Systematic characterization of non-coding RNAs in triple-negative breast cancer.

Abstract Triple‐negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer with negativity for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2). Non‐coding RNAs (ncRNAs) make up most of the transcriptome and are widely present in eukaryotic cells. In recent years, emerging evidence suggests that ncRNAs, mainly microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs), play prominent roles in the tumorigenesis and development of TNBC, but the functions of most ncRNAs have not been fully described. In this review, we systematically elucidate the general characteristics and biogenesis of miRNAs, lncRNAs and circRNAs, discuss the emerging functions of these ncRNAs in TNBC and present future perspectives in clinical practice.


| INTRODUC TI ON
In the past few decades, the morbidity of human breast cancer has increased continuously and has led to a great threat to women's lives. According to the statistics gathered by the American Cancer Society, there will be more than 271 000 new cases of breast cancer and approximately 42 260 deaths in 2019. 1 Being a heterogeneous disease, breast cancer can be classified into several main subclasses based on the expression status of oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2) and antigen ki-67 (Ki-67). 2 Among known subclasses, triple-negative breast cancer (TNBC) is the most aggressive subtype, which is characterized by negativity for ER, PR and HER2. Great efforts have been made to understand the mechanisms of TNBC carcinogenesis, especially focus on the role of non-coding RNAs (ncRNAs).
Non-coding RNAs make up most of the transcriptome, while protein-coding RNAs only account for 3% of the genome; the remaining 97% is composed of "dark matter" of transcripts with molecular functions. 3 It has been proven that the genome "dark matter" can be transcribed into various RNA species, most of which do not encode proteins, namely, ncRNAs, but exert significant functions mainly responsible for phenotypic regulation. 4 The emerging functions of ncRNAs have been generally determined in the cancer research field. Currently, studies of ncRNA-related cancer are commonly concentrated on miRNAs, lncRNAs and circRNAs. Here, we review the general characteristics and functions of ncRNAs and discuss their underlying mechanisms in the carcinogenesis and development of TNBC.

| C ATEG ORIE S AND G ENER AL CHAR AC TERIS TIC S OF N CRNA s
Depending on the number of nucleotides (nt), ncRNAs can be cat-  3 Although circRNA belonging to the lncRNA family, researchers tend to discuss them separately distinguishing from lncRNAs due to their unique structure. The general characteristics and functions of common ncRNAs are summarized in Table 1.

| Biogenesis of miRNAs
MicroRNAs are endogenous, non-coding small RNAs with approximately 20-24 nt in length which mainly participate in regulating gene expression. In the canonical biogenesis pathway, genes encoding miRNAs are transcribed into long initial transcript by RNA polymerase Ⅱ, namely pri-miRNAs, 300-1000 base pairs (bp) in length. 5 The pri-miRNAs are cleaved to pre-miRNAs (about 70 bp in length) with a stem-loop structure under the action of RNase III Drosha. 6 Next, the pre-miRNAs are transported from the nucleus to the cytoplasm by the Ran-GTP-dependent transporter Exportin 5. 7 The pre-

| MiRNAs cause mRNA degradation
MicroRNAs play significant regulatory roles in eukaryotes by binding to corresponding mRNA transcripts, leading to their degradation at mRNA level and/or translational repression. There is currently no clear conclusion on how miRNAs induce mRNA degradation. Two different perspectives have been issued currently: (a) ordinary mechanism of mRNA degradation. This view holds that miRNAs merely act as guides, they do not directly degrade mRNAs through AGO proteins, but only label and send mRNAs that need to be degraded to the normal mRNA degradation mechanism. Giraldez et al 8 found that miR-430 induced the deadenylation of its targets, resulting in the recruitment to processing bodies, where mRNAs were decapped and degraded. (b) MiRISC-mediated mRNA degradation. This view holds that a variety of related enzymes and complexes, such as AGO proteins, decapping enzymes and their cofactors, all of which may be involved in miRNA-mediated degradation of mRNAs. As a recognized mechanism, miRNAs bind to AGO proteins in miRISCs to recognize their mRNA targets. AGO proteins interact with a GW182 protein, in turn, interacts with cytoplasmic poly(A)-binding protein (PABP) and with the cytoplasmic deadenylase complexes CCR4-NOT and PAN2-PAN3, which catalyse the deadenylation of the mRNA targets, and then, deadenylated mRNAs are decapped and rapidly degraded by 5' to 3' exoribonuclease 1 (XRN1). 9-11

| MiRNAs repress protein translation
Several studies have proven that miRNA-mediated gene silencing is first manifested through repressing translation and subsequently consolidated by mRNA deadenylation and decay. 12,13 Interestingly, Beilharz et al 14 observed that miRNA-mediated mRNA deadenylation contributed to translational repression in mammalian cells as well. Multiple perspectives have been issued to explain this mechanism, such as inhibition of ribosome assembly and degradation of neopeptide, 15 but there are still no unified views up to now. The emerging recognition is that miRNAs inhibited translation initiation by interfering with the activity and/or assembly of the eukaryotic initiation factor 4F (eIF4F) complex, consisting of the cap-binding protein eIF4E, the adaptor protein eIF4G and the box protein 6 (DEAD) box RNA helicase eIF4A. 16 Whatever, it is now widely accepted that both mRNA degradation and translational repression participate in miRNA-mediated gene silencing.

| Unconventional functions of miRNAs
Although negative regulation of target gene expression is the most common mechanism of miRNAs participating in biological functions, other non-canonical mechanisms also play irreplaceable roles. It has been proven that miRNAs can interact with non-AGO

| MiRNAs in TNBC
MicroRNAs have been exhibited to act as multiple roles in TNBC, including functioning as diagnostic and prognostic biomarkers and exerting multifaceted effects on tumour progression, such as the modulation of tumour growth, metastasis and manipulation of chemoresistance, and regulation of metabolism (Table 2).  On the other hand, oncogenic miRNAs also play crucial roles in TNBC. MiR-498, miR-374a-5p and miR-761 were significantly overexpressed in TNBC and promoted cell proliferation and migration by downregulating PTEN, ARRB1 and TRIM29, respectively. [37][38][39] Moreover, the miR-106b-25 cluster mediated oncogenesis in breast cancer by activation of NOTCH1 by targeting NEDD4L in both ER+ and TNBC breast cancer cells, suggesting that the miR-106b-25/ NEDD4L/NOTCH1 axis played a crucial role in breast cancer. 40 Cantini et al described a novel algorithm named ClustMMRA, which was applied to explore clustered miRNAs potentially driving cancer molecular subtypes. Using ClustMMRA to analyse breast cancer patient data, the authors identified miR-199/miR-214 as a novel cluster that promoted TNBC cell proliferation and further validated these findings in vitro. 41

| MiRNAs affect migration and metastasis
Although the aetiology and onco-genetic mechanism of TNBC  respectively. [48][49][50] RhoA is a classic small GTPase that is generally thought to be essential for the formation of stress fibres and invasion of cancer cells.
Xiong et al 51

| MiRNAs regulate the sensitivity to therapeutic drugs
Chemotherapy remains an indispensable therapeutic strategy for TNBC, but the acquisition of chemoresistance is the primary obstacle for successful treatment. Accordingly, tremendous effort has been made in recent years to elucidate the mechanisms of TNBC chemoresistance with the aim of identifying new molecular targets. 56 It has become evident that ncRNAs act as significant regulators in the development of TNBC chemoresistance.
Cisplatin is a common anti-cancer drug applied in clinical practice. Overexpression of miR-638 increased sensitivity to cisplatin by regulating the expression of BRCA1 in TNBC. 57 In contrast, miRNAs could also promote resistance to chemotherapy.
Cisplatin-resistant MDA-MB-231 cell-derived miR-423-5p increased the resistance of recipient cells in an exosomal-dependent manner. 58 The bidirectional effects of miRNAs on chemoresistance have also been demonstrated for paclitaxel and doxorubicin.

| Exosomes-mediated miRNAs in TNBC
Exosomes are becoming a novel hotspot in the field of cancer research, which are a subclass of extracellular vesicles involved in intercellular communication. 65 Not surprisingly, exosomes-mediated miRNAs play significant roles in tumorigenesis of TNBC.

| LONG NON -CODING RNA s 4.1 | Biogenesis of lncRNAs
LncRNA is one of ncRNAs subclass that most researched in the field of cancer research. In mammalian genome, more than 58 000 lncR-NAs have been identified, and approximately 30 000 lncRNAs have been curated in GENCODE v29. 69 As we all know, lncRNAs exist widely in human organism, and it is vital to human gene expression modulation and physiological and pathological processes. Most types of lncRNAs are transcribed by RNA polymerase Ⅱ, capped at the 5' end, polyadenylated at the 3' end and edited by a series of splicing processes which occurs in the nucleus. Besides, there are other mechanisms participating in the process of lncRNA maturation. For instance, precursor lncRNAs (pre-lncRNAs) are cleaved by RNaseP to achieve mature ends. 70 LncRNAs are pervasively interspersed in the genome based on their various transcriptional origins, including whole or partial natural antisense transcripts, coding genes, between coding genes, within introns, promoter and enhancer ( Figure 2). Due to the complexity and diversity of lncRNAs, the biogenesis and regulation mechanism of different lncRNAs have not been completely summarized. In the future years, by virtue of the advanced techniques, including chromatin isolation by ChIRP-Seq, CRISPR and CLIP, [71][72][73] it is worthy of believing that the mechanism of biogenesis and synthesis of lncRNAs will be further deeply cognized.

| LncRNAs act as ceRNA
Long ncRNAs have diversity of regulatory functions, which can modulate chromatin remodelling, transcriptional regulation and post-transcriptional processing, translation. 74,75 Although multiple functions of lncRNAs have been observed, competing endogenous F I G U R E 2 Mechanism of lncRNA biogenesis. LncRNA is transcribed by RNA polymerase II, capped at the 5' end, polyadenylated at the 3' end and edited by a series of splicing events that occur in the nucleus. This figure preliminarily summarizes the diverse biogenesis of lncRNAs based on their transcriptional origin, namely, promoter-associated lncRNAs, enhancer-associated lncRNAs, exonic lncRNAs, intronic lncRNAs, bidirectional lncRNAs, intergenic lncRNAs, antisense lncRNAs and sense lncRNAs RNA (ceRNA) or miRNA sponge is the most focused function. For instance, LINC01123 was found to be upregulated in non-smallcell lung cancer and associated with poor prognosis. By a series of functional experiment, researchers revealed that LINC01123 might increase c-MYC mRNA expression by sponging miR-199a-5p, which was a direct transcriptional regulator for c-MYC. 76 Even if the function of lncRNAs as ceRNA has been widely accepted, the mechanism concerning these remain unknown to some extent and require to further deeper investigation.

| LncRNAs are involved in chromatin action
There are lots of lncRNAs accumulating in the nucleus to regulate chromatin architecture, participate in chromatin remodelling and promote the recruitment of chromatin modifiers. For example, the genome multipotent stem cells are equipped with the form of higher-order chromatin architecture, with a variety of intra-and inter-chromosomal interactions. The peculiarity of stem cell pluripotency is directly determined by the promoter region of stem cell core factor genes around the architecture. In many studies, lncR-NAs can take part in the composition of chromatin architecture to establish the stemness state, chiefly bringing distant enhancer elements into proximity of the core promoter. 84 Besides, lncRNAs also can interact with chromatin-modifying enzymes, catalysing covalent changes of histones or DNA on the chromatin to affect genetic expression information, such as GADD45a, DNMT1 and PRC. 85

| LncRNAs regulate mRNA stability
Studies show that lncRNAs can regulate mRNA stability via corresponding miRNAs, recruiting proteins to degrade mRNA and acting as molecular decoys for RBPs taking part in mRNA decay.
For instance, the binding of hnRNP E1 to a nucleic acid structural element located in exon 12 of PNUTS pre-RNA can regulate its alternative splicing. In breast cancer cells, the expression of lncRNA PNUTS was elevated and associated with levels of ZEB mRNAs.
Furthermore, PNUTS also served as a competitive sponge for miR-205 during epithelial-mesenchymal transition. 86 Besides, PDCD4-AS1 regulated breast cancer progression through stabilizing PDCD4 RNA by forming RNA duplex and controlling the interaction between PDCD4 RNA and RNA decay-promoting factors such as HuR. 87 Although the role of lncRNAs in regulating mRNA stability has been rarely observed, it is a novel mechanism that lncRNA-related biological process needs to be further explored in the future.

| LncRNAs in TNBC
Long ncRNAs play crucial roles in human beings, not only do lncRNAs participate in regulating normal mammalian gene expression function or other body biological processes, but also they have notable influences on human diseases, such as various cancers, neuropsychiatric disorders and atherosclerosis. [88][89][90] Apparently, there are gradually increasing explorations in the mechanism of lncRNAs functioning in TNBC in recent years. Accumulated evidence suggested that lncR-NAs can modulate proliferation, invasion, differentiation, chemoresistance of TNBC cells in positive or negative manner (Table 3). Even a few studies indicated that lncRNAs can work as a biomarker for diagnosing and evaluating the prognosis in TNBC.

| LncRNAs mediate migration and metastasis
As long non-coding antisense transcript of NAMPT, NAMPT-AS was upregulated in TNBC and promoted cell migration. NAMPT-AS epigenetically regulated the expression of NAMPT in two different ways. One was that the transcription of NAMPT was activated by NAMPT-AS, thereby recruiting POU2F2. The other was that NAMPT-AS acted as ceRNA to rescue NAMPT degradation from miR-548b-3p. 10

| LncRNAs dominate the sensitivity to chemotherapy
Studies have shown that lncRNA NEAT1 expression is upregulated in cisplatin-and taxol-resistant cells compared with parental cells.
By qRT-PCR assay, it was demonstrated that when knocking down NEAT1 in sh-NEAT1 cells, drug transporter genes, such as ATP7A and ATP7B, were downregulated, and functional analysis indicated that NEAT1 knockdown sensitized cells to chemotherapy through a synergistic effect. 16 BORG, an oncogenic lncRNA, was greatly responsive to cytotoxic drug treatment, particularly doxorubicin. The mechanism of drug BORG-associated resistance relied on its significant activation of the NF-κB signalling pathway through the BORGmediated feed-forward signalling loop and its ability to activate RPA1, making BORG-expressing TNBC sensitive to doxorubicininduced cytotoxicity. 17 In addition, upregulation of lncRNA HCP5 contributed to cisplatin resistance in TNBC, and inhibition of HCP5 reversed resistance to cisplatin by upregulating PTEN expression. 18 However, tumour suppressive lncRNA GAS5 could enhance the sensitivity of TNBC to paclitaxel and induce apoptosis in TNBC cells by targeting miR-378a-5p/SUFU signalling. 19 Moreover, lncRNAs not only participate in the process of chemoresistance, but can also be used for predicting the response to

| LncRNAs serve as potential biomarkers for diagnosis and prognosis
At present, with the deepening of TNBC research, lncRNAs have generally been revealed as promising diagnostic and prognostic biomarkers. 111

| LncRNAs mediate immunomodulation
Immunotherapy, including PD-1 and/or PD-L1 blockade, is an important cancer therapeutic method to restrict cancer progression. 117  CircMYLK promoted hepatocellular carcinoma progression by upregulating Rab23 via sponging miR-362-3p. 127 To sum up, these studies above support the idea that circRNAs functioning as miRNA sponges may be a common mechanism in cancerous diseases.

| CircRNAs interact with functional proteins
Similar to linear RNAs, which have been reported to interact with proteins, several studies have also exhibited that some circRNAs can work as protein partner such as circANRIL and circFOXO3.
CircANRIL had been revealed to directly bind to PES1, an important 60S-preribosomal assembly factor and next control maturation of ribosomal RNA and modulating pathways of atherogenesis, resulting in regulating the progression of atherosclerosis. 129 Besides, circFOXO3 was found to be highly expressed in non-cancer cells and correlated with cell cycle progression. Functional analysis indicated that overexpression of circFOXO3 can repress cell cycle progression via binding to CDK2 and cyclin-dependent kinase inhibitor 1 (or p21), resulting in the formation of a ternary complex. 130

| CircRNAs can be translated into proteins
Most circRNAs are derived from exons and predominantly present in the cytoplasm, suggesting that they can be loaded into ribosomes for translation into polypeptides. CircZNF609 was derived from the circularization of the second exon of its host gene. A 753 nt ORF was present in circZNF609, which could be translated into a protein in a splicing-dependent and cap-independent manner, providing an novel example of a protein-coding circRNA in eukaryotes. 131

| CircRNAs in TNBC
In recent years, circRNAs have gradually become a novel hotspot in the ncRNAs and cancer research field. However, the functions of circRNAs in cancerous diseases, especially in TNBC, have not been fully understood. The known studies about circRNAs mostly focalized on the mechanisms of ceRNA in TNBC progression. Besides, circRNAs act as biomarkers for diagnosis and prognosis in TNBC is also widely observed (Table 4).

| CircRNAs act as tumour promoters
Derived from gene GFRA1, circGFRA1 was upregulated in TNBC and high expression of circGFRA1 was correlated with poor OS.
Knockdown of circGFRA1 suppressed proliferation and promoted apoptosis via binding to miR-34a and upregulating GFRA1 expression in TNBC. 134 Besides, silencing of circEPSTI1 inhibited cell proliferation and induced apoptosis via sponging miR-4753 and miR-6809 to increase BCL11A in TNBC. 135 It was also revealed that circUBAP2 was correlated with tumour size, advanced TNM stage and worse prognosis in TNBC and promoted tumour progression by sponging miR-661 to upregulate MTA1. 136   Besides, the expression of tDRs from specific tRNA loci has been found to be associated with the observed race disparities in TNBC, such as the nuclear tRNA Gly and tRNA Leu , the mitochondrial tRNA Val and tRNA Pro . 150 In addition to serving as biomarkers, tDRs also participate in the process of TNBC. Cui et al 151

| NcRNAs as diagnostic and prognostic biomarkers in cancer assessment
Non-coding RNAs were demonstrated to have strong diagnostic and prognostic values in multiple cancers, which were revealed by pancancer analysis. 155 had been reported to serve as TNBC-specific diagnostic biomarkers. [21][22][23] Besides, the dysregulated lncRNA ANRIL, HIF1A-AS2 and UCA1 can be detected in plasma of TNBC patients and the 3-lncR-NAs combined signature exhibited excellent diagnostic value. 112 Moreover, even hypermethylation of LINC00299 in peripheral blood of TNBC patients also served as a useful circulating biomarker for TNBC. 113 However, the circulating circRNAs have not been found to be biomarkers in TNBC. Theoretically, circRNAs are covalently closed and generally resistant to the degradation of ribonucleases, suggesting they might be more stabilized in peripheral blood. Thus, detection of the dysregulated circRNAs in the peripheral blood of TNBC patients should next hotspot in the field of ncRNA research.

| NcRNAs as therapeutic tool in cancer treatment
It has been proven that abnormal small ncRNA expression levels in tumour cells can affect tumour development and therefore can be used as effective targeted drugs for the treatment of tumours. 158 Although small ncRNAs have been shown to be promising and effective therapeutic drugs in vitro, due to the degradation of nucleases in the body, naked ncRNAs have a short half-life in the blood, and the low bioavailability of these nucleic acid drugs in vivo is a major challenge. Therefore, ncRNAs need to be transported to the target tissue by a suitable carrier to exert their effect. Various small ncRNA carriers or systems have been proposed and widely explored, including nanoparticles, ncRNA modification and oncolytic adenovirus strategies. 159 Nanoparticle-based small ncRNA carriers are the most common strategies. Shu et al 160

| CON CLUS ION
To conclude, ncRNAs have notable effects on TNBC progression.
We summarize the roles of ncRNAs and their mechanisms in TNBC in Figure 5. Although the functions of miRNAs and lncRNAs on the carcinogenesis and development of TNBC have been widely studied and have well-established roles, many other ncRNAs including circRNAs also have a significant influence on tumour progression but have long been neglected. With more research interests concentrated on the lesser-known ncRNAs and their relationship with TNBC, we believe that the mystery of the ncRNA world will eventually be solved and the clinical practice of TNBC management will be largely improved in the future.

ACK N OWLED G EM ENTS
This work was supported by grants from the Natural Science Foundation of Jiangsu Province of China (BE2017626, BK20181367) and the Foundation of Wuxi Health Commission (QNRC003).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
YZ, CL and JM conceptualized the review. JM and LH wrote the manuscript. LH, HW, RX and YL prepared the figures and tables. YZ and CL critically reviewed and edited the manuscript. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.