Minjuan Wu

Endogenous miRNA Sponge lincRNA-RoR Regulates Oct4, Nanog, and Sox2 in Human Embryonic Stem Cell Self-Renewal

The embryonic stem cell (ESC) transcriptional and epigenetic networks are controlled by a multilayer regulatory circuitry, including core transcription factors (TFs), posttranscriptional modifier microRNAs (miRNAs), and some other regulators. However, the role of large intergenic noncoding RNAs (lincRNAs) in this regulatory circuitry and their underlying mechanism remains undefined. Here, we demonstrate that a lincRNA, linc-RoR, may function as a key competing endogenous RNA to link the network of miRNAs and core TFs, e.g., Oct4, Sox2, and Nanog. We show that linc-RoR shares miRNA-response elements with these core TFs and that linc-RoR prevents these core TFs from miRNA-mediated suppression in self-renewing human ESC. We suggest that linc-RoR forms a feedback loop with core TFs and miRNAs to regulate ESC maintenance and differentiation. These results may provide insights into the functional interactions of the components of genetic networks during development and may lead to new therapies for many diseases.

Critical regulation of miR-200/ZEB2 pathway in Oct4/Sox2-induced mesenchymal-to-epithelial transition and induced pluripotent stem cell generation

Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3′ UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.

microRNA-29b is a novel mediator of Sox2 function in the regulation of somatic cell reprogramming

Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the application of Yamanaka factors (OSKM), but the mechanisms underlying this reprogramming remain poorly understood. Here, we report that Sox2 directly regulates endogenous microRNA-29b (miR-29b) expression during iPSC generation and that miR-29b expression is required for OSKM- and OSK-mediated reprogramming. Mechanistic studies show that Dnmt3a and Dnmt3b are in vivo targets of miR-29b and that Dnmt3a and Dnmt3b expression is inversely correlated with miR-29b expression during reprogramming. Moreover, the effect of miR-29b on reprogramming can be blocked by Dnmt3a or Dnmt3b overexpression. Further experiments indicate that miR-29b-DNMT signaling is significantly involved in the regulation of DNA methylation-related reprogramming events, such as mesenchymal-to-epithelial transition (MET) and Dlk1-Dio3 region transcription. Thus, our studies not only reveal that miR-29b is a novel mediator of reprogramming factor Sox2 but also provide evidence for a multistep mechanism in which Sox2 drives a miR-29b-DNMT signaling axis that regulates DNA methylation-related events during reprogramming.

MiR‐138 Promotes Induced Pluripotent Stem Cell Generation Through the Regulation of the p53 Signaling

Induced pluripotent stem (iPS) cells, especially those reprogrammed from patient somatic cells, have a great potential usage in regenerative medicine. The expression of p53 has been proven as a key barrier limiting iPS cell generation, but how p53 is regulated during cell reprogramming remains unclear. In this study, we found that the ectopic expression of miR‐138 significantly improved the efficiency of iPS cell generation via Oct4, Sox2, and Klf4, with or without c‐Myc (named as OSKM or OSK, respectively), without sacrificing the pluripotent characteristics of the generated iPS cells. Exploration of the mechanism showed that miR‐138 directly targeted the 3′ untranslated region (UTR) of p53, significantly decreasing the expression of p53 and its downstream genes. Furthermore, the ectopic expression of p53 having a mutant 3′‐UTR, which cannot be bound by miR‐138, seriously impaired the effect of miR‐138 on p53 signaling and OSKM‐initiated somatic cell reprogramming. Combined with the fact that miR‐138 is endogenously expressed in fibroblasts, iPS cells, and embryonic stem cells, our study demonstrated that regulation of the p53 signaling pathway and promotion of iPS cell generation represent an unrevealed important function of miR‐138. STEM Cells2012;30:1645–1654

Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomal MicroRNAs Suppress Myofibroblast Differentiation by Inhibiting the Transforming Growth Factor-β/SMAD2 Pathway During Wound Healing

Excessive scar formation caused by myofibroblast aggregations is of great clinical importance during skin wound healing. Studies have shown that mesenchymal stem cells (MSCs) can promote skin regeneration, but whether MSCs contribute to scar formation remains undefined. We found that umbilical cord‐derived MSCs (uMSCs) reduced scar formation and myofibroblast accumulation in a skin‐defect mouse model. We found that these functions were mainly dependent on uMSC‐derived exosomes (uMSC‐Exos) and especially exosomal microRNAs. Through high‐throughput RNA sequencing and functional analysis, we demonstrated that a group of uMSC‐Exos enriched in specific microRNAs (miR‐21, ‐23a, ‐125b, and ‐145) played key roles in suppressing myofibroblast formation by inhibiting the transforming growth factor‐β2/SMAD2 pathway. Finally, using the strategy we established to block miRNAs inside the exosomes, we showed that these specific exosomal miRNAs were essential for the myofibroblast‐suppressing and anti‐scarring functions of uMSCs both in vitro and in vivo. Our study revealed a novel role of exosomal miRNAs in uMSC‐mediated therapy, suggesting that the clinical application of uMSC‐derived exosomes might represent a strategy to prevent scar formation during wound healing.

Long non-coding RNA GAS5 controls human embryonic stem cell self-renewal by maintaining NODAL signalling

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However, most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5), a known tumour suppressor and growth arrest-related lncRNA, is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal, but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis, we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore, we propose that the above pluripotency factors, GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific, our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms.

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation.

H/ACA Box Small Nucleolar RNA 7A Promotes the Self-Renewal of Human Umbilical Cord Mesenchymal Stem Cells.

Human umbilical cord blood derived mesenchymal stem cells (uMSC) are pluripotent cells that have been now considered as a promising candidate for various cell‐based therapies. However, their limited in vitro proliferation ability and the gradual loss of pluripotency set barricades for further usages. Emerging evidence suggests that small nucleolar RNAs (snoRNA) are actively involved in cell proliferation especially in tumor cells, but their roles in stem cells are largely unknown. In this study, we demonstrated that H/ACA box small nucleolar RNA 7A (SNORA7A) is inversely correlated to the decreased proliferation rate during in vitro passaging of uMSC. Further investigations indicate that SNORA7A overexpression can promote uMSC proliferation and self‐renewal. The inhibition of SNORA7A using antisense oligonucleotides significantly reduces the expression and the binding of SNORA7A to DKC1, core protein that essential to form small nucleolar ribonucleo‐particles (snoRNP) complex and catalyze pseudouridines in 28S RNA. And the inhibition also significantly suppresses uMSC proliferation and self‐renewal. Moreover, overexpression of SNORA7A transcripts with mutations of binding regions for snoRNP core proteins and 28S RNA did not induce proliferation and self‐renewal. Besides, SNORA7A also suppresses both the osteogenic and adipogenic differentiation, strengthening its self‐renewal maintaining roles in uMSC. Taken together, our study for the first time showed that H/ACA box snoRNAs are actively involved in MSC proliferation as well as pluripotency control, and we identify SNORA7A as one of the critical snoRNAs that regulate the proliferation and self‐renewal of uMSC through snoRNP recruiting. Stem Cells 2017;35:222–235

Mesenchymal Stem Cells With Modification of Junctional Adhesion Molecule A Induce Hair Formation

The junctional adhesion molecule A (JAM‐A) has been shown to serve a crucial role in the proliferation, differentiation, and tube‐like formation of epithelial cells during angiogenesis. The role of JAM‐A in hair follicle (HF) regeneration has not yet been reported. In this study, we used human JAM‐A‐modified human mesenchymal stem cells (MSCs) to repair HF abnormalities in BALB/c nu/nu mice. The JAM‐A gene and JAM‐A short hairpin RNA were transfected into cultured human MSCs to generate the JAM‐A overexpression MSCs (JAM‐Aov MSCs) and JAM‐A knockdown MSCs (JAM‐Akd MSCs), respectively. These cells were injected intradermally into the skin of nude mice during the first telogen phase of the HF that occurs 21 days postnatally. We found that JAM‐Aov MSCs migrated into the HF sheath and remodeled HF structure effectively. The HF abnormalities such as HF curve and HF zigzag were remodeled, and hair formation was improved 7 days following injection in both the JAM‐Aov MSC and MSC groups, compared with the JAM‐Akd MSC group or negative control group. Furthermore, the JAM‐Aov MSC group showed enhanced hair formation in contrast to the MSC group, and the number of curved and zigzagged HFs was reduced by 80% (p < .05). These results indicated that JAM‐Aov MSCs improved hair formation in nude mice through HF structure remodeling.

Ca2+ and EGF induce the differentiation of human embryo mesenchymal stem cells into epithelial‐like cells

The mesenchymal to epithelial transition (MET) occurs in organ development and anti‐tumorigenesis. We have investigated the effects of calcium (Ca2+) and epidermal growth factor (EGF) on human mesenchymal stem cell (hMSCs) differentiation into epithelial‐like cells. hMSCs lost their biological characteristics after EGF transfection, and MET was achieved by adding 0.4 mmol Ca2+. Western blotting and immunofluorescence showed expression of EGF, keratin, keratin 19 (K19), β1‐integrin, E‐cadherin and phosphorylated focal adhesion kinase (p‐FAK, Ser‐910) increased in hMSCs infected with EGF and exposed to Ca2+, although Smad3 activation was downregulated. hMSCs co‐stimulated with EGF transfection and Ca2+ can therefore differentiate into epithelial‐like cells in vitro.