MicroRNAs (miRNAs/miRs) are 21–24 nucleotide single-stranded non-coding small RNAs that play a crucial role in the regulation of gene expression (1). miRNAs have been shown to play a key role in the cellular response to environmental stresses, such as starvation, hypoxia, oxidative stress and DNA damage. The biological importance of miRNAs has been highlighted in recent decades by the deregulation of miRNA expression in a variety of human diseases, including cancer (2,3). Several studies have found that the dysregulation of miRNA expression is closely linked to tumor initiation, growth and metastasis (4–6). Numerous miRNAs can function as oncogenes or tumor suppressors, causing cancer or suppressing tumor growth (7,8). Several miRNAs have been found to be involved in epigenetic, transcriptional and post-transcriptional processes (9,10). miRNA dysfunction disrupts the expression of oncogenic or tumor-suppressing target genes, which is implicated in cancer pathogenesis. The majority of mature miRNAs are known to interact with the 3′UTR of target mRNAs via complete or incomplete pairing to induce mRNA degradation or translational inhibition (11,12). miRNAs are loaded onto Argonaute (AGO) proteins in the cytoplasm to direct the RNA-induced silencing complex (RISC), which is composed of AGO, DICER, TAR-RNA-binding protein (TRBP) and trinucleotide repeat containing 6 (TNRC6), to bind to the 3′UTR (13). Post-transcriptional gene silencing in the cytoplasm is a well-known function mediated by miRNAs through the miRNA-induced silencing complex (miRISC). Furthermore, certain studies have suggested that it occurs in the 5′-UTR (14) and even in protein-coding sequences (15). However, interactions of miRNAs with other regions, such as gene promoters, have been reported (16,17). miRNAs are active in the nuclei, according to research, and several miRNAs that are prominently localized in the nucleus are associated with transcriptional regulation. To support this, the majority of miRNAs have recently been reported to be imported back into the nucleus following maturation, with abundant putative miRNA target sites in gene promoters in both the sense and antisense orientations, which regulate transcriptions in the nucleus by binding to complementary sequences in the promoters (18–20) of target genes. miRNAs have been found to have two functions: Transcriptional gene activation (TGA) and transcriptional gene silencing (TGS) (21). It is now clear that the transcriptional regulation of nuclear miRNAs can promote the occurrence and progression of a variety of malignant tumors, including gastric, prostate cancer (Pca) and lung (22–24). In recent years, there has been a growing body of evidence indicating an epigenetic interaction between DNA methylation modification and nuclear miRNA expression in cancer (24–26). Several mature miRNAs have been presently identified to be enriched in the nuclei of various tissues and cell types using evolving technologies such as high-throughput sequencing (27). These findings shed new light onto the mechanisms and treatment strategies of miRNAs in the control of tumor gene expression (23). However, the types and numbers of miRNAs found in the nuclei of different cells, as well as their potential biological regulatory functions, remain unknown (28). The understanding of the time frame and associated mechanisms through which nuclear miRNAs regulate transcription in tumor cells is particularly limited. As a result, it is of utmost importance to investigate the regulatory networks of gene expression that not only cover nuclear miRNA expression, but also its immediate effects on different tumor cell types for cancer treatment by targeting or manipulating the expression profile of nuclear miRNAs.

The present review article discusses a number of processes involved in the biogenesis of miRNAs and their transport into the nucleus. The structure, function and roles of miRNA in the regulatory effects on AGO proteins are also discussed. In addition, some software tools for predicting miRNA binding sites in order to identify and predict the corresponding miRNAs are noted and summarized. It is demonstrated that nuclear miRNAs, through epigenetic modifications, mediate the regulatory function of TGA or TGS, as well as their importance in tumorigenesis and development. The aim of the present review was to discuss the endogenous function of nuclear miRNAs in cancer cell growth and to propose potential applications in disease treatment, such as the development of drug targets via miRNAs (29).

The majority of miRNAs have their own promoters that exist in the genome on their own (30). The promoter, in conjunction with RNA polymerase II (RNAP II), transcribes the genes encoding miRNAs into primary miRNA transcripts (pri-miRNAs) (31). A microprocessor complex (32) converts these pri-miRNAs into short 70-nucleotide stem-loop structures known as precursor miRNAs (pre-miRNAs) in the nucleus. The DROSHA RNase III enzyme and the Di George syndrome critical region 8 double-stranded-RNA binding protein comprise the microprocessor complex (DGCR8). DROSHA cleaves the primary miRNAs in order to remove a portion of the unpaired RNA sequences (33).

The nuclear export machinery, which consists of Exportin-5 complexed with GTPase (Ran GTP), transports the pre-miRNAs to the cytoplasm, where they are converted into mature miRNAs, a 22-nt miRNA duplex, by cleavage of the hairpins by the enzyme DICER (34). The asymmetry rule is a strand-selection mechanism in which only one strand is preferentially retained to form the functional miRISC, while the other strand is degraded. In most cases, the miRISC induces mRNA decay and translational suppression by interacting with complementary sequences in the 3′UTR of target gene mRNAs (Fig. 1). Several previous studies have found that miRISC is imported into the nucleus of mammalian cells, and that nuclear RISC (an ~158 kDa complex) is smaller than its cytoplasmic counterpart (a 20-fold larger complex of nearly 3 MDa). Moreover, several RISC components have been identified to function in the nucleus (21). Western blot analysis has revealed the presence of AGO protein, DICER, TRBP and GW182/TNRC6 in the nucleus, where they combine to form multi-protein complexes (35). Furthermore, miRISC plays a role in the transcriptional regulation of a variety of life activities, including cell proliferation, differentiation, development and apoptosis (11).

A large number of nuclear transport receptor proteins are directly involved in the process of miRNA import to the nucleus (36). Importin 8 and exportin 1, importin family members that can recognize nuclear localization sequences in protein cargoes and assist their active transport through the nuclear pore complex, have been found to be involved in the nuclear and cytoplasmic shuttling of miRNAs and RNA interference/RNA activation (RNAa) components (37,38). To function in the nucleus, miRNAs must first form an AGO-miRNA complex with the AGO protein, then bind to importin 8 and TNRC6A (also known as GW182) in the cytoplasmic-processing body (P body) for introduction into the nucleus (35,36). Unlike other members of the cytoplasmic RISC, AGO2 lacks a nuclear localization signal. TNRC6A also contains a nuclear localization signal (NLS) and a nuclear export signal (NES), which can be transported between the cytoplasm and the nucleus by importin 8 or exportin 1 (39,40). As a result, by combining with TNRC6A, which possesses NLS, AGO2 can function as a nuclear-cytoplasmic shuttling protein to re-localize the miRNA to the nucleus. TNRC6A functions as a scaffold in the nucleus, allowing other effector proteins to be recruited (41). TNRC6A directs AGO-miRNA to promoter region of target the gene, eventually cleaving the target RNA or retaining it in the nucleus. As a result, TNRC6A is yet another core protein involved in miRNA-mediated gene transcription (42,43). Furthermore, exportin 1 can transport AGO-TNRC6 complexes back to the cytoplasm. Consequently, inhibiting exportin 1 leads to an accumulation of TNRC6A and AGO2 in the nucleus (44). DICER and TRBP, on the other hand, are not attached to the RISC during their import into the nucleus. Kalantari et al (45) used semi-quantitative mass spectrometry to confirm that the association of AGO2 with GW182/TNRC6 and AGO3 was most well preserved and more stable in both the nuclei and cytoplasm. The interactions of AGO2 with DICER and TRBP, on the other hand, are restricted to the cytoplasm. Although cytoplasm-processed miRNAs can be introduced into the nucleus to regulate gene expression, the time frame and mechanisms through which miRNAs perform their functions in the nucleus remain to be fully elucidated.

AGO proteins are members of the Argonaute family, which is highly specialized and conserved (46). In several organisms, they are the primary participants in the small non-coding RNA-mediated gene silencing pathway, and they are primarily responsible for regulating gene expression (47). The Argonaute family is divided into two groups: The ubiquitously expressed AGO subfamily proteins, also known as the AGO clade, and the gonad-expressed PIWI subfamily proteins, also known as the PIWI clade (48). AGO subfamily proteins are typically associated with miRNAs and short-interfering RNAs (siRNAs) and collaborate in the process of post-transcriptional gene silencing (49). The PIWI clade proteins are only found in another type of small RNA known as PIWI-interacting RNA (piRNA). Unlike the AGO clade, PIWI proteins are primarily expressed in germ cells and help to silence male germline transposons by interacting with piRNAs (50).

The majority of AGO proteins have 700-1,000 amino acid residues and have molecular weights of ~100 kDa, with 80% amino acid sequence identity (51). AGO protein amino acid sequences are highly conserved across species (Fig. 2A). The AGO proteins have an amino-terminal (N), a PIWI/Argonaute/Zwille (PAZ) domain, a middle (MID) domain and a PIWI domain (52–54). It should be noted, however, that the N-terminal domain is required for small RNA loading and double-stranded small RNA unwinding (55). Mammalian cells contain four AGO proteins (AGO1-4), all of which are loaded with siRNA and miRNA duplexes (56). They have similar biochemical preferences for binding to duplex RNA (45,57), but only AGO2 cleaves completely complementary mRNA targets (58).

AGO proteins have been previously shown to play critical roles in germ cell maintenance and division, as well as in transcriptional and translational regulation (59). These proteins are also involved in the repair of double-strand breaks in DNA through homologous recombination, chromatin modifications and alternative splicing. The AGO proteins, on the other hand, are incapable of catalyzing any reaction on their own. They are the core component of the RISC, interacting directly or indirectly with various partners, such as DICER, TRBP and GW182 family proteins, which are responsible for binding to short RNA and the cleavage or inhibition of target mRNA translation (60). A growing body of evidence suggests that AGO not only functions as a critical regulator of gene expression in the cytoplasm, but also mediates the repression of miRNA transcription in the nucleus, with importin 8 acting as an essential co-factor and assisting in the localization of AGO2 in the nucleus. miRNAs have also been found to colocalize in the nucleus with the AGO-TNRC6A complex and to exhibit gene-silencing activity (39). AGO1 and AGO2 proteins, in particular, promote histone H3 lysine 9 (H3K9) methylation and play critical roles in miRNA-mediated gene regulation (61). Notably, while previous research provided knowledge of the nuclear functions of AGO clade proteins, the mechanisms underlying the transport of cytoplasmic AGO proteins into the nucleus remain largely unknown.

Furthermore, the association of AGO-bound miRISC with various cellular organelles or structures remains unknown. Data on the cellular localization of the numerous steps involved in the assembly, function and recycling of these complexes are limited. It is well understood that AGO-mediated epigenetic modification is a common phenomenon that plays a role in the transcriptional regulation of nuclear miRNAs. However, further and more in-depth knowledge of the types and quantities of modified proteins recruited by the AGO complex, such as epigenetic enzymes and transcription factors (TFs) is required.

A number of mature miRNAs have been found to be enriched in the nucleus using high-throughput profiling technologies, such as microarray and deep sequencing. The number and types of nuclear miRNAs found in different species, tissues and cells are also distinct, emphasizing the specificity and importance of nuclear miRNA regulation (Table I).

The database RNALocate (http://www.rna-society.org/rnalocate/) is the first database which comprehensively focuses on RNA subcellular localization. miRNA symbols from the miRBase database are also provided in RNALocate. This database also includes some miRNAs with nuclear localization (62). Other databases, such as microPIR2 (https://tools4mirs.org/software/mirna_databases/micropir2/) (63), Tools4miRs (https://tools4mirs.org/) (64) and miRWalk2.0 (http://zmf.umm.uni-heidelberg.de/mirwalk2) (65) provide predicted and validated information of miRNA-target interaction. Furthermore, the program mirSTP (miRNA transcription Start sites Tracking Program) was created to predict the transcription start sites (TSSs) of miRNAs. mirSTP can be found at http://bioinfo.vanderbilt.edu/mirSTP/ (66). The knowledge of nuclear-localized miRNAs is critical for locating the core promoters of miRNAs and integrating the control of miRNA transcription into complex regulatory networks.

In Table I, a list of various nuclear miRNAs identified in various studies (35,67–74) is presented.

The mechanism of nuclear miRNA-mediated transcription regulation is similar to that of piRNA/PIWI-mediated transposon silencing (75). piRNAs assemble with PIWI members of AGO proteins to form piRNA-induced RNA silencing complexes (piRISCs) and modulate key signaling pathways at the transcriptional or post-transcriptional level in the piRNA/PIWI pathway (76). Studies have demonstrated that the nuclear PIWI-piRNA complexes at the cytoplasmic PIWI proteins silence their targets post-transcriptionally via piRNA-directed cleavage and the transcriptional ping-pong cycle. PIWI is translocated to the nucleus after piRISC assembly and represses transposons co-transcriptionally by inducing the formation of local heterochromatin at target transposon loci (77). CG9754 is a critical downstream pathway protein of PIWI/piRNA that works with PIWI to repress transposon transcription (78). Its effect on PIWI/piRNA-guided transcriptional silencing is realized after histone H3K9 is methylated as a result of its binding to the target DNA or RNA and the inhibition of genetic transposition by PIWI (79). The overexpression of the PIWI/piRNA pathway in tumor cells can lead to dysregulated methylation at H3K9, and some tumor suppressor genes may be inhibited (80). Similarly, the transcriptional activation or inhibition of genes by nuclear miRNAs is primarily accomplished through the mediation of epigenetic modifications such as H3K9 methylation and H3 lysine 4 (H3K4) acetylation. AGO proteins, in particular, as well as proteins from the epigenetic machinery, alter the structure of chromatin (Fig. 3). This process is critical for tumor cell growth, proliferation, invasion and migration, which has become a hot topic in tumor regulation research.

Following maturation, the majority of miRNAs are imported back into the nucleus, according to sequencing and bioinformatics analyses (81). miRNAs guide AGO proteins to promoter targets (82), such as the TATA-box motif or regions associated with transcription factors, owing to an abundance of putative miRNA target sites in gene promoters (83,84). The recruitment of AGO proteins (primarily AGO2) and the enrichment of RNAP II at regulated gene promoters then alter histone epigenetic modification and transcription factor binding. During the initiation of transcription, the DNA double helix unwinds. miRNAs typically mediate acetylation at H3K9 and histone H3 lysine 27 (H3K27), as well as methylation at H3K4, which stabilizes the promoter region and increases the recognition and binding efficiency of RNAP II, thereby activating gene transcription (85–87). Some miRNAs, such as miR-26a-1, miRNA-558, miR-195-5p and miR-24-1, have been shown to induce target gene expression. Recent research, for example, has demonstrated that nucleus-enriched miR-195-5p may regulate Foxo3 expression in porcine ovarian granulosa cells, thereby regulating follicular development and atresia. The mechanism of action is considered to involve miR-195-5p binding to the Foxo3 promoter TATA box, transcription activation, histone acetylation and hypomethylation and transcriptional regulation. Of note, it was found that AGO2, rather than other AGO proteins, regulates the effect of miR-195-5p on Foxo3 transcription (88). However, PcG protein recruitment increases H3K27 trimethylation (H3K27 me3) and decreases H3K4 trimethylation (H3K4 me3), and DNA methylation is frequently observed at some miRNA-targeted promoter regions. The chromatin structure would then be altered, and the promoter region would be tightened to establish a non-permissive transcriptional status (89,90). Several studies have found that nuclear miRNAs exert an inhibitory effect on transcriptional regulation. The lysosome is the critical organelle of autophagy, according to a study on autophagy and lysosome biogenesis, and the TF EB (TFEB) is the primary regulator of lysosome biogenesis. Dephosphorylated TFEB enters the nucleus and binds to the palindromic sequence (GTCACGTGAC) at the promoter of autophagy and lysosomal biogenesis-related genes, i.e., the coordinated lysosomal expression and regulation (CLEAR) element, to activate transcription. The miR-30b-5 represses the transcription of TFEB-dependent downstream genes by targeting CLEAR, the TFEB binding motif, further inhibiting the flux of autophagy and lysosome biogenesis (91).

miRNAs are now known to be potent gene regulators. However, there is a limited understanding of the time frame and mechanisms through which miRNAs regulate transcription (92). Several in vitro studies have been conducted over the past few decades, which have involved the transfection of pre-miRNAs or mature mRNA mimics into immortalized and cancer cell lines. However, further research is required to determine how well these findings reflect endogenous miRNA functions in vivo. Emerging evidence regarding miRNAs has demonstrated the significance of the regulatory mechanisms of nuclear miRNA expression in cancer (93,94). The critical role of nuclear miRNAs in tumor initiation, growth and metastasis is discussed below.

Previous research has suggested that changes in miRNA expression and function may contribute to cancer formation and progression, as well as to cellular responses to various types of stress (95,96). The Let-7 family, for example, influences tumor differentiation, migration, invasion and proliferation, and its members are dysregulated in various types of cancer. They can be activated or deactivated, and they can function as tumor suppressors or oncogenes (97). miR-124 has the potential to be a potent tumor suppressor in cancers with a high myc expression. A previous study discovered that miR-124 had an RNA-activating function, which resulted in the direct induction of p27 protein levels by binding to and inducing transcription on the p27 promoter region, causing G1 arrest. In breast and ovarian cancers, miR-124 can not only reverse the myc/p27/phospho-Rb protein signature to target cell proliferation, but it can also block integrin 1 and increase sensitivity to etoposide (98). Another family of onco-suppressor miRNAs, miR-34, may target cell cycle-related genes (including BCL2, MET, MYC, AXL and CDK4) and has tumor-suppressive properties that mediate apoptosis, cell cycle arrest and senescence. miR-34a, in particular, has been shown to be a direct transcriptional target of p53, which is frequently mutated in epithelial ovarian carcinomas (99,100). Furthermore, the miR-21 and miR-17-92 clusters are potent carcinogenic factors. miR-21 is a robust modulator of tumor behavior and malignant transformation. The expression of miR-21 in non-small cell lung cancer (NSCLC) tissues was regulated by the transcriptional factor nuclear factor (NF)-B binding to its promoter element. As a result, inhibiting NF-B via RNA silencing protects cells from cisplatin by decreasing miR-21 expression (101). Clusters of miR-17-92, on the other hand, are linked to cell proliferation, migration and angiogenesis. According to the findings of previous studies, miR-17-5p may inhibit the proliferation and induce the apoptosis of NSCLC H460 cells by targeting TGFR2 (102,103).

A number of factors, including matrix metalloproteinase 14 (MMP-14), circular RNAs (circRNAs_ and Cockayne syndrome protein B (CSB), play a role in the miRNA-mediated regulation of tumorigenesis (26,104,105). MMP-14, a membrane-anchored MMP that promotes tumorigenesis and aggressiveness, has been found to be highly expressed in neuroblastoma (NB) and gastric cancer. Xiang et al (26) identified one binding site of miRNA-584-5p (miR-584-5p) within the MMP-14 promoter through mining computational algorithm program and Argonaute-chromosome interaction dataset. Their results indicate that promoter-targeting miR-584-5p exerts tumor suppressive functions in NB through repressing the transcription of MMP-14. Zheng et al (106) identified adjacent binding sites of the myeloid zinc finger 1 (MZF1) and miRNA-337-3p (miR-337-3p) in the MMP-14 promoter by mining computational algorithm programs and chromatin immunoprecipitation datasets. According to the findings, miR-337-3p binds directly to the MMP-14 promoter to repress MZF1-mediated MMP-14 expression, thereby suppressing GC progression (106). circ-PLEKHM3 was identified as one of the most significantly downregulated circRNAs in ovarian cancer tissues by Zhang et al (107). circPLEKHM3 overexpression was found to inhibit cell growth, migration and epithelial-mesenchymal transition (EMT). Mechanistically, when circPLEKHM3 was downregulated in ovarian cancer, the competitive binding of miR-9 was attenuated, resulting in a decrease in miR-9-targeted gene expression, which promoted tumor cell proliferation (107). It has been have demonstrated that the binding sites of Let-7 and miR-29 in the proximal 3′UTR of CSB are highly conserved. Let-7 and miR-29 suppress CSB expression by directly targeting the 3′UTR of CSB in lung cancer cells, resulting in NSCLC cell apoptosis (104). The importance of the regulatory mechanisms of nuclear miRNA expression in cancer has been demonstrated by emerging evidence exhibiting genetic and epigenetic dysregulations of the machinery of miRNA biogenesis and the regulatory mechanisms of miRNAs.

miRNAs have been shown to be extensively deregulated in human cancers over the past few decades, highlighting their critical role in tumor initiation, growth and metastasis. Similarly, the regulatory mechanisms that control miRNA expression are strongly linked to cancer diagnosis, prognosis and treatment, as well as to cancer pathogenesis. miRNAs typically suppress target gene expression by partially binding to complementary sequences in the 3′-UTR of target mRNAs, inhibiting translation and degrading the target mRNAs. However, additional evidence suggests that miRNA-mediated gene expression regulation also has other functions, and the 5′-UTR coding sequence or promoter of the genes could be the target region. Several small RNAs within miRNA response elements, such as circRNAs and long non-coding RNAs, have been shown to interact with miRNAs to regulate target gene expression. Current research on the regulation of miRNAs, however, is still focused on the traditional cytoplasmic pathways and how miRNAs specifically control specific types of cancer. The present review article summarized the series of processes involved in the entry of miRNAs into the nucleus, particularly new mechanisms of nuclear miRNA expression regulation in tumors. Mature miRNAs that are carried into the nucleus and the target gene promoter region by AGO proteins to activate epigenetic modification and regulate transcription perform the majority of the regulatory functions of miRNAs in the nucleus. A better understanding of nuclear miRNA regulation and the potential underlying mechanisms may aid in the understanding of the association between miRNAs and tumorigenesis. These new signs of progress will lead to the development of more precise therapeutic targets and the identification of novel biological markers for the diagnosis of tumors and cancers, as well as new avenues for future research on a variety of malignant diseases.