Dicer- and Bulge Stem Cell-Dependent MicroRNAs During Induced Anagen Hair Follicle Development.
ABSTRACT: MicroRNAs (miRNAs) are a major class of conserved non-coding RNAs that have a wide range of functions during development and disease. Biogenesis of canonical miRNAs depend on the cytoplasmic processing of pre-miRNAs to mature miRNAs by the Dicer endoribonuclease. Once mature miRNAs are generated, the miRNA-induced silencing complex (miRISC), or miRISC, incorporates one strand of miRNAs as a template for recognizing complementary target messenger RNAs (mRNAs) to dictate post-transcriptional gene expression. Besides regulating miRNA biogenesis, Dicer is also part of miRISC to assist in activation of the complex. Dicer associates with other regulatory miRISC co-factors such as trans-activation responsive RNA-binding protein 2 (Tarbp2) to regulate miRNA-based RNA interference. Although the functional role of miRNAs within epidermal keratinocytes has been extensively studied within embryonic mouse skin, its contribution to the normal function of hair follicle bulge stem cells (BSCs) during post-natal hair follicle development is unclear. With this question in mind, we sought to ascertain whether Dicer-Tarpb2 plays a functional role within BSCs during induced anagen development by utilizing conditional knockout mouse models. Our findings suggest that Dicer, but not Tarbp2, functions within BSCs to regulate induced anagen (growth phase) development of post-natal hair follicles. These findings strengthen our understanding of miRNA-dependency within hair follicle cells during induced anagen development.
Project description:MicroRNAs (miRNAs) regulate the expression of many mammalian genes and play key roles in embryonic hair follicle development; however, little is known of their functions in postnatal hair growth. We compared the effects of deleting the essential miRNA biogenesis enzymes Drosha and Dicer in mouse skin epithelial cells at successive postnatal time points. Deletion of either Drosha or Dicer during an established growth phase (anagen) caused failure of hair follicles to enter a normal catagen regression phase, eventual follicular degradation and stem cell loss. Deletion of Drosha or Dicer in resting phase follicles did not affect follicular structure or epithelial stem cell maintenance, and stimulation of anagen by hair plucking caused follicular proliferation and formation of a primitive transient amplifying matrix population. However, mutant matrix cells exhibited apoptosis and DNA damage and hair follicles rapidly degraded. Hair follicle defects at early time points post-deletion occurred in the absence of inflammation, but a dermal inflammatory response and hyperproliferation of interfollicular epidermis accompanied subsequent hair follicle degradation. These data reveal multiple functions for Drosha and Dicer in suppressing DNA damage in rapidly proliferating follicular matrix cells, facilitating catagen and maintaining follicular structures and their associated stem cells. Although Drosha and Dicer each possess independent non-miRNA-related functions, the similarity in phenotypes of the inducible epidermal Drosha and Dicer mutants indicates that these defects result primarily from failure of miRNA processing. Consistent with this, Dicer deletion resulted in the upregulation of multiple direct targets of the highly expressed epithelial miRNA miR-205.
Project description:Several in vitro studies have suggested that canonical microRNA (miRNA) biogenesis requires the DICER cofactors TARBP2 and PRKRA for processing of pre-miRNAs to mature miRNAs. To investigate the roles of TARBP2 and PRKRA in miRNA biogenesis in vivo, and to determine possible functional redundancy, we first compared the phenotypes of Tarbp2 and Prkra single and double mutants. In contrast to Dicer -/- embryos, which die by embryonic day 7.5 (E7.5), single Tarbp2 -/- and Prkra -/- mice survive beyond E7.5 and either die perinatally or survive and exhibit cranial/facial abnormalities, respectively. In contrast, only a few Tarbp2 -/- ; Prkra -/- double mutants survived beyond E12.5, suggesting genetic redundancy between Tarbp2 and Prkra during embryonic development. Sequencing of miRNAs from single-mutant embryos at E15.5 revealed changes in abundance and isomiR type in Tarbp2 -/- , but not Prkra -/- , embryos, demonstrating that TARBP2, but not PRKRA, functions in miRNA biogenesis of a subclass of miRNAs, and suggesting that functional redundancy between TARBP2 and PRKRA does not involve miRNA biogenesis.
Project description:The discovery that microRNAs (miRNAs) play important roles in regulating gene expression via posttranscriptional repression has revealed a previously unsuspected mechanism controlling development and progenitor-cell function (reviewed in ); however, little is known of miRNA functions in mammalian organogenesis. Processing of miRNAs and their assembly into the RNA-induced silencing (RISC) complex requires the essential multifunctional enzyme Dicer . We found that Dicer mRNA and multiple miRNAs are expressed in mouse skin, suggesting roles in skin- and hair-follicle biology. In newborn mice carrying an epidermal-specific Dicer deletion, hair follicles were stunted and hypoproliferative. Hair-shaft and inner-root-sheath differentiation was initiated, but the mutant hair follicles were misoriented and expression of the key signaling molecules Shh and Notch1 was lost by postnatal day 7. At this stage, hair-follicle dermal papillae were observed to evaginate, forming highly unusual structures within the basal epidermis. Normal hair shafts were not produced in the Dicer mutant, and the follicles lacked stem cell markers and degenerated. In contrast to decreased follicular proliferation, the epidermis became hyperproliferative. These results reveal critical roles for Dicer in the skin and implicate miRNAs in key aspects of epidermal and hair-follicle development and function.
Project description:<h4>Background</h4>MicroRNAs (miRNAs) are a large family of endogenous, non-coding RNAs, about 22 nucleotides long, which regulate gene expression through sequence-specific base pairing with target mRNAs. Extensive studies have shown that miRNA expression in the skin changes remarkably during distinct stages of the hair cycle in humans, mice, goats and sheep.<h4>Results</h4>In this study, the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fibre-producing goat breed. In total, 63,109,004 raw reads were obtained by Solexa sequencing and 61,125,752 clean reads remained for the small RNA digitalisation analysis. This resulted in the identification of 399 conserved miRNAs; among these, 326 miRNAs were expressed in all three follicular cycling stages, whereas 3, 12 and 11 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. We also identified 172 potential novel miRNAs by Mireap, 36 miRNAs were expressed in all three cycling stages, whereas 23, 29 and 44 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. The expression level of five arbitrarily selected miRNAs was analyzed by quantitative PCR, and the results indicated that the expression patterns were consistent with the Solexa sequencing results. Gene Ontology and KEGG pathway analyses indicated that five major biological pathways (Metabolic pathways, Pathways in cancer, MAPK signalling pathway, Endocytosis and Focal adhesion) accounted for 23.08% of target genes among 278 biological functions, indicating that these pathways are likely to play significant roles during hair cycling.<h4>Conclusions</h4>During all hair cycle stages of cashmere goats, a large number of conserved and novel miRNAs were identified through a high-throughput sequencing approach. This study enriches the Capra hircus miRNA databases and provides a comprehensive miRNA transcriptome profile in the skin of goats during the hair follicle cycle.
Project description:The periodic regrowth of rabbit fur is economically important. Here, we aimed to characterise the histological traits and microRNA (miRNA) expression profiles in the skin tissue of Wan Strain Angora rabbits at different weeks after plucking. Haematoxylin-eosin staining showed that hair follicles were in the telogen phase in the first week, while they were in the anagen phase from the fourth to twenty-fourth weeks. In addition, two small RNA libraries derived from skin samples of Wan Strain Angora rabbits at telogen and anagen stages yielded over 24 million high-quality reads. Specifically, 185 miRNAs were differentially expressed between the telogen and anagen phases. The function of the differentially expressed miRNAs was explored by comparing them with known mammalian miRNAs and by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of their predicted targets. Five new functional miRNAs were validated using quantitative real-time PCR. Moreover, the fibroblast growth factor 5 (FGF5) gene was verified to be a target of conservative_NC_013672.1_9290 and conservative_NC_013675.1_10734. We investigated differential miRNA profiles between the telogen and anagen phases of the hair cycle and our findings provide a basis for future studies focusing on the mechanisms of miRNA-mediated regulation of rabbit hair follicle cycling.
Project description:Small RNA-induced gene silencing is essential for post-transcriptional regulation of gene expression; however, it remains unclear how miRNA/siRNA efficiency is regulated. Here we show that TARBP2 is SUMOylated at K52, which can be enhanced by its phosphorylation. This modification can stabilize TARBP2 via repressing its K(48)-linked ubiquitination. We find that TARBP2 SUMOylation does not influence the overall production of mature miRNAs, but it regulates miRNA/siRNA efficiency. SUMOylated TARBP2 recruits Ago2 to constitute the RNA-induced silencing complex (RISC)-loading complex (RLC), and simultaneously promotes more pre-miRNAs to load into the RLC. Consequently, Ago2 is stabilized and miRNAs/siRNAs bound by TARBP2/Dicer is effectively transferred to Ago2. Thus, these processes lead to the formation of the effective RISC for RNA interference (RNAi). Collectively, our data suggest that SUMOylation of TARBP2 is required for regulating miRNA/siRNA efficiency, which is a general mechanism of miRNA/siRNA regulation.
Project description:<h4>Background</h4>During goat embryonic morphogenesis and postnatal initiation of hair follicle (HF) regeneration, dermal papilla (DP) cells play a vital role in hair formation. Growing evidence shows that microRNAs (miRNAs) participate in HF development and DP cell proliferation. However, the molecular mechanisms have not been thoroughly investigated.<h4>Result</h4>In this study, we utilized miRNA sequencing (miRNA-Seq) to identify differentially expressed miRNAs at different HF cycling stages (anagen and telogen). MiRNA-Seq has identified 411 annotated miRNAs and 130 novel miRNAs in which 29 miRNAs were up-regulated and 32 miRNAs were down-regulated in the anagen phase compared to the telogen phase. Target gene prediction and functional enrichment analysis indicated some major biological pathways related to hair cycling, such as Wnt signaling pathways, ECM-receptor interaction, VEGF signaling pathway, biosynthesis of amino acids, metabolic pathways, ribosome and oxidative phosphorylation. Also, we explored the function of chi-miR-30b-5p in regulating hair growth cycle. Similar to the HF cycling, DP cells were isolated from skin and used to investigate miRNA functions. The MTT and EdU assays showed that the viability and proliferation of DP cells were inhibited or promoted after the transfection of chi-miR-30b-5p mimic or inhibitor, respectively. Bioinformatics analysis revealed CaMKII? as a candidate target gene of chi-miR-30b-5p, and the dual-luciferase and western blot assay demonstrated that chi-miR-30b-5p bound to the 3'UTR of CaMKII? and further inhibited its translation.<h4>Conclusion</h4>Chi-miR-30b-5p was found to be highly expressed in the telogen than that in the anagen phase and could inhibit the proliferation of DP cells by targeting CaMKII?. Our study provides new information on the regulatory functions of miRNAs during HF development.
Project description:In this study, the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fiber-producing goat breed. In total, 63,109,004 raw reads were obtained by Solexa sequencing and 61,125,752 clean reads remained for the small RNA digitalization analysis. This resulted in the identification of 399 conserved miRNAs; among these, 326 miRNAs were expressed in all three follicular cycling stages, whereas 3, 12 and 11 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. We also identified 172 potential novel miRNAs by Mireap, 36 miRNAs were expressed in all three cycling stages, whereas 23, 29 and 44 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. Gene Ontology and KEGG pathway analyses indicated that five major biological pathways (Metabolic pathways, Pathways in cancer, MAPK signalling pathway, Endocytosis and Focal adhesion) accounting for 23.08% of target genes among 278 biological functions, indicating that these pathways are likely to play significant roles during hair cycling. the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fiber-producing goat breed
Project description:The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro-RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR-31 markedly increases during anagen and decreases during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and midanagen phases of the hair cycle results in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signaling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes, both in vitro and in vivo. Using luciferase reporter assay, we show that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. Thus, by targeting a number of growth regulatory molecules and cytoskeletal proteins, miR-31 is involved in establishing an optimal balance of gene expression in the hair follicle required for its proper growth and hair fiber formation.
Project description:Deregulation of microRNA (miRNA) expression has been documented in diffuse large B-cell lymphoma (DLBCL). However, the impact of miRNAs and their machinery in DLBCL is not fully determined. Here, we assessed the role of miRNA expression and their processing genes in DLBCL development. Using microarray and RT-qPCR approaches, we quantified global miRNAs and core components of miRNA-processing genes expression in 75 DLBCLs (56 de novo and 19 transformed) and 10 lymph nodes (LN). Differential miRNA signatures were identified between DLBCLs and LNs, or between the de novo and transformed DLBCLs. We also identified subsets of miRNAs associated with germinal center B-cell phenotype, BCL6 and IRF4 expression, and clinical staging. In addition, we showed a significant over-expression of TARBP2 in de novo DLBCLs as compared with LNs, and decreased expression of DROSHA, DICER, TARBP2 and PACT in transformed as compared with de novo cases. Interestingly, cases with high TARBP2 and DROSHA expression had a poorer chemotherapy response. We further showed that TARBP2 can regulate miRNA-processing efficiency in DLBCLs, and its expression inhibition decreases cell growth and increases apoptosis in DLBCL cell lines. Our findings provide new insights for the understanding of miRNAs and its machinery in DLBCL.