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pre-mRNAs. These circRNAs consist of both exons and introns. It is predominately located in the
[19]
nucleus . This means they may provide transcript and splicing functions [20,21] . Finally, ciRNAs are formed
[19]
only by the head-to-tail joining of intronic sequences and located primarily in the nucleus . The
mechanism of formation of the first two circRNAs is relatively similar. There are currently three mechanism
hypotheses for the origination of EciRNAs and EIciRNAs: intron pairing-driven circularization, RNA
[22]
binding protein (RBP)-dependent circularization, and lariat-driven circularization [Figure 1]. For intron
pairing-driven circularization, complementary sequences in introns promote circRNA circularization by
base pairing to bring the 3' splice acceptor site and the 5' splice donor site closer together in spatial
conformation. Sequences known to promote intron circularization are Alu repeat elements . For RBP-
[23]
dependent circularization, it can promote circRNA formation by binding to intronic sequences or specific
motifs within flanking introns near the splice site and indirectly bridging the distance between upstream
and downstream exons, thus promoting loop formation . Representative RBPs include FUS and
[25]
[24]
[26]
MBL . For lariat-driven circularization, partial folding of exons may occur during the transcription process
of pre-mRNA, leading to the formation of lariat. These folds cause exons or introns that were originally far
[27]
apart to come closer, thereby promoting the formation of circRNA . CiRNA formation is less similar to
circRNAs containing exons, in that it relies on undegraded lariats that contain important sequences such as
GU repeats at the 5' splice site and C elements enriched close to the branch site, allowing the 5' end site to
form a 2'-5' linkage with the 2'-OH to form circRNA . Most circRNAs belong to the exon-containing
[28]
types, and their conservation is higher than other types of circRNAs.

The vast majority of circRNAs have corresponding linear parental genes in organisms, which makes it
difficult to be distinguished easily. Currently, the main approach is first to enrich circRNAs based on their
unique stable structure by digesting total RNA with RNase R, and then to identify specific circRNAs
through the detection of specific junction sequences by RNA high-throughput sequencing . Although
[29]
these methods can identify many BSJ sequences, their detection sensitivity is low once the BSJ sequence is
very short. In recent years, some emerging long-read sequencing technologies have gradually developed,
such as PacBio and Oxford Nanopore, which can better distinguish circRNA from its corresponding linear
[30]
transcripts . Furthermore, Chiang et al. have recently developed a FL-circAS nanopore long-read
sequencing technology, which can decipher circRNAs from the aspects of their expression, formation, and
function. This technology will promote the further development of circRNA research .
[31]

THE FUNCTION OF CIRCULAR RNA IN TNBC
Sponging miRNA
In accordance with the base complementary pairing principle, miRNA inhibits or facilitates mRNA
[32]
translation by binding to non-translational regions in target genes . RNA that can competitively bind to
common miRNAs to inhibit their activity on target genes is known as competitive endogenous RNA
(ceRNA) . Research has revealed that circRNAs carry a large amount of miRNA-responsive sequences,
[33]
which can serve as effective ceRNAs. They bind to miRNA to adsorb it and effectively prevent its binding to
[34]
the target genes, thereby providing regulatory effects on target genes .

Research on circRNAs in TNBC primarily concentrated on understanding the function of miRNA
sponging. For example, CiRS-7 may be the most distinctive circRNA, containing over 70 conserved binding
sites of miR-7, and has a stable expression in many tumors including TNBC . Recent research shows that
[35]
CiRS-7 also has miR-1299 binding sites besides miR-7. Experiments identified that downregulation of
CiRS-7 expression inhibits migration and invasion of TNBC cells in vitro and suppresses their metastasis to
the liver and lung in vivo. CiRS-7 regulates the expression of matrix metalloproteinases (MMP) family
[36]
members through sponging miR-1299 . Li found that circCRIM1 is upregulated in TNBC, which