Novel glycylated sugar alcohols protect ESC-specific microRNAs from degradation in iPS cells.

Excessive accumulation of embryonic stem cell (ESC)-specific microRNAs occurs in both ESCs and induced pluripotent stem cells (iPSC); yet, the mechanism involved is unknown. In iPSCs, we for the first time found that novel glycylated sugar alcohols, particularly glycylglycerins, are tightly bound with ESC-specific microRNA precursors (pre-miRNA), such as pre-miR-302. Among these isolated glycylglycerins, we further identified that 1,3-diglycylglycerin and 1,2,3-triglycylglycerin are two major compounds bonded with negatively charged nucleic acids via electro-affinity and subsequently forming sugar-like coats in the hairpin-like double helix structures of pre-miRNAs. As a result, such glycylglycerin-formed coating serves as a protection layer against miRNA degradation. Moreover, we found that the pH value of iPSC cytosol determines the charges of these glycylglycerins. During iPSC differentiation, the cytosol pH is increased and hence neutralizes the charges of glycylglycerins, consequently leading to fast miRNA degradation. Therefore, the current findings not only explain how ESC-specific miRNAs are preserved and accumulated in iPSCs and ESCs but also demonstrate an important function of glycylglycerins in protecting the structural integrity of highly degradable miRNAs, providing a useful means for maintaining miRNA/siRNA function as well as developing the related RNA interference (RNAi) applications.

microRNAs and Fragile X Syndrome.

Fragile X syndrome (FXS) is one of the major causes for autism and mental retardation in humans. The etiology of FXS is linked to the expansion of the CGG trinucleotide repeats, r(CGG), suppressing the fragile X mental retardation 1 (FMR1) gene on the X chromosome, resulting in a loss of fragile X mental retardation protein (FMRP) expression, which is required for regulating normal neuronal connectivity and plasticity. Recent studies have further identified that microRNAs are involved in the mechanisms underlying FXS pathogenesis at three different developmental stages. During early embryogenesis before the blastocyst stage, an embryonic stem cell (ESC)-specific microRNA, miR-302, interferes with FMR1 mRNA translation to maintain the stem cell status and inhibit neural development. After blastocyst, the downregulation of miR-302 releases FMRP synthesis and subsequently leads to neuronal development; yet, in FXS, certain r(CGG)-derived microRNAs, such as miR-fmr1s, are expressed and accumulated and then induce DNA hypermethylation on the FMR1 gene promoter regions, resulting in transcriptional inactivation of the FMR1 gene and the loss of FMRP. In normal neuronal development, FMRP is an RNA-binding protein responsible for interacting with miR-125 and miR-132 to regulate the signaling of Group 1 metabotropic glutamate receptor (mGluR1) and N-methyl-D-aspartate receptor (NMDAR), respectively, and consequently affecting synaptic plasticity. As a result, the loss of FMRP impairs these signaling controls and eventually causes FXS-associated disorders, such as autism and mental retardation. Based on these current findings, this chapter will summarize the etiological causes of FXS and further provides significant insights into the molecular mechanisms underlying microRNA-mediated FXS pathogenesis and the related therapy development.

Regulation of somatic cell reprogramming through inducible mir-302 expression.

Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation.

Concise review: Deciphering the mechanism behind induced pluripotent stem cell generation.

Regenerative medicine using spluripotent/multipotent stem cells holds a great promise in developing therapies for treating developmental abnormalities, degenerative disorders, and aging-related illness. However, supply and safety of the stem cells are two major problems with today's regenerative medicine. Recent development of induced pluripotent stem cells (iPSCs) has overcome the supply shortages by allowing the reprogramming of patients' body cells to embryonic stem cell (ESC)-like pluripotent cells. Still, the potential tumorigenicity of iPSCs remains as an obstacle. During early embryogenesis ESCs can be generated without tumor formation; therefore, understanding the mechanisms underlying ESC generation may help us to prevent iPSC tumorigenicity. Previous studies have shown that an ESC-enriched noncoding RNA, miR-302, induces somatic cell reprogramming (SCR) to form iPSCs, suggesting its pivotal role in stem cell generation. Recent research further revealed that miR-302-induced SCR involves an epigenetic reprogramming mechanism similar to the natural zygotic reprogramming process in the two- to eight-cell-stage embryos. These findings indicate that miR-302, as a cytoplasmic gene silencer, inhibits the translation of multiple key epigenetic regulators, including AOF1/2, methyl-CpG binding proteins 1 and 2, and DNA (cytosine-5-)-methyltransferase 1, to induce global DNA demethylation, which subsequently triggers the activation of the previously defined factors Oct4, Sox2, and Nanog to complete the reprogramming process. The same mechanism was also found in the event of somatic cell nuclear transfer. Based on these advanced understandings, this review describes the currently established SCR mechanism--as compared to the natural process of early ESC formation--and demonstrates how stem cell researchers may use this mechanism to improve iPSC generation.

Mechanism of repeat-associated microRNAs in fragile X syndrome.

The majority of the human genome is comprised of non-coding DNA, which frequently contains redundant microsatellite-like trinucleotide repeats. Many of these trinucleotide repeats are involved in triplet repeat expansion diseases (TREDs) such as fragile X syndrome (FXS). After transcription, the trinucleotide repeats can fold into RNA hairpins and are further processed by Dicer endoribonuclases to form microRNA (miRNA)-like molecules that are capable of triggering targeted gene-silencing effects in the TREDs. However, the function of these repeat-associated miRNAs (ramRNAs) is unclear. To solve this question, we identified the first native ramRNA in FXS and successfully developed a transgenic zebrafish model for studying its function. Our studies showed that ramRNA-induced DNA methylation of the FMR1 5'-UTR CGG trinucleotide repeat expansion is responsible for both pathological and neurocognitive characteristics linked to the transcriptional FMR1 gene inactivation and the deficiency of its protein product FMRP. FMRP deficiency often causes synapse deformity in the neurons essential for cognition and memory activities, while FMR1 inactivation augments metabotropic glutamate receptor (mGluR)-activated long-term depression (LTD), leading to abnormal neuronal responses in FXS. Using this novel animal model, we may further dissect the etiological mechanisms of TREDs, with the hope of providing insights into new means for therapeutic intervention.

Loss of mir-146a function in hormone-refractory prostate cancer.

The pattern of microRNA (miRNA) expression is associated with the degree of tumor cell differentiation in human prostate cancer. MiRNAs bind complementarily to either oncogenes or tumor suppressor genes, which are consequently silenced, resulting in alterations of tumorigenecity. We have detected eight down-regulated and three up-regulated known miRNAs in androgen-independent human prostate cancer cells compared to those in androgen-dependent cells, using miRNA microarray analyses. These identified miRNAs showed the same expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive noncancerous prostate epithelium as determined by fluorescent in situ hybridization assays in human prostate cancer tissue arrays. One of the eight down-regulated miRNAs, mir-146a, was selected and constitutively expressed to examine its effects on suppression of prostate cancer transformation from androgen-dependent to -independent cells as determined by in vitro tumorigenecity assays. Transfection of mir-146a, which perpetually express the miRNA, suppressed >82% of the expression of the targeted protein-coding gene, ROCK1, in androgen-independent PC3 cells, consequently markedly reducing cell proliferation, invasion, and metastasis to human bone marrow endothelial cell monolayers. Given that ROCK1 is one of the key kinases for the activation of hyaluronan (HA)-mediated HRPC transformation in vivo and in PC3 cells, mir-146a may function as a tumor-suppressor gene in modulating HA/ROCK1-mediated tumorigenecity in androgen-dependent prostate cancer.

Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state.

Renewal of stem cells differs from cancer cell growth in self-controlled cell division. The mir-302 microRNA (miRNA) family (mir-302s) is expressed most abundantly in slow-growing human embryonic stem (ES) cells, and quickly decreases after cell differentiation and proliferation. Therefore, mir-302s was investigated as one of the key factors essential for maintenance of ES cell renewal and pluripotency in this study. The Pol-II-based intronic miRNA expression system was used to transgenically transfect the mir-302s into several human cancer cell lines. The mir-302-transfected cells, namely, miRNA-induced pluripotent stem (mirPS) cells, not only expressed many key ES cell markers, such as Oct3/4, SSEA-3, SSEA-4 ,Sox2, and Nanog, but also had a highly demethylated genome similar to a reprogrammed zygotic genome. Microarray analyses further revealed that genome-wide gene expression patterns between the mirPS and human ES H1 and H9 cells shared over 86% similarity. Using molecular guidance in vitro, these mirPS cells could differentiate into distinct tissue cell types, such as neuron-, chondrocyte-, fibroblast-, and spermatogonia-like primordial cells. Based on these findings, we conclude that mir-302s not only function to reprogram cancer cells into an ES-like pluripotent state but also to maintain this state under a feeder-free cultural condition, which may offer a great opportunity for therapeutic intervention.

MiR-302-Mediated Somatic Cell Reprogramming and Method for Generating Tumor-Free iPS Cells Using miR-302.

Human induced pluripotent stem cells (iPSCs) by four factors have the risks of teratoma formation and potential tumorigenicity. To overcome this major hurdle, we examined the mechanism(s) by which the cell cycle genes of embryonic cells were regulated. Naturally occurring embryonic stem cells (ESCs) possess two unique stemness properties: pluripotent differentiation into all cell types and self-renewal with no risk of tumor formation. Despite overwhelming reports describing iPSC pluripotency, there have been no observations of tumor prevention mechanism that suppresses tumor formation similar to that in naturally occurring ESCs. The ESC-specific microRNA (miRNA), miR-302, regulates human iPSC tumorigenicity through co-suppression of both cyclin E-CDK2 and cyclin D-CDK4/6 cell cycle pathways during G1-S phase transition. MiR-302 also silenced BMI-1, a cancer stem cell marker gene, to promote the expression of two senescence-associated tumor suppressor genes, p16Ink4a and p14/p19Arf. Together, the combinatory effect of reducing G1-S cell cycle transition and increasing p16/p14(p19) expression resulted in a relatively attenuated cell cycle rate similar to that of 2-to-8-cell-stage embryonic cells in early mammalian zygotes (20-24 h/cycle), as compared to the fast proliferation rate of iPSCs induced by four defined factors Oct4-Sox2-Klf4-c-Myc (12-16 h/cycle). In addition to the prevention of stem cell tumorigenicity, the mechanism underlying miR-302-mediated iPSCs also includes the initiation of global genomic DNA methylation, activation of ESC-specific gene expression, and inhibition of developmental signaling. Overall, we have established an effective protocol to express the intronic miR-302 cluster, according to its own natural biogenesis mechanism to generate tumor-free iPSCs for use in biology and therapy.

Intron-mediated RNA interference and microRNA biogenesis.

Nearly 97% of the human genome is non-coding DNA, and introns occupy most of it around the gene-coding regions. Numerous intronic sequences have been recently found to encode microRNAs, which are responsible for RNA-mediated gene silencing through RNA interference (RNAi)-like pathways. microRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. This flexible characteristic is different from double-stranded siRNAs (small interfering RNAs) because a much more rigid complementarity is required for siRNA-induced RNAi gene silencing. miRNAs were firstly discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development. Currently, varieties of miRNAs are widely reported in plants, animals and even microbes. Intronic microRNA is a new class of miRNAs derived from the processing of gene introns. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of type II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNAs are able to induce RNA interference in not only human and mouse cells but also zebrafishes, chicken embryos and adult mice, demonstrating the evolutionary preservation of the intron-mediated gene silencing through miRNA functionality in cell and in vivo. These findings suggest an intracellular miRNA-mediated gene regulatory system, fine-tuning the degradation of protein-coding messenger RNAs.

Androgen receptor regulates CD168 expression and signaling in prostate cancer.

Dysregulation of the androgen receptor (AR) and its signaling in the prostate often occurs during normal aging or after androgen ablation, consequently leading to the development of hormone-refractory prostate cancer (HRPC). Hyaluronan (HA) plays an important role in this transformation of androgen-independent cancer. Previous studies have shown that activation of the receptor for hyaluronan-mediated motility, CD168, was correlated with the Gleason's score, cancer stage, transformation and metastasis in >90% of HRPC patients. However, the relationship between loss of AR dependency and HA-mediated CD168 signaling remains unclear. We report here that AR regulates normal CD168 expression and its downstream signaling in androgen-dependent (AD) prostatic epithelial cell lines. Furthermore, we observed that the concurrent treatments of HA and dihydrotestosterone (DHT), a native androgen, significantly promoted the tumorigenicity of AD prostate cancer cell lines, which showed elevated rates of cell proliferation, invasion and metastasis to the human bone marrow endothelial cell layer. Inhibition of CD168 downstream Rho-activated protein kinases completely prevented this type of tumorigenicity. These findings suggest that the interaction of androgen and AR is essential for regulating HA-mediated cancer progression via the CD168/ROCK signal transduction pathway and also indicate that the loss of AR regulation not only causes CD168 overexpression but it also activates HA-mediated CD168 signaling in malignant cancer progression and metastasis of HRPC.

Intron-mediated RNA interference and microRNA (miRNA).

MicroRNAs (miRNAs) are small single-stranded regulatory RNAs capable of interfering with messenger RNAs (mRNAs) through complete or partial complementarities. Partial complementarity gives miRNAs a flexibility which is useful for construction of new therapies against cancer polymorphisms and viral mutations. Varieties of miRNAs have been reported in diverse species; and they are believed to induce RNA interference (RNAi), a post-transcriptional gene silencing mechanism. Recently, many intronic sequences have been shown to encode microRNAs. Intronic miRNA, a new class of miRNAs, is derived from introns by RNA splicing and Dicer processing, and it differs uniquely from previously described intergenic miRNA in that intronic miRNAs require type II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of intronic miRNAs have been identified; however, their functions and applications have not been reported. Here, we show for the first time that intron-derived miRNAs are able to induce RNA interference in many cells demonstrating the evolutionary preservation of this post-transcriptional regulatory system in vivo.

The microRNA (miRNA): overview of the RNA genes that modulate gene function.

MicroRNAs (miRNAs), widely distributed, small regulatory RNA genes, target both messenger RNA (mRNA) degradation and suppression of protein translation based on sequence complementarity between the miRNA and its targeted mRNA. Different names have been used to describe various types of miRNA. During evolution, RNA retroviruses or transgenes invaded the eukaryotic genome and inserted in the non-coding regions of DNA, conceivably acting as transposon-like jumping genes, providing defense from viral invasion and fine-tuning of gene expression as a secondary level of gene modulation in eukaryotes. When a transposon is inserted in the intron, it becomes an intronic miRNA, taking advantage of the protein synthesis machinery, i.e., mRNA transcription and splicing, as a means for processing and maturation. Recently, miRNAs have been found to play an important, but not life-threatening, role in embryonic development. They might play a pivotal role in diverse biological systems in various organisms, facilitating a quick response and accurate plotting of body physiology and structures. Based on these unique properties, man-made intronic miRNAs have been developed for in vitro evaluation of gene function, in vivo gene therapy and generation of transgenic animal models. The biogenesis and identification of miRNAs, potential applications, and future directions for research are presented, hopefully providing a guideline for further miRNA and gene function studies.

Identification and Isolation of Novel Sugar-Like RNA Protecting Materials: Glycylglycerins from Pluripotent Stem Cells.

Pluripotent stem cells are a resourceful treasure box for regenerative medicine. They contain a large variety of novel materials useful for designing and developing new medicines and therapies directed against many aging-associated degenerative disorders, including Alzheimer's disease, Parkinson's disease, stroke, diabetes, osteoporosis, and cancers. Currently, identification of these novel stem cell-specific materials is one of major breakthroughs in the field of stem cell research. Particularly, since the discovery of induced pluripotent stem cells (iPSC) in year 2006, the methods of iPSC derivation further provide an unlimited resource for screening, isolating, and even producing theses novel stem cell-specific materials in vitro. Using iPSCs, we can now prepare high quality and quantity of pure stem cell-specific agents for testing their therapeutic functions in treating various illnesses. These newly found stem cell-specific agents are divided into four major categories, including proteins, saccharides, nucleic acids, and small molecules (chemicals). In this article, we herein disclose one of the methodologies for isolating and purifying glycylglycerins-a group of glycylated sugar alcohols that protect hairpin-like microRNA precursors (pre-miRNA) and some of tRNAs in pluripotent stem cells. In view of such a unique RNA-protecting feature, glycylglycerins may be used to preserve and deliver functional small RNAs, such as pre-miRNAs and small interfering RNAs (siRNA), into human cells for eliciting their specific RNA interference (RNAi) effects, which may greatly advance the use of RNAi technology for treating human diseases.

Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a post-transcriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest in clinical and circumstantial malfunction; for example, type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of non-protein-coding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, their functions and applications have not been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice cells, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Mechanism and Method for Generating Tumor-Free iPS Cells Using Intronic MicroRNA miR-302 Induction.

Today's researchers generating induced pluripotent stem cells (iPS cells or iPSCs) usually consider their pluripotency rather than potential tumorigenicity. Oncogenic factors such as c-Myc and Klf4 are frequently used to boost the survival and proliferative rates of iPSCs, creating an inevitable problem of tumorigenicity that hinders the therapeutic usefulness of these iPSCs. To prevent stem cell tumorigenicity, we have examined mechanisms by which the cell cycle genes are regulated in embryonic stem cells (ESCs). Naturally, ESCs possess two unique stemness properties: pluripotent differentiation into almost all cell types and unlimited self-renewal without the risk of tumor formation. These two features are also important for the use of ESCs or iPSCs in therapy. Currently, despite overwhelming reports describing iPSC pluripotency, there is no report of any tumor prevention mechanism in either ESCs or iPSCs. To this, our studies have revealed for the first time that an ESC-specific microRNA (miRNA), miR-302, regulates human iPSC tumorigenicity through cosuppression of both cyclin E-CDK2 and cyclin D-CDK4/6 cell cycle pathways during G1-S phase transition. Moreover, miR-302 also silences BMI-1, a cancer stem cell gene marker, to promote the expression of two senescence-associated tumor suppressor genes, p16Ink4a and p14/p19Arf. Together, the combinatory effects of inhibiting G1-S cell cycle transition and increasing p16/p14(p19) expression result in an attenuated cell cycle rate similar to that of 2-to-8-cell-stage embryonic cells in early zygotes (20-24 h/cycle), which is however slower than the fast proliferation rate of iPSCs induced by the four defined factors Oct4-Sox2-Klf4-c-Myc (12-16 h/cycle). These findings provide a means to control iPSC tumorigenicity and improve the safety of iPSCs for the therapeutic use. In this chapter, we review the mechanism underlying miR-302-mediated tumor suppression and then demonstrate how to apply this mechanism to generate tumor-free iPSCs. The same strategy may also be used to prevent ESC tumorigenicity.

The MicroRNA.

MicroRNAs (miRNAs), widely distributed, small regulatory RNA genes, target both messenger RNA (mRNA) degradation and suppression of protein translation based on sequence complementarity between the miRNA and its targeted mRNA. Different names have been used to describe various types of miRNA. During evolution, RNA retroviruses or transgenes invaded the eukaryotic genome and were inserted itself in the noncoding regions of DNA, conceivably acting as transposon-like jumping genes, providing defense from viral invasion and fine-tuning of gene expression as a secondary level of gene modulation in eukaryotes. When a transposon is inserted in the intron, it becomes an intronic miRNA, taking advantage of the protein synthesis machinery, i.e., mRNA transcription and splicing, as a means for processing and maturation. MiRNAs have been found to play an important, but not life-threatening, role in embryonic development. They might play a pivotal role in diverse biological systems in various organisms, facilitating a quick response and accurate plotting of body physiology and structures. Based on these unique properties, manufactured intronic miRNAs have been developed for in vitro evaluation of gene function, in vivo gene therapy, and generation of transgenic animal models. The biogenesis of miRNAs, circulating miRNAs, miRNAs and cancer, iPSCs, and heart disease are presented in this chapter, highlighting some recent studies on these topics.

Isolation and Identification of Gene-Specific MicroRNAs.

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducing RNA interference (RNAi)-related gene silencing, we have developed an intronic miRNA expression system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hence successfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanism has been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA so obtained is transcribed by type-II RNA polymerases, coexpressed within a primary gene transcript, and then excised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII) endonucleases further process the spliced introns into mature miRNAs. Using this intronic miRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitro but also in zebrafishes, chicken embryos, and adult mice in vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design various intronic pre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

The miR-302-Mediated Induction of Pluripotent Stem Cells (iPSC): Multiple Synergistic Reprogramming Mechanisms.

Pluripotency represents a unique feature of embryonic stem cells (ESCs). To generate ESC-like-induced pluripotent stem cells (iPSCs) derived from somatic cells, the cell genome needs to be reset and reprogrammed to express the ESC-specific transcriptome. Numerous studies have shown that genomic DNA demethylation is required for epigenetic reprogramming of somatic cell nuclei to form iPSCs; yet, the mechanism remains largely unclear. In ESCs, the reprogramming process goes through two critical stages: germline and zygotic demethylation, both of which erase genomic DNA methylation sites and hence allow for different gene expression patterns to be reset into a pluripotent state. Recently, miR-302, an ESC-specific microRNA (miRNA), was found to play an essential role in four aspects of this reprogramming mechanism-(1) initiating global genomic DNA demethylation, (2) activating ESC-specific gene expression, (3) inhibiting developmental signaling, and (4) preventing stem cell tumorigenicity. In this review, we will summarize miR-302 functions in all four reprogramming aspects and further discuss how these findings may improve the efficiency and safety of the current iPSC technology.

Transgene-Like Animal Models Using Intronic MicroRNAs.

Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude for a human body for etiological and pathological research of diseases. All pharmaceutically developed medicines must be proven to be safe and effective in animals before approval by the Food and Drug Administration (FDA) to be used in clinical trials. To this end, the transgenic animal models of diseases serve as the front line of drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA)-related research. MiRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts (mRNAs) that contain either complete or partial complementarity to the miRNA, are useful for the design of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to be derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis spp.), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By the insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerases type II (Pol-II), coexpressed with the encoding gene transcripts, and excised out of the encoding gene transcripts by intracellular RNA splicing and processing mechanisms. In conjunction with retroviral transfection, the designed hairpin-like pre-miRNA construct has also been placed in the intron regions of a cellular gene for tissue-specific expression, specifically regulated by the gene promoter of interest. Because the retroviral vectors are integrated into the genome of its host cells, we can select and propagate the most effective transgenic animals to form a stable model line for further research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA expression system reported previously, which has been proven to be useful for studying miRNA function as well as the related gene regulation in vivo.

Transcriptional control of Shh/Ptc1 signaling in embryonic development.

In vivo profiling of signal-directed gene expression patterns is a major bottleneck in studying developmental biology. A signal molecule initiates its specific gene expression pattern through the activation of certain transcription factor (TF); however, tissue heterogeneity often masks this pattern due to intercellular complexity of other signal transduction pathways. To decipher the synergistic regulation of signal-directed gene expression in the tissue level, we report here a unique transcriptional responsive element (TRE) existing in the 5'-upstream promoter regions (5'-UPR) of the genes responding to the Shh/Ptc1 signal transduction pathway during feather placode development in chicken embryos. By locating the TRE homologue and its interactive TF, we were able to reveal the gene expression pattern of the Shh/Ptc1 signaling. We firstly demonstrated that homology profiling of the 5'-UPR of the genes, Gli1, TGF-beta2 and Msx2, responding to the Shh/Ptc1 signaling showed a more than 70% conserved region. Computer alignment of the consensus sequences in the conserved region revealed a 37-nucleotide TRE sequence, containing two regulatory elements homologous to human and mouse Gli-binding sites. Activation of this newly identified Shh/Ptc1-responsive TRE by active Smo signaling in chicken hepatoepithelial carcinoma cells elicited a strong synergistic expression of the Shh/Ptc1-downstream genes. Based on previous bioinformatics and the present experimental findings, we successfully established an in vivo signaling model for the Shh/Ptc1-directed embryonic feather morphogenesis.