Xiangrong Liu1†
Dimitri Papukashvili2†Zhixiang Wang1†
Yan Liu1Xiaoxia Chen1
Jianrong Li1Zhiyuan Li1Linjie Hu1Zheng Li1
Nino Rcheulishvili2Xiaoqing Lu1*Jinfeng Ma1*- 1Shanxi Province Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, China
- 2Department of Pharmacology, School of Medicine, Southern University of Science and Technology, Shenzhen, China
Breast cancer (BC) remains the most prevalent malignancy due to its incidence rate, recurrence, and metastasis in women. Conventional strategies of cancer detection– mammography and tissue biopsy lack the capacity to detect the complete cancer genomic landscape. Besides, they often give false- positive or negative results. The presence of this and other disadvantages such as invasiveness, high-cost, and side effects necessitates developing new strategies to overcome the BC burden. Liquid biopsy (LB) has been brought to the fore owing to its early detection, screening, prognosis, simplicity of the technique, and efficient monitoring. Remarkably, microRNAs (miRNAs)– gene expression regulators seem to play a major role as biomarkers detected in the samples of LB. Particularly, miR-21 and miR-155 among other possible candidates seem to serve as favorable biomarkers in the diagnosis and prognosis of BC. Hence, this review will assess the potential utility of miRNAs as biomarkers and will highlight certain promising candidates for the LB approach in the diagnosis and management of BC that may optimize the patient outcome.
Introduction
Breast cancer (BC) is the most malignant and emergent tumor among women cancer patients (1, 2). Millions of women are diagnosed with BC and over half a million deaths are reported annually (3, 4). There are four main molecular subtypes of BC– luminal A, luminal B, human epidermal growth factor receptor 2 (Her2)-enriched, and basal-like (5). The first two subtypes usually have a more favorable prognosis. The most common subtype- luminal A is characterized by the expression of estrogen and progesterone receptors (ER/PR) while the luminal B subtype is additionally characterized by the absence of Her2. In the Her2-positive subtype, the Her2 gene is highly expressed and the cell proliferation rate is also high. Basal-like subtype does not express any of the mentioned markers and accounts for up to 20%. Additionally, there is one more subtype– normal-like subtype which represents the rarest BC and accounts for only up to 10% of all BCs. The normal-like subtype is characterized by the expression of ER, PR, and Her2 and clinically it is between basal-like and luminal A subtypes (6). The early diagnosis and advancements in treatment are the main focus of BC research. Mammography and tissue biopsy remain the standard screening methods until now (7). However, a number of disadvantages exist. E.g., the false-positive result of mammography requires additional analysis that may lead to potential side effects (8) while the eventual result may be negative. Except for the mentioned, mammography is related to exposure to ionizing radiation (9). Mammography imaging often is not sufficient for evaluation and requires further analysis. It is often performed along with tissue biopsy. This requires the imaging to be done before and after the biopsy to ensure the accuracy of sampling and biopsy marker placement. This technique is advantageous for its specificity to the suspicious tissue which is detected on a mammogram. Nonetheless, the disadvantages include a painful, long time of the procedure, invasiveness, and high-cost (10). Moreover, it is not adequately comprehensive to obtain the complete landscape of BC (11). Fortunately, a relatively new approach liquid biopsy (LB) that is a non-invasive and simple technique compared with surgical biopsies enables obtaining the important information of tumor via simple body fluids-based samples, mainly blood. The other advantages comprise the short time of the procedure, precise and real-time results, serial sampling, and monitoring. This enhances its application for the early diagnosis that enables better management of BC, better outcome, and, most importantly, less mortality (11). Indeed, the application of LBs has revolutionized the existing standard clinical approach and may play a critical role in diagnosing and monitoring the tumor as well as the response to the treatment. LB next-generation sequencing (NGS)-based FoundationOne Liquid CDx test was approved by Food and Drug Administration (FDA) in 2020 for application for diagnosis via detecting multiple tumor biomarkers in plasma (12). LB is undoubtedly considered one of the most perspective detection approaches for many cancers including BC. Indeed, LB allows the detection of circulating tumor components such as cancer cells, RNA, or circulating tumor DNA (ctDNA) in liquid specimens (13). Despite their low concentrations in the liquid samples, they still can be used as indicators and biomarkers of cancer. This characteristic makes LB a sensitive, advanced, alternative, reliable, and cost-effective approach for the diagnosis and screening of BC (14). Remarkably microRNAs (miRNAs) detected in LB specimens seem to be promising measurable indicators of BC. The change in their levels reflects the various conditions of the body. miRNAs are small non-coding RNAs that serve as gene-regulator molecules in the body. As miRNAs are implicated in various signaling pathways, alteration of their levels indicates certain conditions, e.g., various cancers (11, 15–17). Indeed, proof-of-concept studies demonstrated that the composition of miRNAs and their abundance in the blood are altered in cancer patients (11, 18, 19). Additionally, Li et al. have studied miRNAs in BC patients and demonstrated that 13 miRNAs were found to be differentially expressed in patients with metastatic BC (20). Kim et al. successfully monitored the expression levels of miR-21 and miR-155 in the urinary samples of mice injected with BC cells (21). Hence, in this review, we summarize recent and relevant data on circulating miRNAs (c-miRNAs) for their potential role in the diagnostics of BC to become a part of the LB approach in clinics. A schematic illustration of the potential utility of miRNAs detected in body fluids as biomarkers for BC diagnosis and prognosis is given in Figure 1.
Figure 1 Schematic illustration of body fluids-based miRNA utility as biomarkers in breast cancer diagnosis and prognosis. The body fluids are the source of miRNAs that can be obtained via liquid biopsy. Bold-type indicates the most promising and studied body fluid for detecting miRNAs in breast cancer. Several listed biofluids have not yet been studied for miRNA detection as biomarkers in breast cancer, still, they represent potential liquid biopsy candidates.
miRNA role in cancers
Since the identification of miRNA in 1993, it is gaining growing attention. Currently, over 3000 miRNAs are found in almost all human body fluids. These miRNAs are implicated in various processes and play a significant role in the progression or regression of a number of diseases (22–26). miRNAs vary in their function as they have a strong capacity of regulating multiple gene expression. miRNAs have a significant impact on tumorigenesis, apoptosis, embryogenesis, cell proliferation, and differentiation among many other biological processes (27). miRNAs regulate gene expression via the post-transcriptional mechanism (6). The biogenesis of miRNA consists of several steps and takes place in the nucleus and cytoplasm of the cell. First, the primary miRNA (pri-miRNA) is transcribed mostly from introns into the nucleus and cleaved by endonuclease Drosha which results in the formation of precursor miRNA (pre-miRNA). Pre-miRNA is then translocated into the cytoplasm by Exportin 5 (XPO5) where it undergoes further processing and the mature miRNA duplex is formed. Argonaute (AGO) protein family recognizes miRNA and leads to the loading of miRNA guide strand into the RNA-induced silencing complex (RISC). The passenger strand is degraded and approximately 22 nucleotide-length miRNA containing seed region is ready to bind the specific region of the target messenger RNA (mRNA) molecule on the 3’untranslated region (3’UTR) that consequently leads to the silencing of the target gene (22, 28). Remarkably, miRNAs are implicated in the regulation of key pathways that are usually genetically altered in various cancers. As cancer represents a disease when the normal cells become abnormal via acquiring the ability of uncontrollable growth and division, miRNAs that are regulators of cell proliferation, differentiation, and apoptosis, play a crucial role in this condition. Indeed, several signaling pathways have been identified to be actively involved in cancers– RTK/RAS/MAP pathway, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (29), Wingless (WNT)/β-catenin (30), etc.
OncomiRs, tumor suppressors
According to the role in cancer, miRNAs elicit either oncogenic or tumor-suppressive functions via silencing target genes. Therefore, they may be considered oncomiRs or tumor suppressor miRNAs (6). miRNAs that are associated with cancer due to their upregulated levels are called oncomiRs (31, 32). Interestingly, tumor angiogenesis is the formation of new blood vessels that helps the tumor to grow via supplying with the blood (33) and plays a key role in cancer development. It is a complex process and comprises activation, migration, proliferation, and differentiation of the endothelial cell. There are numerous factors that control each of these steps positively or negatively (34). A number of miRNAs may act as pro-angiogenic or anti-angiogenic factors. Evidently, upregulation of miR-155 increases proliferation, invasion, migration, and tube formation in triple-negative BC (TNBC) and other cancers via targeting tumor suppressor von Hippel-Lindau (VHL) (35). TNBC is a type of BC with no commonly found BC receptors– PR, ER, and Her2. Elevated miR-210 expression increases the endothelial cell migration and its levels in BC represent a prognostic factor (34, 36). miR-182 promotes invasion and proliferation via inducing vascular endothelial growth factor A (VEGF-A) in BC (37). miR-10b is the first miRNA with oncogenic action that was discovered in metastatic BC cells. Its expression levels are positively correlated with metastasis and invasion as the action of miR-10b induces overexpression of RAS homolog family member C (RHOC) via inhibiting homeobox D10 (HOXD10) mRNA translation (38). Another miRNA miR-21 also influences the oncogenic mechanism via inhibiting tumor suppressor genes. It promotes the growth and metastasis of tumor cells. Most importantly, miR-21 is the most commonly found oncomiR in BC. Its upregulation is directly correlated with the poor prognosis of BC (38, 39) and angiogenesis (34, 40). The significance of miR-21 is conditioned by a number of target genes that are affected by miR-21 (38). On the contrary, certain miRNAs act like tumor suppressors inhibiting tumor cell growth and preventing metastasis. miR-335 is one of the tumor suppressor miRNAs that inhibits tumor growth via inducing apoptosis (41). miR-16, miR-26a, and miR-101, among a number of other miRNAs, are reported to elicit tumor-suppressive properties in BC (15).
Role of miRNAs in breast cancer
A number of miRNAs have been reported to have a regulatory function in the pathways implicated in the development of BC. Indeed, certain expression levels of certain miRNAs, e.g., miR-222, are increased in the serum of patients with BC (6). Hannafon et al. have demonstrated remarkably high concentrations of miR-21 and miR-1246 in exosomes obtained from plasma of BC patients compared with healthy controls (42). Si et al. have assessed the impact of miR-21 on tumorigenesis via transfecting anti-miR-21 antagomiR into the MCF-7 BC cells and demonstrated that the cell growth was inhibited via augmented apoptosis in MCF-7 cells as well as in xenograft mouse model. Interestingly, the decreased cell proliferation was associated with the downregulation of B-cell lymphoma 2 (Bcl-2) protein (43). Zhao et al. observed a similar outcome after miR-21 knockdown via antagomiR-21 in a mouse breast tumor model. Moreover, the results revealed that anti-miR-21 suppressed angiogenesis via inhibiting HIF-1A/VEGF/VEGFR2-associated signaling pathway (44). Wang et al. have demonstrated the augmented levels of miR-21 in BC patients compared with patients with benign breast tumors and healthy subjects while significantly reduced levels of miR-21 were found in the patients after surgery. Moreover, inhibition of this miRNA impeded tumor progression in BC. Remarkably, leucine zipper transcription factor-like 1 (LZTFL1) was found to be a direct target of miR-21 (45). Therefore, miR-21 may be considered an important pro-angiogenic miRNA (46). miRNAs such as the cluster miR-17-92 are evidenced to be implicated in BC angiogenesis and are called oncomiR-1 (33). miRNAs involved in the angiogenesis of cancers are listed in Table 1. There is a number of other miRNAs implicated in various stages of BC metastasis. These miRNAs include miR-9, miR-10a, miR-10b, miR-93, miR-125b, miR-155, miR-181d-5p, miR-191, miR-200, miR-205, miR-221/222, miR-374a, miR-375, miR-378e, etc. (31). There are anti-angiogenic miRNAs that act as tumor suppressors, e.g., miR-7, miR-29b, miR34a, miR124, miR153, miR141, miR-148a, miR-152-3p, miR-205, miR-497, etc. (55). miR-155 is found to be abnormally overexpressed in BC tissues and is associated with advanced tumor stages as well as metastasis (56). On the contrary, miR-497 is found to diminish the tumor development and formation of endothelial cell tube in BC and it was found to be downregulated in BC (57). miR-497 could be considered anti-angiogenic miRNA. Hence, a number of miRNAs are important components of BC angiogenesis which also makes them potential therapeutic targets (55). miRNAs induce tumor progression or suppression via regulating gene expression implicated in certain signaling pathways. For instance, miRNAs of the miR-200a-c family represent tumor suppressor miRNAs. miR-200a is found to be under-expressed in BC while its increased levels interfere with WNT/β-catenin signaling which is one of the leading pathways implicated in tumor cell proliferation (58). Pathways including PI3K/AKT, GAS6/MERTK, RTK/RAS/MAP-kinase, and TGF-β signaling are being affected by certain miRNAs that leads to the regulation of tumor progression or suppression (58). According to the abovementioned, there are a number of miRNAs that can be used as potential therapeutic targets for BC. miRNAs involved in inhibiting angiogenesis via targeting pro-angiogenic genes are listed in Table 2.
Table 1 Pro-angiogenic miRNAs in breast cancer and their target genes.
Table 2 Anti-angiogenic miRNAs in breast cancer and their target genes.
Liquid biopsy in breast cancer
The main barrier to advancing BC treatment is the complex heterogeneity of breast tumors (66). For BC diagnosis, a solid biopsy is a well-established technique in clinics that provides information about tumor histology and subtype. Nonetheless, mishaps in the detection of alternative biomarkers, insufficient tissue obtention, or incompatibility with long-term monitoring, among other limitations still exist (5). Compared with solid biopsy, LB is a more comprehensive diagnostic test that uses blood, urine (67), saliva, and other biological fluids (68, 69). After the sampling circulating tumor nucleic acids and cells are extracted and further analyzed (68, 70). The major tumor component that is usually extracted for the identification of tumor characteristics is circulating tumor cells (CTCs) (71). Except for the CTCs, cell-free DNA (cfDNA), ctDNA (72, 73), and c-miRNAs among other circulating non-coding RNAs (74) can be used as LB markers (13). The advancement of NGS and digital genomic approaches made biomarkers such as c-RNAs, circulating extracellular vesicles, tumor-educated platelets, proteins, etc. possible to detect in biofluids (71, 75, 76). Detection and identification of specific biomarkers are essential for the early diagnosis of BC and other cancers as they can provide additional insight into diagnosis, prognosis, and the response to treatment. In order to achieve this, LB represents a favorable approach. Additionally, in LB, invasiveness and residual disease are minimized that also enables and simplifies the longitudinal monitoring of BC patients. LB potentiates obtaining the complete molecular picture of the tumor and, thus, enhances more precise clinical decisions (5, 77). Indeed, a number of studies successfully consolidate the significance of LB in BC (78–81) and other cancers too (82, 83). Biomarkers detected via LB in biofluid samples provide an attractive alternative to biomarkers identified in tissues. The number of body fluids and the existence of all the mentioned potential biomarkers in it as tumor components warrant their promising utility in BC diagnosis, prognosis, and discrimination of different BCs. E.g., serum biomarkers are especially attractive because of the simplicity of sample collection (13). The schematic illustration of the importance and feasibility of LB in BC diagnosis and prognosis is given in Figure 2.
Figure 2 Advantages of liquid biopsy utility in breast cancer management via using various samples of body fluids. The biofluids written in bold indicate the most studied samples for miRNA detection in breast cancer. Several listed biofluids have not yet been studied for miRNA detection as biomarkers in breast cancer, still, they represent potential liquid biopsy candidates.
Diagnostic and prognostic function of miRNAs in breast cancer
Current biomarkers for BC such as CA15.3 and BR27.29 lack enough sensitivity which necessitates seeking the new biomarkers (84). miR-19a, miR-21, miR-24, miR-155, and miR-181b, are found to be overexpressed in patients with BC and downregulated after the surgery or treatment. This also exacerbates the concept of miRNAs’ application as a biomarker for BC diagnosis (45, 84). Henaghan et al. have studied the miRNA expression in the serum of patients with various cancers. The study revealed that certain miRNAs were specific to certain cancer types. E.g., miR-195 was abnormally increased in patients with BC (85). The downregulation of miR-329 in serum and tissue of patients with BC is associated with metastasis to lymph nodes while upregulation of miR-200 is correlated with metastasis to the brain, liver, and lung (84). Indeed, there are a number of studies that demonstrate the potency of certain miRNA utilization as BC biomarkers in various samples (56, 86–93). Apparently, miRNAs maintain the healthy balance of signaling pathways for the normal function of the body. The dysregulation of their normal expression may lead to the progression of the BC. This makes the c-miRNAs attractive biomarker candidates. Application of miRNAs as non-invasive biomarkers in BC allows not only timely diagnosis and successful monitoring of the disease (38), but it also has a discriminatory capacity for, e.g., metastatic and non-metastatic cancers (75). Moreover, in some cases, the miRNA panel demonstrates better discrimination (75, 94). Many individual miRNAs or miRNA combinations associated with certain tumors have been successfully detected in various biofluids such as uterine (95) and cervical (96, 97) fluids, serum, bile (98, 99), etc. Song et al. have screened breast milk for cfDNA and miRNAs and demonstrated that both of these molecules are stably present in breast milk. As they are derived from breast cells they might serve as biomarkers sampled non-invasively (100). Interestingly, levels of miR-155 which is strongly associated with BC were increased in urine samples of BC patients while the expression levels of other miRNAs– miR-21, miR-125b, and miR-451 were significantly lower in the urine samples compared with healthy controls (101). Interestingly, nipple aspirate fluid (NAF) as one of the best samples for detecting miRNAs deserves attention. NAF is an intraductal mammary physiological fluid and represents a source of many biomarkers including miRNAs (102). A number of advantages including its obtainability in the majority of women, the origin of BC, simplicity, and non-invasiveness of sampling, make it a valuable source of miRNAs and LB target in BC diagnosis (102, 103). There are other body fluids where miRNAs have been detected and associated with certain cancers or disorders– saliva (104–106), sweat (107), tear (108–111), and stool (112, 113). Besides, fingernails are also found to be a promising sample for the detection of miRNAs (114). Importantly, the characteristics such as the area under curve (AUC), sensitivity, and specificity should be considered while selecting the most suitable biomarker for BC diagnosis. AUC evaluates the diagnostic capability of studied candidate miRNA in BC detection. When the AUC is close to one, the diagnostic value of miRNA is higher as there are substantial differences between BC and negative control samples. Additionally, while assessing the miRNA diagnostic potential, sensitivity, and specificity that indicate the accuracy of biomarkers also need to be considered (115, 116). E.g., a study revealed that four plasma miRNA panel comprising miR-24, miR-206, miR-373, and miR-1246 could discriminate BC patients from healthy subjects with 96% specificity, 98% sensitivity, and 97% accuracy (117, 118) while the combination of miR-142-5p and miR-320a could distinguish luminal A subtype from healthy individuals with 100% sensitivity, 93.80% specificity, and AUC 0.94 (117). The abovementioned information indicates that c-miRNAs found in various body fluids are strongly correlated with the health state and may serve as attractive biomarker candidates for the diagnosis and prognosis of BC (116) (Figure 3). Broad information on c-miRNAs with a significant role in BC is combined in Table 3. Apparently, the technique of detecting miRNAs for liquid biopsy is based on the following: after obtaining the liquid sample of a BC patient, total RNA is extracted, cDNA is synthesized, and RT-qPCR is performed to detect the relative expression of target miRNAs.
Figure 3 Representative miRNA candidates with the potential of biomarker function in breast cancer diagnosis and prognosis.
Table 3 Summary of recent studies since 2015 on altered levels of circulating miRNAs in breast cancer.
miR-10b
miR-10b via binding to the mRNA of HOXD10 gene in 3’UTR inhibits its translation and gives rise to the upregulation levels of RHOC, therefore, invasion, and metastasis (38), as well as angiogenesis, are promoted in BC (48). Indeed, a number of studies have demonstrated that the levels of miR-10b are overexpressed in the serum of BC patients which makes this particular miRNA a promising target for developing a non-invasive and cost-efficient BC LB detection approach (129, 144, 209, 212). Ibrahim et al. have studied the expression levels of certain miRNAs in plasma during different steps– diagnosis, chemotherapy, and after the tumor resection in BC patients. According to their finding, it is suggested that the upregulation of miR-10b might be the diagnostic marker of BC (144). Mangolini et al. have also demonstrated the significance of miR-10b as a biomarker of tumor aggressiveness. Particularly, the study showed that serum levels of miR-10b-5p were increased significantly along with the tumor stage while there was no substantial difference observed between the miR-10b-5p expression levels in stage I and control subjects (209). Despite the evidence, there are more studies needed to strengthen the idea of miR-10b utility as a BC biomarker via the LB approach.
miR-16
miR-16 is evidenced to be overexpressed in the blood of BC patients (127, 186). Indeed, Kim et al. have demonstrated the upregulation of miR-16 along with several more miRNAs in EVs of early-stage BC patients compared with the healthy subjects which indicates the involvement of miR-16 in tumor initiation. Their results suggested the promising outcome of the tested miRNA signatures for BC early diagnosis (127). On the contrary, Hu et al. showed no difference in the expression levels of miR-16 in BC patients and healthy subjects (177). The current evidence warrants the need for more and comprehensive studies on this miRNA to validate its role as a biomarker for LB in BC patients.
miR-17
Despite a number of studies focusing on c-miR-17 expression regulation, there is no consistency in its expression levels in BC patients (176, 187, 194, 195). Hesari et al. have studied the changes in the levels of miR-17 in the serum of BC patients compared with control subjects and found that its upregulation is associated with augmented cell proliferation and poor survival time in patients (176). Swellam et al. have also revealed the concordance of the increased miR-17-5p serum levels and clinical stages that indicates its implication in cancer progression. Furthermore, the detection technique of miR-17-5p along with miR-155 and miR-222 with RT-PCR was superior to the tumor markers such as carcinoembryonic antigen (CEA) and Cancer antigen 15-3 (CA15.3) for BC early diagnosis (187). Conversely, Jurkovicova et al. have shown decreased levels of plasma miR-17 in BC patients compared with healthy controls (194). Hence, according to the current evidence, it is difficult to postulate whether this particular miRNA can serve as a biomarker candidate for the diagnosis and prognosis of BC.
miR-20b
It is suggested that miR-20b plays a critical role in BC tumorigenesis. miR-20b is shown to have pro-angiogenic functions via inhibiting HIF-1A, STAT3 (49), and PTEN (50). Besides, it is also evidenced that its circulating levels are downregulated in BC patients (174, 195). This makes miR-20b-5p an interesting biomarker target for LB in BC.
miR-21
Noteworthily, miR-21 is abundantly found oncomiR in BC and its augmented level is strongly associated with the poor prognosis of BC (38, 39). Based on recent studies miR-21 represents a key target for most scientists in terms of its dysregulated circulating levels in BC. Indeed, a number of studies have demonstrated that c-miR-21 levels are found to be significantly augmented in BC patients compared with the healthy controls (90, 123, 127, 144, 145, 150, 162, 167, 169, 173, 177, 185, 186, 191, 201, 204, 207, 213). Interestingly, both– miR-21-3p and miR-21-5p are found to be upregulated in BC (177). Moreover, miR-21 seems to promote tumor progression via inducing cell growth, invasion, and metastasis (40). Therefore, miR-21 is indeed suggested to be one of the most attractive candidates for LB approach development in BC. According to the abovementioned information miR-21 also represents a pro-angiogenic molecule (46). Indeed, the study demonstrated that the miR-21 knockdown with anti-miR-21 resulted in the suppression of angiogenesis and cell proliferation via inhibiting the HIF-1A/VEGF/VEGFR2-associated signaling pathway in mice with implanted 4T1 murine BC cells (44). Yu et al. have also validated the advantages of miR-21-3p and miR-21-5p as potential biomarkers for BC early detection (173). Similarly, Matamala et al. have hypothesized miR-21-5p as a biomarker candidate for non-invasive BC detection (207). Subsequently, miR-21 is the most studied oncomiR in BC and the results of various studies are consistent. Thus, miR-21 deserves more attention that may change the standards in BC detection and become the first approved c-miRNA-based biomarker for BC detection.
miR-30b
The circulating levels of miR-30b are also noticed to be dysregulated in patients with BC. Particularly, its levels are reported to be augmented (120, 130, 161, 199). Indeed, Adam-Artigues et al. investigated the plasma levels of miR-30b and found it to be a valuable non-invasive diagnostic biomarker for BC. miR-30b may aid in the discrimination of stage I BC without clinical manifestation from the plasma samples which are obtained from healthy subjects (120). Zhang et al. have also found the upregulated miR-30b-5p to be a promising biomarker candidate for the diagnosis of BC in patients with very early-stage BC (199). Hence, miR-39b is suggested to be one of the candidates for LB approach development in BC detection, although, more studies are required for the complete validation of this hypothesis.
miR-99a
c-miR-99a is reported to be significantly upregulated in BC patients (120, 125, 143, 147). However, Yu et al. identified a three-miRNA panel that might be a biomarker for early BC detection, and the levels of serum miR-99a-5p were found to be downregulated in BC (173) which causes discrepancy and requires validation with more studies before it is considered as a biomarker candidate for the diagnosis of BC via LB approach.
miR-103a
miR-103a is suggested to be a biomarker for BC detection as its circulating levels are reported to be increased in patients with BC (86, 210). Liu et al. have investigated the circulating levels of miR-103a-3p in patients with BC and found them to be significantly upregulated when compared with the healthy subjects. Moreover, its expression levels in patients with positive Her2 status were higher than in subjects with Her2-negative status and patients with metastases exhibited higher levels of miR-103a-3p compared with the subjects without metastasis (86). Sahlberg et al. have suggested miR-103a-3p along with other miRNAs as a minimally invasive biomarker for tumor relapse in patients with TNBC (210). However, the studies are not sufficient enough until now and more cohorts are vital to validate its application in LB for BC detection and overall management.
miR-125b
Despite the lack of studies on c-miR-125b-5p, there is no inconsistency in its levels in BC patients. It is reported that miR-125b-5p levels are upregulated in the plasma of BC (8, 168, 207). Incoronato et al. have investigated the BC-associated miRNAs and found miR-125b-5p to be upregulated in the plasma of BC patients and could distinguish BC patients from healthy controls (168). miR-125b-5p provides information about prognosis and, therefore, the potential use of this miRNA is suggested as a non-invasive BC biomarker after further validation with more studies is performed.
miR-155
Evidently, miR-155 plays an important role in BC development. Via targeting the VHL gene it exhibits a pro-angiogenic role in BC tumorigeneses. miR-155 is shown to be upregulated in the blood of BC patients by a number of studies (149, 186, 187, 197, 212, 216). Anwar et al. studied the dynamic changes of c-miR-155 expression levels in BC patients and according to the results they concluded that women with BC expressed significantly higher levels of c-miR-155 compared with the healthy subjects while after the surgery and chemotherapy, its levels were found to be downregulated. Moreover, the expression levels were higher in patients with larger-size tumors (149). Fan et al. demonstrated that the serum level of c-miR-155 may serve as a biomarker for BC early diagnosis as well as discrimination of BC molecular subtypes (186). The same results were shown by Hagrass and colleagues who also concluded the potential of miR-155 utility as BC biomarkers (212). Kong et al. have demonstrated that except for promoting the angiogenesis, the upregulation of miR-155 promotes BC metastasis (35). Gao et al. suggested that the augmented levels of miR-155 seem to be associated with the initiation of the tumor but not the metastasis and that miR-155 may serve as a non-invasive biomarker for early-stage BC detection (197). Accordingly, miR-155 undoubtedly deserves further attention for its role in BC non-invasive detection via the LB technique.
miR-200c
There is evidence demonstrating that circulating levels of miR-200 are augmented in BC patients (169, 185, 196), which makes it an attractive biomarker candidate for BC diagnosis/prognosis. Niedźwiecki et al. have investigated the serum levels of miR-200c and its association with TNCB. They compared the expression levels of c-miR-200c between TNBC and ER/PR-positive group and demonstrated that its circulating level was lower in patients with TNBC (179). As there are still discrepancies regarding its levels in BC patients, more studies are warranted for the validation of its utility as a biomarker molecule.
miR-210
Interestingly, miR-210 targets the EFNA3 gene which makes it pro-angiogenic miRNA in BC tumorigenesis (54). Besides, a number of studies also demonstrate that the circulating levels of miR-210 are increased in BC patients (89, 138, 160). Qattan et al. have revealed that the significantly augmented levels of miR-210-3p which is implicated in certain signaling pathways are specific to the TNBC subtype of BC (138). The consistent results of the reported studies support the miR-210 utility as a promising biomarker molecule for the non-invasive detection of BC.
miR-222
It has been observed that the levels of miR-222 are increased in the blood of patients with BC (133, 162, 181, 182). A study has revealed the significantly increased plasma levels of miR-222-3p in inflammatory BC patients (133). Similar results were shown in other studies (162, 181). Zhu et al. have observed the overexpression of miR-222 in BC and demonstrated its potential role as a predictive biomarker of the response to neoadjuvant chemotherapy in HR-positive/Her2-negative BC (182). Evidently, no controversy on the levels of this miRNA in different studies has been noticed. However, in order to strengthen this claim and make miR-222 a reliable biomarker candidate, more studies are required.
miR-1246
Based on the evidence, miR-1246 is also suggested to exhibit a biomarker role in BC detection, thus, it may represent one of the key targets of LB development in BC. miR-1246 is shown to be upregulated in the blood of BC patients (122, 178, 189, 203). However, not all the studies are consistent (118). Thus, more cohorts are required to confirm the reliability of miR-1246 as a potential biomarker candidate for LB development in BC.
let-7
miRNA let-7 (lethal-7) was discovered in C. elegans and, evidently, the abnormal expression levels and dysregulation of c-let-7 are observed in BC patients. Nevertheless, it is noteworthy that these studies are not consistent about the levels of c-let-7 in BC patients as some evidence its upregulation (193) while some show the downregulation (191). Elghoroury et al. have revealed that the downregulation of let-7 expression level is associated with the risk of metastasis in BC patients and, thus, let-7 is a promising biomarker of the BC prognosis and progression (191). Importantly, more studies have demonstrated its decreased levels (133, 144, 150, 191, 208) in the blood of BC patients which makes it an attractive biomarker candidate for LB.
Challenges of liquid biopsy in breast cancer
Despite the number of advantages of LB in BC compared with tissue biopsy (217), some challenges of this approach still exist. E.g., because of the low concentration in the blood, the isolation and detection of CTCs are technically complicated which necessitate developing a specific method for it (75). In case of cfDNA application as a diagnostic biomarker, the major obstacle is the need for knowledge about tumor-specific variants (218). As to the ctDNA, its low concentration in the early stages of cancer makes the detection technically difficult while, on the other hand, the somatic mutations in blood stem cells become the source of the background noise and often lead to inaccurate interpretation of LB results (219). The main challenge for the application of c-miRNAs as biomarkers in BC diagnosis and prognosis is the detection and accurate quantification difficulties that are conditioned by their low abundance and small size in biofluids (220). Other than blood samples, there are insufficient studies on miRNAs detected in various biofluids (NAF, breast milk, saliva, urine, cervical fluid, etc.) for BC detection. Besides, more sensitive detection methods are needed for facilitating LB application in clinical settings. Additionally, the biology of each particular type of BC needs to be considered as different miRNAs exhibit different expression levels depending on the tumor types (116). This makes their use more challenging. Moreover, considering the huge number of miRNAs potentially and actually implicated in different BCs along with their complexity, there still is a lack of evidence to finally implement their utility as LB-detected biomarkers. Although a number of pre-clinical and clinical studies (Table 4) have been performed on c-miRNA dysregulation in BC for LB approach advancement, the most suitable miRNAs or miRNA panels that could be employed in clinics remain controversial. There are conflicting studies on certain c-miRNAs in BC, e.g., there is inconsistent evidence regarding the levels of miR-145. Some studies demonstrate its upregulated levels (126, 141, 170) while the others report its downregulation in BC (144, 148). This makes miR-145 a nonreliable target for the LB approach in BC diagnosis and prognosis. As well, miR-205-5p together with miR-194-5p is evidenced to exhibit a biomarker role in the detection of BC brain metastasis. miR-205-5p expression level is upregulated in 4T1-injected mice plasma compared with the control (124). However, there is no consistency in miR-205 levels in BC detection (215, 216). According to Zhang et al., miR-205 expression levels in the serum of control subjects are higher than in subjects with stage I and II (215) while Shaker et al. demonstrated significantly augmented serum levels of miR-205 in BC (216). Nevertheless, despite the mentioned shortcomings, single as well as panel miRNAs remain remarkably promising biomarker candidates for the early diagnosis and prognosis of BC.
Table 4 Clinical trials on breast cancer involving circulating miRNAs (sorted by study start date).
Summary
The current review aimed to collect the most relevant scientific findings regarding the LB technique in BC detection and discussed the potential attractive miRNAs for developing this non-invasive approach. In order to save time, minimize pain, increase efficiency, and overcome the rest of the obstacles which exist in the current screening methods, the development of LB for the early diagnosis of BC is critical. miRNAs, except for their function– post-transcriptional gene regulation, may also accomplish a biomarker role for the diagnosis and prognosis of BC via detecting their levels in various biofluids. Apparently, single miRNAs or miRNA panels have a great potential of improving the accuracy of clinical diagnosis and prognosis that will undoubtedly have a positive impact on BC management and treatment. Remarkably, some miRNAs including miR-21 and miR-155 have been demonstrated to play a major role in BC progression among other miRNAs which makes them special in BC management. Except for the evidence of increased blood levels of miR-21 and miR-155 in BC patients, it is shown that the urinary levels are also upregulated compared to healthy subjects. This makes these two miRNAs even more attractive targets for developing the LB approach in BC. Although a number of pre-clinical and observational/interventional clinical studies are focused on c-miRNAs for their utility for LB in BC, the consensus about the most suitable miRNA candidate or panel still remains a debate. Currently, there is no miRNA-based LB detection method employed in clinics yet. Therefore, in order to facilitate the development of the LB approach based on miRNAs in BC, more robust and well-designed studies including a large number of subjects are essential. Moreover, the utility of the LB approach is advantageous with its feasibility– minimal invasiveness, minimal pain, short procedure, simplicity, optimal for longitudinal monitoring, and cost-effectiveness. There is a considerable evidence that miRNA signatures may be employed successfully for LB in BC.
Author contributions
The Authors DP, XLiu, and ZW contributed to the study conception, YL, XC, JL, ZhiL, LH, and ZheL contributed in data collection, DP and NR wrote the sections and prepared the original draft of the manuscript, DP worked on the visualization of the manuscript, NR reviewed and edited the manuscript, XLu and JM supervised. All authors contributed to manuscript revision, read, and approved the submitted version.
Funding
This work was supported by the National Natural Science Foundation of China (82002820 and 82072740).
Acknowledgments
Parts of the figures were created with BioRender.com.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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Keywords: liquid biopsy, breast cancer, miRNA, biomarker, biofluids
Citation: Liu X, Papukashvili D, Wang Z, Liu Y, Chen X, Li J, Li Z, Hu L, Li Z, Rcheulishvili N, Lu X and Ma J (2022) Potential utility of miRNAs for liquid biopsy in breast cancer. Front. Oncol. 12:940314. doi: 10.3389/fonc.2022.940314
Received: 10 May 2022; Accepted: 04 July 2022;
Published: 04 August 2022.
Edited by:
Elisa Frullanti, University of Siena, ItalyReviewed by:
Viola Bianca Serio, University of Siena, ItalyElena Lastraioli, University of Florence, Italy
Vasyl Chekhun, National Academy of Sciences of Ukraine (NAN Ukraine), Ukraine
Copyright © 2022 Liu, Papukashvili, Wang, Liu, Chen, Li, Li, Hu, Li, Rcheulishvili, Lu and Ma. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xiaoqing Lu, luxiaoqing860227@163.com; Jinfeng Ma, mjinfeng99@163.com
†These authors have contributed equally to this work and share first authorship