Research Progress on the Functions and Mechanisms of MicroRNAs in Embryonic Stem Cells Wang Shao-Hua, Wang Yang-Ming*(Institute of Molecular Medicine, Peking University, Beijing 100871, China)
Abstract: Since their discovery, embryonic stem cells have held great promise for the treatment of various diseases. They are derived from the inner cell mass of the pre-implantation blastocyst and possess the ability to rapidly proliferate and differentiate into all other somatic cells. Due to these characteristics, embryonic stem cells must be carefully controlled to avoid uncontrolled growth (i.e., tumorigenesis) and unnecessary cell contamination during therapeutic applications. Understanding the key molecular mechanisms that regulate their proliferation and differentiation is essential. Regulation of embryonic stem cell proliferation and differentiation occurs at multiple levels, including cell signaling pathways, higher-order chromatin structures, transcription factors, microRNAs, and long non-coding RNAs. This review summarizes the current understanding of microRNA regulatory functions and mechanisms in embryonic stem cells and discusses future directions in this field. Keywords: embryonic stem cells; microRNAs; self-renewal; cell cycle; metabolism Classification Number: Q522; Q253 Literature Mark Code: A
MicroRNA as important regulators in embryonic stem cells WANG Shao-Hua, WANG Yang-Ming*(Institute of Molecular Medicine, Peking University, Beijing 100871, China)
Abstract: Embryonic stem cells (ESCs) hold great promises for regenerative medicine that may cure a variety of diseases including diabetes and neurodegenerative diseases. Derived from the inner cell mass of preimplantation blastocyst, this cell culture artifact can undergo rapid self-renewal indefinitely while keeping the ability to differentiate into any cells in the body. The key to harness their potential for regenerative medicine is to control their proliferation and differentiation. For this reason we need to understand the molecular mechanisms governing the self-renewal and differentiation of ESCs. Multiple layers of regulation imposed by signaling pathways, chromatin modifications and high-order chromatin structures, transcription factors, microRNAs and large noncoding RNAs have been shown to be important for the self-renewal and differentiation of ESCs. In this short review, we will briefly summarize the current understanding of microRNA functions in ESCs, we will also discuss the major future directions of the field. Key words: embryonic stem cells; microRNAs; self-renewal; cell cycle; metabolism
Embryonic stem cells are derived from the inner cell mass of blastocyst-stage embryos, characterized by their self-renewal and pluripotency, the former referring to the ability to proliferate indefinitely, and the latter referring to the ability to differentiate into all somatic cells of the three germ layers (http://stemcells.nih.gov/info/basics/pages/basics3.aspx). Due to their potential as a cell model simulating early embryonic development and providing cellular materials for regenerative medicine, research on embryonic stem cells has received continuous attention since their discovery. Both self-renewal and differentiation of embryonic stem cells are tightly regulated by a well-ordered molecular regulatory network. These regulatory networks encompass signaling pathways, transcription factors, and epigenetic regulatory factors such as DNA methylation and histone modifications. Recent studies have also found that non-coding RNAs, especially microRNAs, play important roles in the self-renewal and differentiation of embryonic stem cells. Ambros and Ruvkun’s group discovered in 1993 that the small RNA lin-4 can influence the development of C. elegans by regulating the expression of protein-coding genes [1-2], revealing the existence of biologically functional microRNAs for the first time. The subsequent discovery of let-7 [3] and its homologs in mammals [4] further demonstrated the potential importance of microRNAs in various life forms, including humans. In fact, microRNAs have been found in lower multicellular organisms such as Poriferans and Cnidarians [5], and have been preserved through evolution to higher animals. Many microRNAs show high conservation across species, with approximately 55% of microRNAs in C. elegans having corresponding homologous genes in humans [6]. Mature microRNAs are typically 22 nt single-stranded RNAs that bind to mRNAs through base pairing, thereby repressing mRNA translation or mediating mRNA deadenylation leading to degradation [7]. One microRNA can regulate multiple mRNAs, and one mRNA can be regulated by multiple microRNAs, with over 60% of protein-coding genes in mammals being regulated by microRNAs [8-9], indicating that microRNAs are involved in almost all life processes, including development and disease. This article reviews microRNAs in embryonic stem cells and their functions, and for more related studies on the roles of microRNAs in the reprogramming process of induced pluripotent stem cells, please refer to our previous review [10]. 1 MicroRNA Biogenesis Pathways MicroRNAs are located in diverse positions within the genome, being found both within protein-coding genes and non-coding RNAs, as well as in exons and introns. Approximately 50% of microRNAs are located close to other microRNAs in the genome, forming a family that generates a polycistronic transcript (miRNA cluster). Plants and animals have independently evolved their respective microRNA systems; this article mainly discusses the microRNA synthesis pathways in animals [11-12]. In animals, the majority of microRNAs are transcribed by RNA polymerase II and are located within a pri-miRNA transcript that contains a hairpin structure; pri-miRNA is processed by the Microprocessor complex composed of the type III ribonuclease Drosha and its cofactor DGCR8 (DiGeorge syndrome critical region gene 8) in the nucleus, releasing a pre-miRNA with a hairpin structure. This process is generally considered co-transcriptional, meaning it occurs simultaneously with transcription. After formation of pre-miRNA, it is transported out of the nucleus by the transport protein Exportin5, and in the cytoplasm, it is further processed by another type III ribonuclease Dicer with the help of cofactors TRBP/PACT to form approximately 22 nt double-stranded paired microRNAs. One strand of the double-stranded microRNA, the guide strand, enters the Argonaut (Ago) containing RISC complex (RNA-induced silencing complex) to become a mature microRNA, while the other strand, the passenger strand, is typically degraded. The bases at positions 2 to 8 of the mature microRNA’s 5′ end are crucial for recognizing target mRNAs, hence referred to as the seed sequence [7]. Except for a few rare cases [13], microRNAs typically pair with their target mRNAs using the seed sequence, guiding the binding of the RISC complex, thus regulating target mRNAs at the post-transcriptional level: first inhibiting their translation, then degrading the target mRNAs [14]. The binding regions of microRNAs on mRNAs are mainly in the 3′ untranslated region (3’UTR) [7], but recent years have also identified examples of microRNAs functioning through the 5’UTR [15] and CDS regions [16]. In addition to their classical role in inhibiting gene expression, microRNAs have also been found to act as translation activators [17] and transcription regulators [18], as well as antagonists to proteins [19]. The above describes the classical microRNA generation pathway, which is the pathway through which most microRNAs are generated; however, many microRNAs can be produced via other pathways. For instance, some microRNAs associated with Alu repeat sequences can be transcribed by RNA polymerase III [20]. Other microRNA precursors located in introns, termed Mirtrons, can form hairpin structures after being cleaved by the processing complex, thus not requiring Microprocessor processing [21]. Additionally, pre-miRNA-451 can be processed into mature microRNA directly by Ago2 without Dicer processing [22]. 2 MicroRNA Expression and Regulation in Embryonic Stem Cells The expression of microRNAs is finely and complexly regulated, with different cells having different microRNA expression profiles. In embryonic stem cells, core transcription factors, such as Oct4, Sox2, Nanog, and Tcf3, bind to the promoter regions of some microRNA genes, thereby activating or inhibiting their expression [23]. Simultaneously, the promoters of the miR-290 family are regulated by super-enhancers, resulting in extremely high expression levels. Embryonic stem cells also regulate the post-transcriptional processing and maturation of miRNAs. MicroRNA let-7 is highly expressed in differentiated somatic cells but has low expression levels in embryonic stem cells; interestingly, the expression level of its precursor pri-let-7 is comparable in embryonic stem cells and differentiated cells, which is due to the Lin28-Zcchc11-Dis3l2 pathway inhibiting the maturation of pri-let-7 in embryonic stem cells [24]. Currently, research on the regulation of the biogenesis pathways of microRNAs is not very complete, and the research results related to the regulation of let-7 generation indicate that this area of research not only helps develop methods to regulate microRNAs to control cell behavior but may also open up a new research direction like Lin28’s studies. Due to these precise regulations, embryonic stem cells possess a unique miRNA expression profile. Multiple research groups have detected the microRNA expression profiles of pluripotent stem cells, such as mouse embryonic stem cells [25], mouse epiblast stem cells [26], and human embryonic stem cells [23, 27]. Each type of cell expresses about hundreds of microRNAs with expression levels above 10 copies. Interestingly, almost every type of cell has one or several microRNA families that dominate (i.e., account for more than half of the total microRNA expression). Among all pluripotent stem cells, the expression levels of superfamilies with AAGUGCU or AAAGUGC seed sequences are the highest. In mouse embryonic stem cells, several microRNAs derived from the miR-290 and miR-302 families possess AAGUGCU seed sequences, while several microRNAs from the miR-17 and miR-106 families have AAAGUGCU seed sequences. The miR-290 family has the highest expression level in mouse embryonic stem cells, accounting for more than half of all microRNAs, and declines with differentiation. Knockout of the miR-290 family results in partial embryonic lethality in mice, while surviving female mice are infertile [28]. The miR-302 family has the highest expression levels in human embryonic stem cells and mouse epiblast stem cells, also accounting for over 60% of the total microRNAs. They have been reported to play important regulatory roles in various biological processes, and miR-302 knockout mice exhibit multiple defects in neural system differentiation [29]. Due to their identical seed sequences, miR-290 and miR-302 exhibit functional redundancy, thus miR-290/302 double-knockout mice display a significantly severe embryonic lethality phenotype, more severe than that observed in either single knockout phenotype. The miR-17 and miR-106b families are highly expressed in various tissues and cancers, with miR-17 family knockout mice experiencing perinatal mortality, while miR-106b family knockout mice show no obvious defects; however, the double knockout of miR-17 and miR-106b leads to embryonic lethality before E15.5, demonstrating redundancy between them [30]. Notably, microRNAs with AAGUGCU seed sequences are also highly expressed and play essential biological roles in early embryos of lower vertebrates, such as zebrafish and Xenopus, but are not expressed in even lower organisms, suggesting that these microRNAs may be related to the origin of vertebrates [31]. 3 MicroRNAs Regulate Embryonic Stem Cells through Various Pathways Early studies have found that enzymes related to microRNA biogenesis pathways play essential roles in embryonic development. Mice lacking Dgcr8 exhibit severe malformations by E6.5, with no embryos detectable by E10, indicating that the absence of DGCR8 leads to early embryonic lethality [32]. Embryonic stem cells lacking DGCR8 also show defects such as slower cell proliferation and delayed differentiation when induced to differentiate. Knockout of Dicer in mice leads to embryonic death before E8.5 [33]. Dicer knockout embryonic stem cells also exhibit slower proliferation and inability to differentiate normally, with more severe phenotypes than DGCR8 knockout embryonic stem cells; moreover, their epigenetic regulation of repetitive sequences near the centromere is impaired [34-35]. Mice lacking Ago2 also exhibit embryonic lethality before E7.5, and embryonic stem cells lacking Ago1-4 show defects such as slower proliferation and delayed differentiation [36]. These findings indicate that microRNAs play crucial roles in early embryonic development and the maintenance and differentiation of embryonic stem cells. The phenotypes of these knockout cells guide the research direction of microRNAs in embryonic stem cells, as detailed below. 3.1 MicroRNAs Regulate the Cell Cycle of Embryonic Stem Cells The normal cell cycle consists of G1/S/G2/M phases, primarily regulated by cyclin-dependent kinases (Cdks). Different Cdks exhibit activity at different phases, regulating the corresponding E2F transcription factors, thereby controlling the expression of related genes to ensure normal cell cycle progression. Compared to somatic cells, embryonic stem cells have a unique cell cycle. Embryonic stem cells almost do not possess a G1/S restriction point, facilitating their rapid transition from G1 to S phase, resulting in a short G1 phase to ensure rapid proliferation. MicroRNAs play essential regulatory roles in this process. Embryonic stem cells lacking Dgcr8 [32], Dicer1 [34], and Ago1-4 [37] all display slower cell proliferation, prolonged cell cycles, and increased accumulation of cells in G1 phase. Our research group transfected approximately 260 microRNAs into DGCR8 knockout cells and identified 14 microRNAs that could reverse this proliferation defect [38]. Among these 14 microRNAs, 11 possess the seed sequences AAGUGCU or AAAGUGC and are microRNAs highly expressed in embryonic stem cells, with the miR-290 family having the highest expression. Target prediction and dual luciferase reporter assays demonstrated that these microRNAs regulating the cell cycle in embryonic stem cells can directly act on the inhibitory regulatory factors p21/Rbl2/Lats2 that facilitate the transition from G1 to S phase, thereby promoting this transition. In embryonic stem cells with knockouts of DGCR8 and the Rb family, our research group further demonstrated that miR-290/302 promotes the G1 to S phase transition in normal conditions in an Rb family-independent manner, while under nutrient deficiency and contact inhibition, it promotes cell proliferation by preventing the accumulation of embryonic stem cells in G1 phase in an Rb family-dependent manner [39]. Similar to mouse embryonic stem cells, knocking down Dicer or Drosha in human embryonic stem cells also leads to slower cell growth, while the addition of miR-372 or miR-195 can repair this defect [40]. miR-372 is a homolog of miR-290 in humans and can directly target p21. MiR-195 targets the inhibitory regulatory protein Wee1 of the CyclinB/CDK complex, promoting the transition from G2 to M phase. Additionally, transfecting anti-miR-92b into human embryonic stem cells leads to an increase in p57 protein [41], indicating that miR-92b can promote the transition from G1 to S phase by inhibiting p57. Similarly, in human embryonic stem cells, interfering with miR-302 through antisense RNA increases the accumulation of G1 phase cells [42], and using TALE-KRAB [transcription activator-like effector (TALE)-based transcriptional repressor] to inhibit miR-302 expression also leads to cell accumulation in G0/G1 phase and slower growth [43]. Research from multiple groups indicates that miR-302 can regulate embryonic stem cell cell cycles through various targets, such as cyclin D1 [42] and p21 [44]. Interestingly, Dicer1 knockout cells proliferate more slowly and have a greater accumulation in G1 phase than Dgcr8 knockout cells [45]. One possible explanation for this is that Dicer, in addition to participating in microRNA generation, also participates in siRNA generation; another possible explanation is that some non-classical microRNAs that do not rely on Dgcr8 but depend on Dicer processing, such as miR-320 and miR-702, also play regulatory roles in the cell cycle. In fact, adding these two microRNAs to Dicer knockout cells restores their proliferation speed and G1 phase cell proportion to the levels seen in Dgcr8 knockout cells. Further target validation indicates that they can directly act on p21 and p57 to regulate the cell cycle. 3.2 MicroRNAs Control the Fate of Embryonic Stem Cells through Signaling Pathways Embryonic stem cells possess unique signaling pathway systems, and some microRNAs can regulate the fate of embryonic stem cells by modulating these pathways. The NF-κB signaling pathway can induce EMT (epithelial to mesenchymal transition), leading to differentiation of embryonic stem cells; overexpression of NF-κB in embryonic stem cells results in differentiation towards the mesoderm. The miR-290 family can directly target a subunit of NF-κB, p65 [46], which may play an important role in maintaining the pluripotency of embryonic stem cells. The Wnt signaling pathway plays crucial roles in embryonic development, cell differentiation, and organ morphogenesis. The miR-290 family has also been reported to directly target the Wnt signaling pathway inhibitor DKK1, thereby promoting Wnt signaling to maintain the self-renewal of embryonic stem cells [47]. Consistent with this, it has also been found that the miR-372 family can target DKK1 in cancer cells [48]. The TGF-β superfamily is a group of important cytokines that play significant regulatory roles in various life activities such as cell proliferation, differentiation, development, and apoptosis, comprising nearly 40 members that can be classified into 4 categories based on amino acid sequence similarity: TGF-βs, activins and inhibins, bone morphogenetic proteins (BMP), and other scattered components. The Nodal/Activin signaling pathway plays a crucial role in the differentiation of the three germ layers. This signaling pathway includes the activator Nodal and the inhibitor Lefty. Nodal signaling inhibits embryonic stem cells from differentiating into neuroectoderm while promoting differentiation into mesoderm, while Lefty has the opposite effect. Rosa et al. [31] demonstrated that in human embryonic stem cells, miR-302 can directly target Lefty, thus promoting Nodal signaling and inhibiting the differentiation of human embryonic stem cells into neuroectoderm. Interestingly, the homologous gene miR-430 in zebrafish [49] and miR-427 in Xenopus both target Lefty and Nodal, balancing the Nodal signaling pathway. Whether this change in regulatory mechanisms from zebrafish to mammals has evolutionary significance remains unknown. Betel’s group identified a total of 146 targets of miR-302 in human embryonic stem cells through AGO2’s PAR-CLIP (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation), including Lefty1/2; they also include Tob2, Dazap2, and Slain1, all of which can inhibit BMP signaling. These targets explain well how miR-302 can also inhibit the differentiation of human embryonic stem cells into neurons by promoting BMP signaling. Zhang’s group found that miR-200 can also inhibit the differentiation of embryonic stem cells into neurons by targeting another BMP signaling pathway inhibitor, ZEB [50]. They also discovered that miR-96 can directly target the neuron differentiation determinant transcription factor PAX6. PAX6 can activate the expression of a series of neuron differentiation factors during neural differentiation, promoting the differentiation of neuroectoderm. Similarly, in mouse embryonic stem cells, the miR-290 family has also been shown to directly target PAX6 [51]. Another neuron differentiation activator NR2F2 has also been proven to be a target of miR-302 [52]. Thus, it can be seen that microRNAs can regulate the self-renewal and differentiation of stem cells by participating in important cell signaling pathways. The TGF-β signaling pathway also plays a crucial role in repairing DNA damage. The rapid self-renewal of embryonic stem cells leads to their genomes being in a state of replication for extended periods, lacking checkpoints, which may result in embryonic stem cells having more DNA damage. In fact, embryonic stem cells exhibit higher frequencies of DSBs and SSBs. Fortunately, miR-590 can directly target Acvr2a, regulating the activity of the TGF-β signaling pathway, thereby promoting the activity of Rad51b [53] and facilitating the repair of DSBs and SSBs, slowing down the proliferation of embryonic stem cells. However, the role of miR-590 in this context requires further validation, as its expression level in embryonic stem cells is not high. 3.3 MicroRNAs Regulate Differentiation of Embryonic Stem Cells Cells lacking Dicer1 [35] and Dgcr8 [32] cannot effectively differentiate; when induced to differentiate into embryoid bodies in vitro, their pluripotency genes such as Oct4 cannot be properly downregulated, and genes for differentiation into endoderm and mesoderm are almost not expressed. Part of this phenomenon may be due to the inability to effectively activate genes necessary for de novo DNA methylation, such as Dnmt3a and Dnmt3b, in Dicer1 knockout cells, leading to DNA methylation defects at many genomic loci, including the Oct4 promoter [54-55]. However, reintroducing the miR-290 family into Dicer1 knockout cells can effectively correct this defect. It has been reported that miR-290 can inhibit Rbl2, thus preventing its negative regulation of Dnmt3a and Dnmt3b, ensuring normal DNA methylation in cells. However, recent studies have proposed differing viewpoints [56], leaving the exact mechanisms by which miR-290 regulates methylation still unresolved. Additionally, several microRNAs have been identified that induce differentiation [57]: miR-134, miR-296, and miR-470 can directly target Oct4, Sox2, and Nanog; miR-200c, miR-183, miR-203, and miR-145 can inhibit Sox2 and Klf4; the let-7 family directly regulates cMyc, Lin28, and Sall4; miR-125 and miR-181 can target the PRC1 subunit Cbx7, which is unique to embryonic stem cells. More complexly, recent screening experiments using Dgcr8 knockout cells have shown that dozens of microRNAs may play roles during embryonic stem cell differentiation [39, 58], a reasonable explanation being that these microRNAs may regulate embryonic stem cells’ differentiation into different lineages, which requires further experimental validation. 3.4 MicroRNAs Regulate Apoptosis of Embryonic Stem Cells The self-renewal defects of embryonic stem cells lacking microRNAs could be due not only to cell cycle effects but also to increased apoptosis. Embryonic stem cells lacking Ago1-4 show significantly increased apoptosis, along with a marked rise in the apoptosis-inducing gene Bim [59]. In human embryonic stem cells, inhibiting miR-302 expression using TALE-KRAB leads to the aforementioned changes in the cell cycle and also results in significantly increased apoptosis, which may primarily account for the reduced self-renewal ability [43]. Further studies have confirmed that miR-302 can directly target the apoptosis-inducing gene BNIP3L/Nix and upregulate BCL-xL, thus inhibiting apoptosis in human embryonic stem cells. Similarly, in mouse embryonic stem cells, knocking out the miR-290 family does not increase baseline levels of apoptosis; however, when the cells encounter gamma radiation or treatment with doxorubicin, apoptosis significantly increases [60]. In this system, pro-apoptotic factors Caspase2 and Ei24 may be direct targets. Our recent experiments have also demonstrated that miR-294/302 can inhibit apoptosis, counteracting let-7 to maintain self-renewal in embryonic stem cells [61]. In upper epiblast stem cells, knocking out Dicer1 also significantly increases the proportion of apoptotic cells, and transfecting miR-20, miR-92, or miR-302, which have AAGUGCU or AAAGUGC seed sequences, can partially inhibit apoptosis [62]. 3.5 MicroRNAs Regulate Energy Metabolism of Embryonic Stem Cells Compared to many differentiated cells, embryonic stem cells tend to utilize glycolysis rather than aerobic respiration for energy metabolism, which seems to play an important role in their pluripotency. Our research group found that the glycolytic efficiency is significantly reduced in Dgcr8 knockout mouse embryonic stem cells [63], while the efficiency of oxidative phosphorylation is significantly increased. Transfecting miR-290 into Dgcr8 knockout cells can upregulate Pkm2 and Ldha, promoting glycolysis. Further studies revealed that miR-290 can inhibit the repressive transcription factor Mbd2, thereby promoting the expression of its target Myc, which has been shown to enhance glycolysis in various systems. The metabolism of embryonic stem cells is currently a highly researched area, and our group’s important finding is the regulatory role of Mbd2; in fact, hundreds of genes are regulated by Mbd2, and whether other genes involved in embryonic stem cell metabolism are also regulated by microRNAs is an urgent topic to explore. Additionally, it is worth investigating whether the regulation of metabolism is closely linked to the regulation of other features of embryonic stem cells, such as the cell cycle, apoptosis, and differentiation. 4 Outlook Since the discovery of microRNAs over twenty years ago, research on microRNAs in embryonic stem cells is about to cross a decade. Despite many findings, many questions remain to be addressed. Pluripotent stem cells are not a single state; various states have been discovered. For instance, mouse embryonic stem cells are considered to be in a naive state, corresponding to pre-implantation epiblast cells; while human embryonic stem cells and mouse epiblast stem cells are considered to be in a primed state, corresponding to post-implantation epiblast cells. They both possess pluripotency in a broad sense but have different molecular characteristics and are regulated differently, each having advantages in applications in regenerative medicine and as models for early embryonic development [64-65]. As research continues to deepen, more subtle differences and classifications will be discovered. As important regulatory molecules, do microRNAs have functions in determining different pluripotent states? How do they act? Given that many differences in these pluripotent states manifest in epigenetic states, transcription factor binding sites, and components of transcription complexes, whether and how microRNAs influence these aspects remains an unresolved question that requires investigation [66]. Finally, closely related to regenerative medicine is whether microRNAs can be utilized to achieve the initial expectations of embryonic stem cells, namely, to promote the differentiation of stem cells into specific lineages, such as liver or cardiac cells, or to purify functionally relevant cells using microRNAs. Research on these issues may usher in another golden decade for the study of microRNAs in embryonic stem cells.
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