MicroRNAs and Stem-like Properties: The Complex Regulation Underlying Stemness Maintenance and Cancer Development

Embryonic stem cells (ESCs) have the extraordinary properties to indefinitely proliferate and self-renew in culture to produce different cell progeny through differentiation. This latter process recapitulates embryonic development and requires rounds of the epithelial–mesenchymal transition (EMT). EMT is characterized by the loss of the epithelial features and the acquisition of the typical phenotype of the mesenchymal cells. In pathological conditions, EMT can confer stemness or stem-like phenotypes, playing a role in the tumorigenic process. Cancer stem cells (CSCs) represent a subpopulation, found in the tumor tissues, with stem-like properties such as uncontrolled proliferation, self-renewal, and ability to differentiate into different cell types. ESCs and CSCs share numerous features (pluripotency, self-renewal, expression of stemness genes, and acquisition of epithelial–mesenchymal features), and most of them are under the control of microRNAs (miRNAs). These small molecules have relevant roles during both embryogenesis and cancer development. The aim of this review was to recapitulate molecular mechanisms shared by ESCs and CSCs, with a special focus on the recently identified classes of microRNAs (noncanonical miRNAs, mirtrons, isomiRs, and competitive endogenous miRNAs) and their complex functions during embryogenesis and cancer development.


Introduction
Embryonic stem cells (ESCs), adult stem cells, and cancer stem cells (CSCs) represent different typologies of stem cells that can be found in our body. ESCs are derived from the inner cell mass of the blastocyst and own the outstanding property of self-renewal, meaning that they can proliferate indefinitely, maintaining stem-cell characteristics [1]. ESCs are also classified as pluripotent cells since they can differentiate into all three germ layers of the embryo and their derivatives [2,3] (Figure 1a).
The balance between self-renewal and differentiation is governed by a multitude of transcription factors (TFs), signaling and epigenetic changes, and noncoding RNAs [4][5][6][7][8]. Adult stem cells, also known as tissue-specific stem cells, are undifferentiated cells located in different regions of our body. These cells have a limited self-renewal ability, with a reduced differentiation potential and a strong propensity to maintain and repair the tissue in which they reside [9] (Figure 1b). Therefore, they can be considered multipotent or unipotent stem cells. CSCs are a rare subpopulation of cells exhibiting stem-like features, and they are responsible for tumor initiation, development, and progression [10][11][12] ( Figure 1c). The origin of CSCs is not still completely clear; they could be generated from tumor cells that acquire stem-cell properties or from normal stem cells that undergo a mutation during DNA replication [13,14]. CSCs can survive and take over the other cells inside the tumor mass, because they acquire exceptional properties that, in physiological

Noncanonical microRNAs Orchestrate New and Complex Functions in ESCs and CSCs
The biogenesis of microRNAs is a mechanism that has been widely described for several decades. It is well accepted that miRNA biogenesis starts with transcription of a long primary transcript, called a pri-miRNA, that, in the canonical pathway, is processed in the nucleus by Drosha and DCGR8 enzymes (forming the microprocessor complex) and converted into a shorter transcript, called pre-miRNA, after a stem-loop cropping [44] ( Figure 2).
Inside the cytoplasm, the pre-miRNA transcript is further processed by the endonuclease Dicer, which generates a small RNA duplex intermediate (about 22 nucleotides) [45]. The latter, together with the Argonaute (AGO) proteins, forms the RNA-induced silencing complex (RISC), which incorporates one strand of miRNA duplex as a template to complementarily bind a region in the 3 -UTR of the target mRNAs. The binding is mediated by a conserved heptametrical sequence, named seed sequence, typically spanning nucleotides 2-7 at the 5 -end of the microRNA sequence [46]. MicroRNAs work to finely regulate gene expression essentially in all cell processes. Many miRNAs are commonly expressed in ESCs and CSCs, but they fulfill context-specific functions (Table 1).

Figure 2.
Biogenesis of canonical and noncanonical microRNAs. In the top left panel, the initiation phase of canonical miRNA biogenesis is summarized. The top right panel shows the initiation phase of noncanonical miRNAs (mirtrons), whose processing occurs in a Drosha-independent manner and requires the action of spliceosome and debranching enzymes in the nucleus. In the cytoplasm (bottom panel), both canonical and noncanonical miRNAs undergo the same effector phase.  [51].
Not Reported.
Tumor-suppressor gene in most cancer types, such as early-stage breast cancer, follicular thyroid tumor, lung adenocarcinoma, and multiple myeloma. Its downregulation is used as biomarker for early cancer diagnosis [58][59][60][61].

miR-21
Nuclear microRNA involved in the regulation of ESC pluripotency. It directly targets Sox2, decreasing its expression and reducing ESC self-renewal [62,63].
Highly expressed in cancer stem/progenitor cells isolated from ovarian teratocarcinoma PA1 cells, with a potential role to mediate growth and self-renewal of CSCs [64].

miR-29
It contributes to the early differentiation of ESCs, repressing the expression of Tet1 transcript and promoting the upregulation of trophoblast lineage markers [66].
E14Tg2a cell line. Not reported.
ATCC/The Jackson Laboratory.
This could be responsible for LIN28 reactivation in a cancer context [79].
MicroRNA expressed at low levels in hepatocellular carcinoma. Its expression inversely correlates with the levels of the G9A histone methyltransferase, causing worst poor overall survival of patients [81]. Oncogenic miRNA overexpressed in different cancer cells with stem-like properties, such as breast cancer. Its inhibition significantly reduces the proliferation of invasive breast cancer cell lines, while its overexpression promotes the acquisition of stem-like properties [83].

miR-302
Involved in pluripotency maintenance of ESCs, through the downregulation of the inhibitors of G1/S cell-cycle transition [84]. OCT4 and SOX2 bind a conserved region in its promoter, such that its expression follows OCT4 expression during embryogenesis [85].
In P19 mouse embryonic carcinoma cells, it is transcriptionally activated by OCT4 [87]. Pluripotency inducer. It represses the transcripts involved in differentiation processes and maintains high levels of the pluripotency factor OCT4 [88]. Prevention of human induced pluripotent stem-cell tumorigenicity by reduction of the G1-S cell-cycle transition (suppression of Cyclin E-CDK2 and Cyclin D-CDK4/6 expression) and induction of the senescence-associated tumor-suppressor genes [89].
Its ectopic expression represses OCT4 and NANOG. The inhibition of the stemness signatures and tumorigenic properties of glioma-initiating cancer cells is mediated by the drastic downregulation of CXCR4/SDF1 pathway and inhibition of the expression of the cell-cycle-related transcripts E2F1, cyclinA, and cyclin D [90].

let-7 miRNAs
Involved in differentiation of ESCs [37]. The expression of miRNAs belonging to let-7 family is inhibited by LIN28 protein, to maintain the cells in their undifferentiated state [91].

miR-429
EMT suppressor acting during embryo implantation, through the targeting of members belonging to Cadherins family [104].
C57BL6/J and BALB/C mice. [104] Tumor suppressor in colon cancer, thanks to the direct binding to HMGB3 oncogene [105].
Oncogenic cluster involved in different human cancers (hepatocellular carcinoma, lung cancer, etc.), where it acts as an antiapoptotic, proliferation, and EMT-promoting factor [107,108].
MiR-200c is highly enriched in human ESCs (H9 and HUES6 Wicells), where it regulates self-renewal and differentiation, while, in human pancreatic cancer stem cells, isolated from PANC-1 by FACS and expressing CD24, CD44, and ESA markers, it appears as an important EMT regulator [47,52]. MiR-451 is upregulated in differentiating mESCs (E14Tg2a) and it is also involved in multiple cancer types (early-stage breast cancer, follicular thyroid tumor, lung adenocarcinoma, multiple myeloma, etc.), representing a biomarker for early cancer diagnosis and a therapeutic candidate for cancer treatment [57,[59][60][61]. Although, for several years, the function of miRNAs has been linked to a simple repression of target mRNAs, some studies have indicated that they can orchestrate more complex mechanisms of gene expression regulation [113].
Recently, different noncanonical miRNAs were identified that fulfill roles in ESCs and CSCs. Table 2 summarizes some examples of noncanonical miRNAs acting in both contexts.

Type of Noncanonical miRNA MiRNA Name Impact in ESCs
Mirtron miR-novel-41 Predominantly expressed in mouse ESCs. It is derived from the intron of the Man2c1 gene and is conserved between mouse and rat [114].

mir-702
Expressed in mouse ESCs. MiR-702 promotes the proliferation of Dgcr8-deficient ESCs, unlocking the arrest in G1 phase of the cell cycle [70,115].

mir-877
Expressed in mouse ESCs, where its mirtronic identity has been confirmed. It is conserved across mouse, humans, and chimps [115].

H/ACA snoRNAs
H/ACA snoRNA families are differentially expressed during the differentiation of mouse ESCs (E14Tg2a) with retinoic acid. Some of them are abundantly expressed in mESCs, while others are highly expressed in retinoic acid-differentiated cells [118].

Type of Noncanonical miRNA MiRNA Name Impact in CSCs and Cancer Cells
Mirtron miR-6778-5 5'-tail mirtron type that acts as critical regulator for maintenance of CSC stemness in Drosha-silenced gastric cancer cells [119].

miR-140
MiR-140 regulates CSCs in luminal subtype invasive ductal carcinoma. Downregulated in CSC-like cells. It targets the stem-cell factors SOX9 and ALDH1 in ductal carcinoma, regulating CSC self-renewal and tumor formation in vivo [120].
Noncanonical miRNAs resemble the structure and function of canonical miRNAs, but they undergo a different maturation pathway, for example in the initiation phase inside the nucleus [125,126] (Figure 2). Examples of noncanonical miRNAs are the alternative precursors named mirtrons, encoded by introns located in the coding regions of some genes, whose pre-miRNAs are generated by intron splicing machinery, bypassing Drosha processing [127] (Figure 2). There are three classes of splicing-derived miRNAs in mammals: conventional mirtrons, 5 -tailed mirtrons, and 3 -tailed mirtrons [128]. In conventional mirtrons, both ends of the pre-miRNA hairpin are defined by splicing mechanisms; they are exported in the cytoplasm and enter in the canonical pathway at level of Exportin 5 [128,129]. The 5 -end hairpins of 3 -tailed mirtrons feature the 5 -splite site, while the 3 -end features the branch point; in 5 -tailed mirtrons, the hairpin at the 3 -end of the intron is preceded by an unstructured region [130,131]. This indicates that tailed mirtrons have only one end of the pre-miRNA, which is formed by the splicing machinery. Of interest, Dicer recognizes and processes the 3 -end of 5 -extended pre-miRNAs, generating mature 3p and extended 5p miRNAs [132]. The analysis of deep sequencing data of small RNA sequences revealed that some miRNAs, annotated as canonical miRNAs in the available miRNAs databases, are processed through a noncanonical pathway [133]. Dicer processing has been reported as a mechanism almost essential for the biogenesis of both canonical and noncanonical miRNAs, while Drosha/DCGR8 cleavage has a more limited role. Indeed, while Dicer knockout caused the loss of canonical and noncanonical miRNAs, in Dcgr8/Drosha KO ESCs, the biogenesis of noncanonical miRNAs was preserved [134,135]. High-throughput sequencing data obtained from Dgcr8 wild-type, Dgcr8 KO, and Dicer KO cells revealed that mESCs express noncanonical miRNAs, as confirmed later by other studies [70,115,116].
Another class of noncanonical miRNAs is represented by small nucleolar RNA (snoRNA)-derived miRNAs. They are small noncoding RNAs, localized in the nucleolus, with a role in ribosomal RNA (rRNA) biogenesis and in the chemical modification of rRNA [136]. SnoRNA-derived miRNAs can regulate transcription or can bind the 3 -UTR of the target mRNAs, inhibiting their expression and functioning as miRNAs [137,138]. The analysis of short RNAs sequencing data also led to the identification of a new type of non-canonical miRNAs, represented by transfer RNA (tRNA)-derived miRNAs, important regulators of protein translation [139,140]. This new class of noncoding RNAs shares functional features with microRNAs; they undergo Dicer1 processing, form RISC complexes with Argonaute proteins and repress the expression of their target mRNAs [141]. Additional proteins able to generate tRNAs are RNase Z, which produces tRNAs from premature tRNA transcripts, and Angiogenin, which makes tRNAs under stress conditions. Thus, tRNA-processing enzymes, such as RNase Z, could generate functional miRNAlike species [125]. The 5 -tRNA-derived small RNAs can modulate the stem-cell state of mESCs [142]. Some 5 -tRNA-derived small RNAs, upregulated during mESC differentiation, interact with the RNA-binding protein IGF2BP1, leading to the repression of the pluripotency promoter factor c-Myc. tRNA-derived miRNAs have also been well characterized in the cancer context, where they share functional characteristics with microRNAs, repressing mRNA transcripts in a sequence-specific manner [141,143]. Aberrantly upregulated noncanonical miRNAs are responsible for the maintenance of malignant properties of CSCs that express low Drosha levels [119]. Altogether, this evidence revealed that noncanonical miRNAs orchestrate important functions in stem cells, and their deregulation is linked with the development of pathological states that often flow in cancer development.

Nuclear Functions of microRNAs
In the canonical pathway, the mature miRNAs promote translation inhibition of their target mRNAs through RISC in the cytoplasm [144,145]. However, different studies demonstrated that miRNAs can also act in the nucleus. The presence of microRNAs inside the nucleus is justified by the presence of some components of the miRNA pathway in this location. For example, the nuclear miRNA pathway contemplates the presence of Argonaute 2 and catalytically active Dicer in the nucleus. In this district, miRNAs are bound to the AGO2 protein, but the loading of duplex RNA is missing, and components of RISC are absent, indicating that the machinery is different between the nucleus and cytoplasm [146]. Different sizes between cytosolic and nuclear RISC were found; while a large complex has been detected in the cytoplasm (approximately 3 MDa), a smaller complex, only formed by AGO2 and a short RNA, has been identified in the nucleus (158 kDa) [147]. Therefore, it is hypothesized that the AGO2/miRNA complex could be formed outside the nucleus. Then, the formed minimal RISC complex could be imported in the nucleus [148]. The nuclear transport of microRNAs is facilitated by the presence of different nuclear localization signals (AGUGUU motif, consensus ASUS sequence, 5 -UUGCAUAGU-3 and 5 -AGGUUGKSUG-3 motifs) located in their sequence [149,150]. The nucleus-cytoplasm trafficking of mature microRNAs is also mediated by Exportin-1, which serves for the translocation of both mature miRNAs and Argonaute proteins in the nucleus [151,152]. Accumulating evidence has revealed that miRNAs can be shuttled from the cytoplasm to the nucleus, because they are involved in the regulation of biogenesis and function of different noncoding RNAs, included other miRNAs and long noncoding RNAs, as well as in the transcriptional activation or silencing of the target genes [150,153]. Data from cell fractionation and high-throughput sequencing even predicted the genomic DNA-binding sites for nuclear miRNAs, which could play a role in the regulation of transcription [154]. Lastly, microRNAs can enter the nucleus to be modified, interact with nuclear proteins, or participate in mechanisms responsible for chromatin remodeling [155]. For example, miR-21, a microRNA involved in regulation of pluripotency in ESCs (E14Tg2a.4 cell line, Bay Genomics), represents one of the first microRNAs detected in the nuclear and cytoplasmic extracts of HeLa cells [62,63]. This microRNA acts in the REST-miR-21-SOX2 axis in ESCs; miR-21 directly targets Sox2, decreasing its expression and reducing mESC self-renewal. In the undifferentiated state of ESCs, miR-21 is repressed by the transcriptional repressor REST, to avoid the loss of self-renewal. Mir-21 is also highly expressed in cancer stem/progenitor cells (CSPCs), isolated from human ovarian teratocarcinoma PA1 cells by FACS, using an antibody directed against the stem-cell marker CD133 [65]. In CSPCs, mir-21 could promote tumorigenesis using different mechanisms; it could regulate the self-renewal of progenitor cells, could produce growth factors, or could induce the dedifferentiation of non-progenitor cancer cells, all of which lead to an enrichment of the stem-cell population [64]. These effects were evaluated by functional assays such as sphere formation and experiments aimed at evaluating CD133 expression (cell sorting, qRT-PCR) [64,65]. MiR-29b is another example of a miRNA with nuclear localization; while miR-29a is mainly located in the cytoplasm, miR-29b showed a nuclear localization mediated by a hexanucleotide terminal motif (AGUGUU) in its 3 -UTR [156]. The MiR-29 family contributes to early differentiation of ESCs (E14Tg2a cell line) by regulating the expression of TET1, the dioxygenase converting 5 -methylcytosine into 5 -hydroxymethylcytosine [66]. TET1 is highly expressed in undifferentiated mESCs; in this context, mir-29 directly targets the Tet1 transcript, causing the downregulation of the TET1 protein and promoting the upregulation of trophoblast lineage markers. In cancer, miR-29 mainly functions as a tumor suppressor although some studies have described it as an oncogene [67]. In ovarian cancer, miR-29 has been proposed as an important regulator of cancer metabolism. Indeed, miR-29b re-expression in ovarian cancer cells inhibits glycolysis and glucose metabolism, directly targeting AKT2 and AKT3 [67,68]. In melanoma cells (A375 and A375-STA1 wt cells), it mediates antiproliferative effects by downregulating CDK6, a regulator of G1/S phase [67,69]. Additional examples of microR-NAs with nuclear localization are miR-320, miR-30e, and miR-122 as well as many human miRNAs identified in the nucleus of neural stem cells [157][158][159][160]. MiR-320 and miR-702 represent two noncanonical miRNAs able to induce the proliferation of Dcgr8-deficient ESCs (germline-competent wild-type (W4), Dgcr8-deficient (Dgcr8 ∆/∆ ), and Dicer-deficient (Dicer ∆/∆ ) cells). These miRNAs bind the 3 -UTR region of the cell-cycle inhibitors p57 and p21, unlocking the cells from the G1 cell-cycle arrest [70]. In cancer cells, miR-320 has a tumor-suppressor function; it is downregulated in different cancers such as breast cancer, glioma, gastric cancer, retinoblastoma, and human non-small-cell lung cancer, and it represents an important EMT inhibitor by reducing the levels of E-Cadherin and increasing those of N-Cadherin and Vimentin [31, 72,[75][76][77][78]. MiR-30 is one of the four miRNAs able to repress the expression of LIN28 protein in both ESCs (mouse embryonic stem cell lines R1 and C57BL/6J-693, ATCC and The Jackson Laboratory) and LIN28-positive human breast cancer cell line T47D, directly binding its 3 -UTR. This mechanism, finely regulated in ESCs, could cause the reactivation of LIN28 in cancer [79]. MiR-122 is a regulator of ESC differentiation; it acts in the miR-122/FoxA1/HNF4a-positive feedback loop, and its overexpression promotes the hepatic differentiation of mESCs (Cyagen company, Santa Clara, CA, USA) [80]. Mir-122 is expressed at low levels in hepatocellular carcinoma, and its expression inversely correlates with that of the G9A histone methyltransferase, causing worst overall survival of patients [81].

Noncanonical Gene Targeting of microRNAs
The analysis of miRNA targetome provided substantial information regarding the miRNA-target interactome [161]. Different microRNAs can regulate gene expression using atypical targeting mechanisms. A recent study revealed that up to 60% of miRNA-target interactions in cancer cells occur via noncanonical seed pairing [162]. Noteworthy, miRNAs having a low GC content in their seed sequence use the noncanonical gene targeting as dominant mechanism for target recognition. However, noncanonical gene targeting does not significantly reduce target expression, as instead happens for the canonical pathway [163]. Noncanonical gene targeting of microRNAs can occur through "seed-like motifs", which include seed sequences containing mismatches, deletions, or wobble pairings [161,164]. One example is represented by miR-155. The transcriptome-wide identification of miR-155 targets revealed that approximately 40% of miR-155-dependent Argonaute binding does not require a perfect seed match [165]. MiR-155 is expressed in ESCs (mouse ESCs CGR8 cell line, ECACC) and is gradually downregulated during ESC cardiac differentiation [82]. It also represents a well-known oncogenic miRNA, overexpressed in different cancer cells with stem-like properties, such as breast cancer [83]. Soft agar colony formation assay and tumor xenografts revealed that miR-155 inhibition reduced the proliferation of the invasive cell line MDA-MB-231, while its overexpression coincided with the acquisition of stem-like properties, as confirmed by sphere-forming experiments [83]. Another example of noncanonical targeting is represented by the microRNAs miR-134, miR-296, and miR-470. These miRNAs target the coding region of ESC pluripotency genes Nanog, Oct4, and Sox2 in mESCs (E14Tg2a, ATCC) instead of/further than their 3 -UTR [166].
IsomiRs, distinct isoforms generated by miRNA precursor arms, are also considered part of noncanonical gene targeting mediated by miRNAs. IsomiRs can be generated by alternative Drosha and/or Dicer processing of pri/pre-miRNA molecules or by posttranscriptional modifications induced by the nucleotidyltransferase [167,168]. An example of how isomiRs can promote noncanonical gene targeting is provided by miR-124. The processing of pri-miR-124 generates two 5 isomiRs, derived from seed sequences shifted by a single nucleotide, which act by regulating different transcripts, contained in overlapping targetomes [169]. Next-generation sequencing data obtained from hESCs (CyT49 (ViaCyte), H1, and H9 cell lines) led to the identification of some miRNAs, such as miR-302, whose stem loop generates different highly expressed isomiRs, with important roles in hESC self-renewal [170]. For example, in hESCs, an miRNA mimetic for miR-302a-5p caused a reduced expression of OTX2, while isomiR 302a-5p(+3) decreased the expression of tuberous sclerosis protein 1 [170]. Dominant isomiRs are also expressed during different stages of hESC differentiation. In differentiating hESCs (H9, HSF1) and induced pluripotent stem cell lines (hIPS2, all from UCLA Stem Cell Core), editing sites in 24 different miRNAs and major-to-minor arm-switching events in 14 pre-miRNAs have been identified [171]. IsomiRs also play important roles in cancer. They have been used to distinguish different breast cancer subtypes and, thus, act as biomarkers [172]. IsomiR expression changes in gastric tumor tissues; for instance, the processing of the same pre-miRNA can generate differentially expressed 5p and 3p arm miRNAs: one specific for the normal tissue, the other one specific for the tumor tissue [173]. Collectively, these data demonstrate that the functions mediated by miRNAs, which are already very complex, present an additional level of complexity due to the possibility to act through noncanonical gene targeting.

The Stemness Properties of ESCs and CSCs Are Regulated by the Same miRNA Circuits
CSCs, having expression signatures that are specific to ESCs, have been identified in many human tumors (human epithelial, breast, and lung cancers) and mouse cancer models [174]. In ESCs (mouse J1 ES cell line), the specific gene expression signature is orchestrated by different modules such as the core module, which includes the core pluripotency factors, the PRC module, represented by polycomb complex factors, and the MYC-module, including MYC-related factors. ESCs and cancer cells share MYC-module activity, raising the hypothesis that cancer cells can reactivate the ESC gene signature [175]. Similarly, the self-renewal ability of leukemia stem cells is sustained by a transcriptional subprogram more like that of ESCs than adult stem cells [176]. In general, cancer cells (mouse lung squamous carcinoma cells, SqCLCs, and KLN205) and mESCs (Celprogen) are very similar to each other, but highly different from normal cells in terms of spectral variations in protein, lipid, carbohydrate, and nucleic acid components [177]. Histologically poorly differentiated tumors also share features with ESCs; in fact, they overexpress the ESC-specific genes NANOG, OCT4, SOX2, and c-MYC and repress the Polycomb-regulated genes [178]. Invasive breast cancers are also able to secrete embryonic morphogens such as NODAL, an embryonic molecule that guides the transition from naïve to formative pluripotency [179,180]. Recent studies demonstrated that microRNAs could regulate similar circuits in ESCs and CSCs. For example, miR-302 is an ESC-specific miRNA that orchestrates the cell cycle, acting in the G1/S cell phase of Dgcr8 knockout (Dgcr8 ∆/∆ ) ESCs [84]. MiR-302 regulates human iPSCs tumorigenicity, suppressing the expression of Cyclin E-CDK2 and Cyclin D-CDK4/6, promoting the expression of the senescenceassociated tumor-suppressor genes, and reducing G1-S cell-cycle transition [89]. In this section, we provide an overview of recently described microRNAs able to regulate common functions in ESCs and CSCs, with the purpose of highlighting the molecular mechanisms linking these two faces of stemness.

Pluripotency-Regulating miRNAs in ESCs and CSCs: The Role of LIN28/let-7 Axis
Different clusters and families of microRNAs regulate the stemness and pluripotency of ESCs. Two major clusters, represented by mir-290-295 and mir-302-367, are highly expressed in ESCs (J1 ESCs and induced pluripotent stem cell lines), where they regulate cell-cycle progression and are responsible for the induction of stemness properties [4]. These clusters are downstream targets of the pluripotency transcription factors [85]. OCT4 and SOX2 bind a conserved region in mir-302 promoter, such that its expression follows Oct4 expression during embryogenesis. A similar mechanism occurs in P19 mouse embryonic carcinoma cells, where OCT4 is required for the expression and transcriptional activation of mir-302. Indeed, OCT4 binds the putative promoter of mir-302, activating the transcription of the primary mir-302 [87]. Mechanistically, in human pluripotent stem cells (hESC lines H9 from Wicell and chHES-22), miR-302 acts as a pluripotency inducer; this miRNA binds the 3 -UTR of target mRNAs involved in differentiation processes, such as AKT1, and suppresses their expression, thus maintaining high levels of OCT4 and hampering teratoma formation [88]. However, in human glioma-initiating cells (primary cell lines TG1, TG6, and GB1, isolated from human glioblastoma), miR-302 has an opposite effect, because it promotes the exit from the stem-cell-like state. Indeed, its endogenous expression is inversely correlated with NANOG and OCT4, and its ectopic expression in glioma stem cells is sufficient to repress OCT4 and NANOG, as well as tumor aggressiveness [39]. In detail, the suppression of the stem-cell-like signature induced by the miR-302-367 cluster in glioma-initiating cells is mediated by the drastic downregulation of the pathway mediated by the chemokine receptor CXCR4 and its ligand SDF1, because miR-302a binds to the CXCR4 3 -UTR. This pathway, when suppressed, causes the disruption of the network SHH-GLI-NANOG responsible for the acquisition of the stem-cell-like signature, inhibiting the tumorigenic properties of the glioma-initiating cells [39]. The MiR-302-367 cluster also causes a pronounced downregulation of transcripts normally involved in the regulation of cell-cycle progression, such as E2F1, cyclin A, and cyclin D, revealing that its expression had a negative impact on cell infiltration and self-renewal. These observations indicate that, in the cancer context, miR-302 can play a dual role, because, as discussed in the previous section, it can also promote the expression of the senescence-associated tumor-suppressor genes [89].
Pluripotency in ESCs and CSCs is regulated by the overlapping functions of miRNAs and RNA-binding proteins. An example is provided by the RNA-binding protein LIN28. In physiological conditions, LIN28 expression is highly restricted to ESCs (E14Tg2a cell line, Bay Genomics), where it is transiently induced after the exit of ESCs from the naïve ground state and downregulated in differentiated cells [42,181]. LIN28, together with OCT4, SOX2, and NANOG, can also reprogram somatic cells into induced pluripotent stem cells, functioning as important regulator of pluripotency [182,183]. In pathological conditions, it acts as potent oncogene, causing malignant transformation and tumor progression [184,185]. Indeed, LIN28 has a widely recognized role in cancer, where it promotes proliferation, migration, and invasion of human bladder cancer cell lines (5637, SW780, T24, and J82), using the same mechanisms described in mESCs [186]. Indeed, it can act as pluripotency inducer by regulating the biogenesis of microRNAs belonging to let-7 family (Figure 3). In CSCs, this regulation is completely reverted, because the oncogenic protein LIN28 represses the expression of the same miRNAs (lower section of the left panel) causing tumorigenesis. In the right panel (upper section), the mechanism responsible for the transient induction of LIN28 upon ESC exit from the naïve ground state of pluripotency is described. This event is promoted by HMGA2-dependent engagement of OTX2 to Lin28 enhancers. In undifferentiated ESCs, LIN28 represses let-7 biogenesis, while, during the differentiation, it controls the levels of Hmga2, binding a conserved region in its 3 -UTR (let-7-independent mechanism). In the lower section of the right panel, the miRNA/HMGA2 axes acting in CSCs are described; the inhibition of let-7 miRNAs by HMGA2 causes proliferation, migration, and invasion of CSCs, while the repression of Hmga2 expression by specific microRNAs suppresses cancer malignancy.
LIN28 inhibits the maturation of let-7 miRNAs to maintain the cells in the undifferentiated state; therefore, its inhibition could represent a possible mechanism to block tumor progression [91,185]. As an RNA-binding protein, LIN28 recognizes and remodels stable planar structures of four guanines, known as G-quartet structures, which are found in microRNAs and its target mRNAs [187]. During embryogenesis, LIN28 levels are regulated by microRNAs such as let-7, miR-125, miR-9 and miR-30, whose expression inversely correlates with LIN28 expression [79] (Figure 3). This negative correlation could explain the oncogenic function of LIN28; indeed, human breast cancer cell lines, expressing high levels of LIN28, are characterized by the downregulation of these miRNAs [79]. A recent study revealed that the treatment with a LIN28 inhibitor (small compound C1632) can increase the levels of let-7 and suppress the expression of the immune checkpoint protein PD-1/PD-L1, reactivating the antitumor activity in MCF-7, U2OS, and HeLa cancer cells [188]. Moreover, let-7 miRNAs can act as tumor suppressors, since, in non-small-cell lung cancer A549 cells harboring mutant KRAS, their substitution reduced both the stemcell population and the resistance to chemotherapy, causing cytotoxicity and inducing apoptosis and reduced invasiveness of the tumor cells [92,93]. In some types of cancers (i.e., non-small-cell lung cancer), the reduced expression of let-7, caused by irradiation, is responsible for the increased expression of LIN28, which further decreases let-7 levels, thereby promoting resistance to irradiation [189]. Indeed, in cancer cells, the downregulation of microRNAs belonging to the let-7 family is a mechanism frequently adopted to promote tumor progression. For example, the long noncoding RNA H19, normally expressed during ESC differentiation, competitively sponges let-7 microRNAs in breast cancer cells (MDA-MB-231 and SK-BR-3 cells), causing an increase in LIN28 expression. The latter further blocks let-7 biogenesis and, in turn, derepresses H19 expression, forming a double-negative feedback loop that promotes breast cancer stem-cell maintenance [94]. In multiple myeloma, the LIN28B/let-7 axis modulates the expression of MYC, which in turn is a let-7 target, suggesting a novel mechanism for therapeutic targeting of the tumor [95]. However, in rare cases, microRNAs belonging to the let-7 family can also act as oncogenes, increasing cancer migration, invasion, and progression [92]. For example, an anchorageindependent assay revealed that let-7a3 overexpression in lung cancer cells caused an increase in aggressiveness [96]. Similarly, let-7f-5p and let-7e-5p were highly expressed in oral cavity squamous cell carcinomas, with let-7f-5p upregulated in nonaggressive tumors and let-7e-5p in aggressive ones [97]. Some tumors are also characterized by LIN28 loss. For example, in glioblastoma stem cells LIN28 is undetectable, while let-7 miRNAs and their targets are expressed. In this context, another RNA-binding protein, named insulin-like growth factor 2 mRNA-binding protein 2 (IMP2), binds to let-7 miRNAs, preventing the repression of let-7 targets [190].

Pluripotency-Regulating miRNAs in ESCs and CSCs Targeting the Architectural Protein HMGA2
In neuroendocrine pancreatic cancer cells, LIN28 can induce the stem-like genes, suppressing let-7 miRNAs and derepressing HMGA2 [191]. HMGA2 is an architectural protein, expressed early during embryogenesis and in ESCs (E14Tg2a cell line, Bay Genomics), whose suppression hampers the exit of ESCs from the pluripotent ground state [6]. Interestingly in mESCs (E14Tg2a cell line Bay Genomics), Hmga2 is regulated by let-7-indipendent mechanisms. Indeed, Lin28a binds highly conserved elements located in Hmga2 mRNA to properly control Hmga2 accumulation during differentiation [42]. HMGA2 expression is undetectable in adult tissues, and it is significantly overexpressed in different cancers as hepatocellular carcinomas, esophageal squamous cell carcinoma, tongue squamous cell carcinoma, and thyroid carcinoma [192][193][194][195][196]. It represents another ESC-specific factor that can induce tumorigenesis using multiple mechanisms. For example, HMGA2 induces the proliferation of cancer cells (ovarian cancer, leukemia, breast cancer, and colorectal cancer) promoting cell-cycle entry and inhibiting apoptosis, but it can also exhibit effects on pathways involved in DNA repair and epithelial-to-mesenchymal transition [197][198][199][200][201]. These mechanisms are often mediated by different microRNAs. HMGA2 3 -UTR is directly targeted by miR-142-3p, inducing a decrease in HMGA2 protein and suppressing breast cancer malignancy [202]. MiR-9 is another type of microRNA that mediates antitumor activities on hepatocellular carcinoma progression directly targeting HMGA2 [203]. Similarly, miR-1249 suppresses the growth, metastasis, and angiogenesis of colorectal cancer cells (HCT116, HT29, SW480, SW620, HCT8, and DLD-1) by targeting VEGFA and HMGA2 [204]. MiR-125b-5p also inhibited cell proliferation, migration, and invasion of esophageal squamous cell carcinoma partially by downregulating HMGA2 [205]. HMGA2 3 -UTR also contains repressive regulatory binding sites for let-7 miRNAs, which are responsible for HMGA2 mRNA decapping and degradation, allowing the correct tissue-type differentia-tion of the normal mesenchymal tissues [206]. The TRIM71 protein, which regulates early development and differentiation, can act as a tumor suppressor by post-transcriptionally repressing LIN28B and modulating the let-7/Hmga2 axis [207]. The reduction in let-7 miRNAs represents one of the main mechanisms responsible for HMGA2 overexpression in atypical teratoid/rhabdoid tumors; therefore, the reconstitution of let-7 miRNA levels or HMGA2 knockdown may represent good therapeutic strategies for cancer treatment [208]. In different mesenchymal tumors, chromosomal rearrangements and breakpoints can generate truncated HMGA2 mRNA transcripts, which are devoid of the 3 -UTR regions, thus altering miRNA-mediated regulation of HMGA2 expression [206,209,210].

EMT-Regulating miRNAs in ESCs and CSCs
Epithelial-to-mesenchymal transition is a well-characterized process that can occur in physiological and pathological conditions such as embryonic development, tissue repair, wound healing, and cancer [50]. Typically, in EMT, the epithelial cells lose their typical traits and acquire mesenchymal features [211]. Epithelial cells are highly plastic cells, which, during embryogenesis and in certain physiological conditions, need to transiently repress their epithelial features (loss of epithelial junctions, repression of epithelial genes) and acquire mesenchymal properties (expression of mesenchymal genes, elongation of cell shape, migratory and invasive phenotype) [212]. This mechanism favors cell migration to a different location. During embryonic development, EMT allows the correct differentiation of cells and the remodeling of tissues. In the cancer context, this allows tumor cells to dissociate from the primary tumor mass and disseminate to distant organs. In the new location, the cells reactivate the epithelial features, causing metastasis [213]. EMT is a process initiated by paracrine signals produced by stromal cells, among which the most famous are the transforming growth factor (TGF)-β, Wnt, Notch, and Sonic Hedgehog [214,215]. All EMT factors are controlled at the transcriptional and translational level by transcription factors and microRNAs [216]. The EMT gene signatures and the relative interactomes (miRNAs, transcription factors, and proteins) are contained in a database, named EMTome, which can be used as a portal for research studies [213]. Many microRNAs regulate the expression of the EMT-transcription factors, such as SNAIL1/SNAIL2, bHLH (E47, E2-2, and TWIST1/TWIST2), and ZEB1/ZEB2, which mainly function as E-Cadherin repressors [217]. In hESCs (H9 and H1 cell lines, WiCell Institute), the zinc-finger E-box-binding homeobox (Zeb) transcription factor is targeted by miR-200 family members, which are highly expressed in ESCs but downregulated in a Wnt-dependent manner during EMT [48,49]. During hESC differentiation (MC50 from Dr. Robert Schreiber and modified A2lox cells), the miR-200 family downregulates the expression of the transcription factor ZEB1 and its target E-Cadherin, to define the proper cell fate [49,50]. In ESCs, miR-200 also acts against the transcription factor Snail1 to regulate EMT [49]. Altogether, these mechanisms indicate that miR-200 members have an inhibitory role in EMT, limiting it spatially and temporally. In the cancer context, members of the miR-200 family are often repressed, such that EMT could occur without spatial and temporal limits. In fact, in colon cancer cells (Caco-2, LS174T, LoVo, HT-29, HCT116, SW480, and SW620) and human colorectal cancer tissues, NANOG directly represses the transcription of mir-200b/c genes, modulating EMT to mesenchymalepithelial transition plasticity [53]. Similarly, ZEB1 can promote tumor cell dissemination and metastasis, repressing the expression of the miR-200 members [54]. Accordingly, a recent study revealed that miR-200 removal in an insulinoma mouse model, as well as the depletion of miR-200 sites in endogenous Zeb1, caused beta-cell dedifferentiation, EMT initiation, and tumor invasion [55]. MiR-200 can also be sponged by lncRNAs, such as lncATB, which induces EMT, restoring TWIST1 expression and causing poor prognosis in breast cancer [56]. In breast tumors, miR-200 family can exhibit a dual role; miRNAs belonging to this family have been found upregulated in breast tumors, while they are downregulated in more aggressive breast cancer molecular subtypes, revealing that the levels of miR-200 members are correlated with the nature of the tumors [218]. Another important cluster of microRNAs regulating EMT during embryonic development and cancer initiation is represented by a microRNA cluster on chromosome 19 (C19MC). This is the largest human microRNA cluster, which contains 46 miRNAs [219]. Bioinformatics analysis showed that some of the C19MC miRNAs share the "AAGUGC" seed sequence with members of the miR-302-372 family and that their putative targets could be involved in reprogramming and apoptosis induction [220]. C19MC is expressed in placenta, ESCs, and cancer. The transcriptional activation of the entire C19MC cistron in human villous trophoblasts resulted in the suppression of EMT-related genes and in the induction of the OCT4 and FGF4 expression [98]. This large cluster of microRNAs is a transcriptional hallmark of different types of cancers (type A and AB thymomas, hepatocellular carcinoma, undifferentiated embryonal sarcoma of the liver, embryonal tumor with multilayered rosettes, etc.) and undergoes recurrent chromosomal breakpoints [99][100][101][102][103]. For example, rearrangements of the chromosomal band 19q13.4 are a typical cytogenetic feature of the thyroid adenomas, and the miRNA gene cluster C19MC is near the breakpoint region [221]. MiR-429 is another EMT suppressor that acts during embryo implantation, by targeting a member of cadherin family, named Pcdh8 [104]. MiR-429 acts as a tumor suppressor in colorectal cancer, thanks to its direct binding to HMGB3, a strong oncogene overexpressed in cancer tissues [105]. As previously mentioned, high-mobility group proteins also play an important role in EMT. HMGA2 promotes EMT by activating specific signaling pathways, as MAPK/ERK, TGFβ/SMAD, PI3K/AKT/mTOR, NF-κB, and STAT3 [201]. The TGF-β molecule can induce EMT by promoting the expression of the embryonic protein HMGA2 that, together with SMADs, regulates different EMT transcription factors [222]. During EMT, HMGA2 promotes the binding of the de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter, inducing the hypermethylation and silencing of the tumor-suppressor E-Cadherin (CDH1); this causes tumor cell invasion [222]. In endometrial cancer, the overexpressed lncRNA miR-210-HG sponges miR-337-3p/137, increasing HMGA2 expression and modulating the malignancy of the tumor via TGF-β/Wnt pathway [223]. Collectively, these data indicate that EMT can be considered a further mechanism commonly shared by ESCs and CSCs. Interestingly, the same classes of microRNAs and proteins, which are normally expressed during embryonic EMT, can be reactivated in pathological conditions, contributing to cancer development and progression.

Regulation of miRNAs through Competitive Endogenous RNAs in ESCs and CSCs
The functions mediated by microRNAs can be regulated by a further class of RNAs, which emerged in recent years, called competitive endogenous RNAs (ceRNAs). These RNA molecules can influence the functions mediated by microRNAs, competing with them inside the cells and providing an additional mechanism of post-transcriptional regulation [224]. The ceRNA-miRNA network can act in both physiological (cell differentiation, regeneration mechanisms, and neural and muscle developmental processes) and pathological conditions (cardiovascular and neurodegenerative diseases, and cancer) [224,225]. The regulatory network mediated by ceRNAs is quite complex because they cross-regulate each other by sponging shared miRNAs [226]. In ESCs, lncRNAs function as ceRNAs to regulate the expression of mRNAs by competitively binding miRNAs. For example, the lncRNA LINC-ROR forms a feedback-loop with transcription factors and microRNAs in self-renewing hESCs (H1 and X-01 cell lines, Zhejiang University) to regulate ESC maintenance and differentiation [227]. LINC-ROR shares miRNA response elements with the core transcription factors NANOG, OCT4, and SOX2 and it prevents the binding of these transcription factors to miRNAs (miR-145), inhibiting the suppression of their expression. Similarly, lnc-NAP, a lncRNA activated by the pluripotency factors NANOG, OCT4, and SOX2, could inhibit the effects mediated by miR-139-5p, impairing its binding to Nanog 3 -UTR and causing Nanog de-repression in mESCs (C57BL/6J ESCs and B6D2F1 iPSCs) and embryos [228]. Aberrations in the ceRNA network can lead to pathological conditions and cancer development [229]. LINC-ROR, acting as a ceRNA in ESCs, is involved in the occurrence and development of different human tumors (breast cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma), and it represents a potential biomarker with clinical significance that can be used as therapeutic target [230]. In colon cancer stem cells, as in ESCs, LINC-ROR acts as a ceRNA to prevent miR-145-mediated suppression of NANOG, OCT4, and SOX2 TFs to regulate cell proliferation and chemosensitivity [231]. LINC-ROR sponge activity against miR-145 also leads to the derepression of ZEB2, inducing EMT in hepatocellular carcinoma and promoting metastasis [232]. In human gastric cancer cells (AGS and MGC-803 cell lines), LINC-ROR sponges miR-212-3p, promoting the proliferation, migration, and invasion of CSCs, as demonstrated by the CCK-8 assay, transwell assays, and a xenograft mouse model [233]. LINC-ROR can also function as a ceRNA for some members of the let-7 family. MTT, wound healing, and matrigel invasion assays, as well as sphere-formation experiments, revealed that the sponge activity of LINC-ROR against let-7 miRNAs contributed to the stem-cell properties of pancreatic cancer cells [234]. In retinoblastoma tissues, LINC-ROR is activated by H3K27 acetylation and sponges miR-32-5p, modulating EMT and regulating Notch signaling [235]. LncRNA XIST, expressed in differentiating ESCs (F1 2-1 ESC line), promotes metastasis and EMT in colorectal cancer, sponging miR-125b-2-3p [236,237]. Likewise, many lncRNAs involved in colorectal cancer act as ceRNAs and regulate the expression of the EMT transcription factors such as ZEB1, 2/E-Cadherin, and Wnt/β-Catenin signaling [238]. CeRNAs have been described as critical components of the TGFβ-induced EMT pathway, and they represent potential targets to disrupt EMT during tumorigenesis [226].
As previously mentioned for LINC-ROR, competitive endogenous RNAs also play important roles in CSCs. For example, the actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), functioning as an endogenous RNA, competitively binds to miR-384, regulating the expression of the Activin Receptor A type I (ACVR1) and inhibiting the stemness of pancreatic cancer cells and tumorigenicity in nude mice [239]. Similarly, the transcription factor E2F6 can also function as a ceRNA, inhibiting the effects mediated by the tumor suppressor miR-193a and promoting the stemness of ovarian cancer cells (HeyC2) through the upregulation of the ovarian cancer stemness marker c-KIT [240]. In thyroid cancer cells (TPC-1 and K-1, ATCC), the long noncoding RNA H19, acting as a ceRNA, inhibits the effects mediated by miRNA-3126-5p, increasing the expression of the estrogen receptor β and inducing cancer stem-like properties [241]. Similarly, H19 is responsible for glycolysis and maintenance of breast cancer stem cells thanks to its ability to bind to let-7 miRNA, releasing hypoxia-inducible factor 1α and increasing the expression of pyruvate dehydrogenase kinase 1, protein highly expressed in breast cancer stem cells [242].
Circular RNAs (circRNAs) are another class of RNAs that can sponge microRNAs, thus regulating their functions in embryogenesis [243,244]. CircRNAs are single-stranded RNA molecules generated by back-splicing reactions [245]. A circRNA map of transcripts has been associated with naïve and primed pluripotency of hiPSCs generated from cord blood MSCs, and numerous studies also revealed that they regulate stem-cell differentiation [246,247]. RMST and FIRRE have been identified as lncRNAs, that are processed as circular lncRNAs during hESC differentiation (H9 cell line, WiCell) [248]. Similarly, the two circular RNAs, circBIRC6 and circCORO1C, have been associated with the pluripotent state of hESCs (H9 cell line), where they interact with miR-34a and miR-145 to modulate hESC pluripotency and differentiation [249]. CircRNAs are also involved in human cancer development (liver, lung, colorectal, breast, prostate, bladder, ovarian, kidney, and gastric cancers, hematological malignancies, and tumors of the central nervous system) and progression, and, to date, they are considered potential diagnostic and prognostic biomarkers [250]. MiOncoCirc is the first database containing circRNAs directly detected in tumor tissues, which allows identifying circRNA candidates as biomarkers for prostate cancer [251]. The circRNAs expressed in ESCs are often found overexpressed in cancer. For example, RMST is overexpressed in medulloblastoma, while circBIRC6 promotes non-smallcell lung cancer cell progression by targeting miR-145 and hepatocellular carcinoma by sponging miR-3918 [252][253][254]. An oncogenic role has also been described for circCORO1C, which promotes the progression of laryngeal squamous cell carcinoma, competitively binding to let-7c-5p and inducing EMT [255]. CircRNAs are also aberrantly expressed in CSCs. In breast cancer stem cells, they could be involved in stemness inhibition, acting as miRNA sponges. In fact, the circRNA VRK1 could inhibit the expansion and self-renewal ability of the breast CSCs, representing a good target for the therapeutic treatment of the tumor [256]. In CSC-enriched colorectal cancer spheroid cells, a circRNA-miRNA-mRNA axis has been identified and it is able to modulate stemness-related pathways. Two circR-NAs (hsa_circ_0066631 and hsa_circ_0082096), downregulating the expression of several microRNAs (miR-140-3p, miR-224, miR-382, miR-548c-3p, and miR-579), inhibit the degradation of transcripts involved in different pathways regulating CSC stemness [257]. In glioma stem cells, circATP5B competitively sponges miR-185-5p, upregulating the expression of the homeobox gene HOXB5, which induces the proliferation of glioma stem cells through JAK2/STAT3 signaling [258]. In the same context, the circRNA cARF1 competitively binds to miR-342-3p, upregulating the expression of the transcription factor ISL2, which induces the expression of U2AF2 and causes an oncogenic effect [259]. In human liver cancer, the circRNA CircMEG3 inhibits the growth of the liver cancer stem cells repressing the expression of the m6A methyltransferase METTL3 and Cbf5, which in turn represses the telomerase activity [260].
Collectively, these data indicate that the mechanisms of post-transcriptional regulation can be highly complex and can involve different classes of RNAs, able to regulate each other.

Impacts of Tissue-Specific miRNAs on Adult Stem Cells and CSCs
The identity and function of certain tissues is guaranteed by the presence of microR-NAs having tissue-or time-specific expression patterns [261]. Tissue-specific microRNAs (TS miRNAs) are a class of miRNAs expressed in specific tissues of our body. Their expression is regulated by non-tissue-specific transcription factors, whose binding sites are located near to the transcription start site of the TS miRNAs [262]. In our body, different miRNAs show a tissue-specific expression pattern. For example, miR-122 is specifically expressed in the liver; miR-9, miR-124, and miR-128a/b are specifically expressed in brain; miR-7, miR-375, miR-141, and miR-200a are specifically expressed in the pituitary gland and intestine; miR-142 is specifically expressed in hematopoietic cells and the colon; miR-1 is strongly expressed in human adult heart, with low levels in liver and midbrain; miR-143 is particularly abundant in the spleen [263][264][265][266]. TS miRNAs have been also selectively identified in adult stem cells, as in the case of the mesenchymal stem cells (MSCs, Lonza) (miR-196b, -196a, -615, -501, -449, -17-3p, -497, and -486) and liver-resident stem cells, isolated from human cryopreserved normal adult hepatocytes (HLSCs, Lonza) (miR-7, -95, -204, and -650) [267]. Of interest, cell-derived microvesicles isolated from MSCs also contain patterns of miRNAs specifically expressed by MSCs (miR-103-1, -140, -143-5p, and -340), which are specific to cell origin and represent a peculiar signature for adult stem cells [267]. Moreover, the different expression of exosome-delivered microRNAs between somatic stem cells and CSCs also represents a good way to specifically identify the CSCs [268]. Indeed, the aberrant expression of microRNAs normally expressed in adult stem cells can be responsible for cancer development. In this section, we recapitulate the impacts of miRNAs on adult stem cells and CSCs, with particular attention paid to microRNAs differentially expressed between neural stem cells (NSCs) and brain tumor-initiating cells, intestinal stem cells (ISCs) and colon CSCs and mammary stem cells (MaSCs) and breast CSCs.

MiRNAs Expressed in Neural Stem Cells and Brain Tumor-Initiating Cells
NSCs are adult stem cells, located in the nervous system that, during embryogenesis, guarantee nervous tissue development, whereas, during adult life, they are reduced in number, remain quiescent, and are limited to specific areas of the brain [269]. Recent studies suggest that, in the adult tissue, NSCs are involved in neuronal plasticity, aging, disease, and regeneration [269]. MicroRNAs expressed in physiological condition in adult stem cells of the brain are often up-or downregulated in neural CSCs, causing tumor progression. For example, microRNAs expressed by neural stem cells such as miR-7, -124, -125, -18, -9, -10, and -130, orchestrating the differentiation of stem cells toward mature neuronal lineages during embryogenesis, are also expressed by neural cancer stem cells, where they exhibit tumor suppressor or oncogenic functions [270]. Indeed, microRNAs play crucial roles in brain tumor-initiating cells; for example, the aberrant upregulation of miR-221 and the downregulation of the brain-enriched miRNAs miR-128, miR-181a, miR-181b, and miR-181c in the human glioblastoma cells lines (DBTRG-05MG, U118, U87, A172, LN18, M059J, M059K, LN229, T98G, and U138MG from ATCC) are responsible for tumor initiation and development [271,272]. Similarly, miR-21 is upregulated in glioblastoma cell lines (A172, U87, U373, LN229, LN428, and LN308) compared with nonneoplastic glial cells and contributes to tumor malignancy by inhibiting the expression of apoptosis-related genes [273]. MiR-340 is an additional example of microRNA expressed in human NSCs (H9, Invitrogen) and normal brain tissue, which is downregulated in glioma-initiating cells, contributing to proliferation and diffuse invasion of glioblastoma cells [274]. Forty-three microRNAs (including miR-34a and miR-221/222 and novel miRNAs) have also been found deregulated in three separate CD133 + human glioblastoma cell lines compared to CD133 + normal NSCs [275]. This expression profile allowed distinguishing CSCs and NSCs that share the expression of the stem-cell marker CD133 [275]. The pro-neural microRNA miR-218 has been also described as a tumor-suppressor miRNA, whose decreased expression correlates with the aggressiveness of glioma-initiating NSCs [276]. MicroRNAs are also differentially expressed between human glioblastoma CSCs and their paired autologous differentiated tumor cells, as in the case of miR-21 and miR-95, which are significantly deregulated in glioblastoma CSCs [277].

MiRNAs Expressed in ISCs and Colon CSCs
ISCs are adult stem cells with roles in intestinal mucosa barrier homeostasis and repair, with self-renewal and differentiation ability [245]. The Drosophila adult intestine has been used as a model to detect TS miRNAs expressed in ISCs [278]. MiR-958 is a TS miRNA, expressed in ISCs, that is transiently downregulated in stress conditions, causing an expansion of stem-cell number and controlling tissue regeneration [278]. Normal colon stem cells (Human T4056 cells, Applied Biological Materials Inc., Richmond, BC, Canada) also express high levels of miR-137, which is downregulated in colon CSCs (EpCAM + /CD44 + /CD66a − , human SW480 cells, ATCC) [279]. In normal colon stem cells, this microRNA targets doublecortin-like kinase 1 (DCLK1) mRNA, and its stable expression suppresses their uncontrolled cell proliferation and tumorigenicity. In colon CSCs, the DCLK1 transcript is highly expressed because of miR-137 downregulation [279].
MicroRNAs in intestinal epithelial stem cells are also regulated by microbiota and chemoprotective dietary agents; perturbation in adult stem cells, caused by diet composition, is considered a trigger event for colon tumorigenesis [266,280]. MiR-375 is a non-tissue-specific microRNA, significantly suppressed by the microbiota in ISCs, which appears to be an important regulator of stem-cell proliferation [266]. Of interest, miR-375 knockdown increases the proliferation ability of the intestinal epithelial stem cells, and, although this microRNA is also expressed in other tissues, its sensitivity to the microbiota is a peculiar feature of ISCs [266]. In colorectal cancer cells (HT29, HCT116, and CaCO2), miR-375 also functions as a tumor suppressor, targeting the JAK2/STAT3 and MAP3K8/ERK pathways [281].

MiRNAs Expressed in MaSCs and Breast CSCs
Adult MaSCs are multipotent stem cells, located in the mammary gland, with selfrenewal and differentiation ability, responsible for tissue development, homeostasis, and expansion [282]. MaSCs express a unique miRNA signature, mainly featuring the expression of the primate-specific miRNA cluster (19q13.4) [283]. Of interest, the expression of C19MC, which harbors around 50 mature miRNAs, also represents a peculiar hallmark of triplenegative breast cancers; it is upregulated in tamoxifen-resistant cells, with miRNA-519a as the most highly upregulated miRNA [284,285]. Mir-31 is another example of microRNAs highly expressed in MaSCs, which is also enriched in mammary tumors [286]. Mir-31 is regulated by NF-kB signaling, and it promotes MaSC proliferation and expansion by modulating different pathways, such as Wnt/β-catenin [286]. Similarly, miR-146a/b is highly expressed in MaSCs and breast CSCs, where it reduces the adaptive response mechanism and promotes the exit from the quiescent state, inducing resistance to chemotherapies [287]. MiR-489 is highly expressed in MaSCs and its overexpression inhibits mammary gland development, with a specific impact on cells with a high proliferation rate (CD49f hi CD61 hi populations in the tumors), inhibiting tumor growth and metastasis [288]. MaSCs are also characterized by the high expression of miR-205 and miR-22 and by the downregulation of let-7 and miR-93 [289]. MiR-205 is aberrantly expressed in breast cancer tissues, and a decrease in its expression correlates with the aggressiveness of the breast cancer phenotype; therefore, its downregulation is associated with poor prognosis and can be used as tumor biomarker [290]. Studies conducted in conditional mammary gland-specific transgenic mouse models revealed that miR-22 promotes the expansion of the stem-cell compartment, inducing tumor development and aggressive metastatic disease [291]. Lastly, miR-93 is also significantly downregulated in chemoresistant breast cancer cell lines (BCap37, Cell Bank of the Chinese Scientific Academy; Bats-72 and Bads-200 established by PTX treatment of parental BCap37 cells) and tumor samples, where it inhibits cell proliferation, inducing G1/S cell-cycle arrest and increasing the chemosensitivity to the pharmacological treatment [292].

Conclusions
Growing evidence reveals that the mechanisms governing the post-transcriptional regulation of gene expression can be highly complex, and they feature the action of different RNA molecules. The discovery of new classes of microRNAs strongly changed the old concept according to which these short noncoding RNA molecules could only regulate the translation of their direct mRNA targets. The functions mediated by the new classes of noncanonical miRNAs, together with their ability to act using noncanonical gene targeting, further complicate the process of gene expression regulation.
MicroRNAs orchestrate numerous functions in ESCs, such as pluripotency, selfrenewal, differentiation, and EMT [4,35]. These processes are spatially and temporally regulated, so that the cells can correctly develop and guarantee a proper embryogenesis during development. During adult life, the lack of this fine regulation can be responsible for the dedifferentiation and deregulation of normal cells, causing their transformation into cancer cells and the acquisition of stem-like properties [175][176][177]. Collectively, the results discussed in this review highlight that microRNAs can orchestrate similar circuits in ESCs and cancer cells (Table 1). Another example in support of this emerging idea is represented by the mir-23a-24-27a cluster and miR-125a/b family. The mir-23a-24-27a cluster is activated in ESCs (E14Tg2a cell line, Bay Genomics), where it protects the cells against BMP4-induced apoptosis during differentiation [106]. This cluster also functions as an oncogene in several human cancers, such as human hepatocellular carcinoma and lung cancer, where it acts as an antiapoptotic and proliferation-promoting factor and reduces E-Cadherin expression to induce EMT [107,108]. MiR-125a and miR-125b target the BMP4-coreceptor DIES1 in ESCs (E14Tg2a cell line, Bay Genomics), downregulating BMP4 signaling and promoting the exit of ESCs from the naïve state [109][110][111]. The suppression of these microRNAs is essential for the maintenance of the stem-cell properties of hepatoblasts and probably cancer cells, although this remains to be discovered [293]. MiR-125a and miR-125b could have a double role, because, in CSCs of hepatocellular carcinoma, they also inhibit cancer-associated macrophages, limiting tumor progression [112]. Importantly, the findings collected so far indicate that microRNAs, functioning as oncogenes or tumor suppressors, can be used as tumor biomarkers or therapeutic targets [294,295]. In some tumors, such as multiple myeloma, preliminary basic studies and associated clinical works have explored the value of microRNAs as potential biomarkers [296]. In lung cancer, oncolytic virotherapy and nanotherapy have been used to deliver microRNAs into cancer cells, although they have not been included in cancer treatments so far [297]. Several clinical studies are evaluating the utility of miRNA-blood based analyses for the early detection of cancer, while others are investigating the prognostic and predictive value of these molecules [298]. Currently, it is difficult to convert the results obtained from research studies into clinical trials, because microRNAs are widely expressed in different tissues and have wide-ranging effects, whereas some limitations are also linked to the technologies used for their delivery. Many years of hard work are still necessary to solve this gap.