is centered on new findings pertaining to the significance of miRNAs in breast cancer chemoresistance. miRNAs regulate numerous signaling pathways and regulatory networks, therefore even little changes in miRNA expression can have a big impact on the development and the progression of the disease. Targeting miRNAs-either reducing or enhancing their expression-seems promising to develop novel, more effective, and customized treatments, boost therapeutic efficacy, and predict patient response to various treatments. However, to fully explain all the miRNAs that are altered in tumors based on profiling data would be beyond the scope of this review. Numerous organizations are exploring the use of microRNAs as potential therapeutics. In vivo and translational investigations are currently the focus of increased research. Evidence exists that points to miRNAs as possible therapeutic agents, particularly when used in conjunction with anti-cancer chemotherapeutics. This could take the form of mimics that support miRNA function and expression or antagonists that block miRNA expression. By affecting the expression of endogenous microRNAs in cancer cells, miRNA mimics or anti-miRNAs can potentially change chemotherapy's efficacy. Two Types of microRNA delivery techniques employed in cancer therapy.  (213). Another phase 1 clinical trial including individuals with liver cancer or metastatic cancer with liver problems is MRX34 (a mimic of the tumor suppressor miR-34). Healthy volunteers and patients with advanced or metastatic liver cancer (hepatocellular carcinoma) are being tested for the safety and effectiveness of MRX34 in this study (214). Future possibilities for these novel medicines are encouraging given the encouraging preliminary findings from both trials. Challenges in the field of miRNA therapy-As mentioned above, the miRNAs can be delivered by either local or systemic approaches. It might not be a suitable strategy, for advanced cancer. However, miRNA cancer therapy works well with systemic delivery. Figure 6 summarizes the various constraints to miRNA delivery. For instance, poor miRNA penetration is caused by the leaky nature of aberrant tumor vasculature (215). The rapid cleavage of naked miRNAs by serum nucleases of the RNase A type poses another challenge (216). Additionally, there is a rapid renal clearance, notably for naked miRNA (217). When utilizing big NPs (>100 nm), reticuloendothelial system (RES) clearance would rise in the liver, spleen, lung, and bone marrow, leading to nonspecific absorption by innate immune cells such monocytes and macrophages (218).
Additionally, the systemic miRNA distribution triggered the innate immune system, as with other nucleic acid types, which resulted in undesired toxicities. Immune system activation includes the release of inflammatory cytokines and Type I IFNs via Toll-like receptors (TLRs) (219). Anti-inflammatory miRNA treatment, however, may prevent the activation of inflammatory pathways (220). On the other hand, some miRNAs work through TLRs to trigger neurodegeneration. For instance, Lehmann et al. (2012) demonstrated that miRNA let7b can cause neurotoxicity by activating TLR7 signaling in neurons (221). Therefore, a significant issue for miRNA systemic cancer therapy is the incidence of miRNA-related neurotoxicity. Additionally, increased miRNA uptake in cancer cells is a problem, and methods to address this issue include increasing endosomal escape and releasing miRNA payloads into the cytoplasm.
Off-target effects brought on by the miRNA mode of action are yet another challenge for miRNA delivery systems. These compounds may have undesirable side effects since they may bind to the 3′-UTR of a number of genes and decrease their expression (222). A developed method to lessen these adverse effects is the use of multifunctional co-delivering systems (223). Furthermore, it was demonstrated that under specific circumstances, such as hypoxia, the activity of miRNA processing enzymes such RISC reduces, which lowers the expression of tumor suppressor miRNAs (224). De Carvalho Vicentini et al. (2013) suggest that altering the expression or activity of these enzymes is another method for suppressing miRNA (225).

Conclusion
The discovery, development, and enhancement of miRNAs as potential medicines for the treatment of breast cancer patients have received significant funding, yet this branch of translational research is still in its infancy. Numerous attempts have been made to tailor cancer therapies using miRNA, but little progress has been made in improving clinic-oncological outcomes using miRNA targeting. miRNA therapies are now facing several developmental obstacles. This study is constrained by the fact that most of the research done thus far provides information about invitro trials, with very few studies coming from sources other than animal or breast cancer cell lines. Clinical trials assessing the clinical FIGURE 6 Challenges in miRNA delivery. effectiveness, risk profiles, and premium benefit are necessary for addition to the generally accepted scientific technique to support the initial findings of these recent investigations. An in-depth discussion of how clinical trial research has transformed BC patient care over the last four decades is provided in the current review. This research has produced novel, individualized therapeutic approaches, minimally invasive surgical techniques for the breast and axilla, and improved clinico-oncological results for patients who might otherwise have died from their disease in earlier times. The personalization of BC patient care appears to be closer than ever thanks to ongoing trials evaluating cutting-edge targeted therapies like immune checkpoint modulation (39, 226) and the use of poly(adenosine diphosphate-ribose) polymerase inhibitors (or PARP inhibitors) in the treatment of early-stage breast cancer in BRCA mutation carriers (227).
Hence, before we can use miRNAs in the therapeutic setting, numerous obstacles remain to be overcome. The delivery method is the key impediment. We might be able to get over this obstacle with the use of chemical alterations, viral vectors, or nanoparticles. Despite these delivery issues, it is possible that miRNAs will play a significant role in cancer therapy, including BC, in the future. A novel approach to treating breast cancer that combines miRNA therapies with conventional chemotherapeutic techniques and drug targets is possible, but further study is needed before this promising paradigm can be implemented in the clinic. Thus, this review emphasizes how important it is to prioritize clinical trials and therapeutic interventions to advance the precision oncology movement's goal of "curing" breast cancer.