Pro-inflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/ S111, thereby suppressing FOXA2 transactivation activity, leading to decreased NUMB expression and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.
Bladder exstrophy epispadias complex (BEEC) is a severe congenital anomaly; however, the genetic and molecular mechanisms underlying the formation of BEEC remain unclear. TP63, a member of TP53 tumor suppressor gene family, is expressed in bladder urothelium and skin over the external genitalia during mammalian development. It plays a role in bladder development. We have previously shown that p63−/− mouse embryos developed a bladder exstrophy phenotype identical to human BEEC. We hypothesised that TP63 is involved in human BEEC pathogenesis. RNA was extracted from BEEC foreskin specimens and, as in mice, ΔNp63 was the predominant p63 isoform. ΔNp63 expression in the foreskin and bladder epithelium of BEEC patients was reduced. DNA was sequenced from 163 BEEC patients and 285 ethnicity-matched controls. No exon mutations were detected. Sequencing of the ΔNp63 promoter showed 7 single nucleotide polymorphisms and 4 insertion/deletion (indel) polymorphisms. Indel polymorphisms were associated with an increased risk of BEEC. Significantly the sites of indel polymorphisms differed between Caucasian and non-Caucasian populations. A 12-base-pair deletion was associated with an increased risk with only Caucasian patients (p = 0.0052 Odds Ratio (OR) = 18.33), whereas a 4-base-pair insertion was only associated with non-Caucasian patients (p = 0.0259 OR = 4.583). We found a consistent and statistically significant reduction in transcriptional efficiencies of the promoter sequences containing indel polymorphisms in luciferase assays. These findings suggest that indel polymorphisms of the ΔNp63 promoter lead to a reduction in p63 expression, which could lead to BEEC.
Bladder exstrophy epispadias complex is a severe congenital abnormality. The affected babies' bladders are born open, leaking urine constantly. Treatment involves multiple major reconstructive surgeries and the need for lifelong care for the complications of the disease. Although a number of studies have suggested a genetic cause of the disease, the genetic and molecular mechanism underlying the formation of BEEC remains unknown. One gene, TP63, plays a crucial role in the early bladder development. Two different genetic promoters of TP63 produce different forms of the protein with opposing properties. We have shown mice lacking p63 displayed a deformity complex identical to human BEEC. There are no genetic mutations in the p63 protein in BEEC, so genetic variants in the promoter could alter protein expression. Our hypothesis was that loss of p63 expression due to sequence polymorphisms in a promoter is a risk factor for BEEC. We found promoter sequence variants that were statistically associated with the disease and the sequence variant location varied between Caucasian and non-Caucasian patients. This is particularly important as Caucasian populations have a higher risk of BEEC. These findings provide an explanation of BECC and a base for further study of TP63 related genes in this disease.
Epigenetic regulation plays an important role in stem cell self-renewal, maintenance and lineage differentiation. The epigenetic profiles of stem cells are related to their transcriptional signature. Enhancer of Zeste homlog 2 (EZH2), a catalytic subunit of epigenetic regulator Polycomb repressive complex 2 (PRC2), has been shown to be a key regulator in controlling cellular differentiation. EZH2 is a histone methyltransferase that not only methylates histone H3 on Lys 27 (H3K27me3) but also interacts with and recruits DNA methyltransferases to methylate CpG at certain EZH2 target genes to establish firm repressive chromatin structures, contributing to tumor progression and the regulation of development and lineage commitment both in embryonic stem cells (ESCs) and adult stem cells. In addition to its well-recognized epigenetic gene silencing function, EZH2 also directly methylates nonhistone targets such as the cardiac transcription factor, GATA4, resulting in attenuated GATA4 transcriptional activity and gene repression. This review addresses recent progress toward the understanding of the biological functions and regulatory mechanisms of EZH2 and its targets as well as their roles in stem cell maintenance and cell differentiation.
EZH2; polycomb repressive complex; embryonic stem cells; adult stem cells; chromatin modification; methylation
The title compound, [Pb2(C2O4)(NO3)2(C10H8N2)2(H2O)2], was synthesized hydrothermally. The binuclear complex molecule is centrosymmetric, the inversion centre being located at the mid-point of the oxalate C—C bond. The PbII ion is heptacoordinated by the O atom of one water molecule, two oxalate O atoms, two nitrate O atoms and two 2,2′-bipyridine N atoms, forming an irregular coordination environemnt. Intermolecular O—H⋯O hydrogen bonds between water molecules and oxalate and nitrate ions result in the formation of layers parallel to (010). π–π interactions between pyridine rings in adjacent layers, with centroid–centroid distances of 3.584 (2) Å, stabilize the structural set-up.
Small non-coding RNAs such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA) exist in almost all kingdoms of organisms and have recently emerged as master regulators of gene expression to affect a diverse range of important biological processes. They exert their functions largely through two related but opposing mechanisms: RNA interference (RNAi) mediated by siRNA, miRNA and piRNA, and RNA activation (RNAa) mediated by small activating RNA (saRNA) and miRNA, leading to silencing and overexpression of target genes respectively. Dysregulation of these mechanisms have been implicated in a variety of human diseases including urological and andrological diseases. Importantly, both mechanisms can be readily harnessed for therapeutic purposes for a variety of diseases by using small RNA molecules as the “ribodrug”. In this review, we highlight recent advances in the applications of small RNA as therapeutics for urological cancer, male infertile and erectile dysfunction.
Small RNA; microRNA; RNAi; RNAa; urology; andrology; prostate cancer; bladder cancer; erectile dysfunction
Small RNA molecules, such as microRNA and siRNA, have emerged as master regulators of gene expression through their ability to suppress target genes in a phenomenon collectively called RNA interference (RNAi). There is growing evidence that small RNAs can also serve as activators of gene expression by targeting gene regulatory sequences. This novel mechanism, known as RNA activation (RNAa), appears to be conserved in at least mammalian cells and triggered by both endogenous and artificially designed small RNAs. RNAa depends on Argonaute proteins, but possesses kinetics distinct from that of RNAi. Epigenetic changes are associated with RNAa and may contribute to transcriptional activation of target genes, but the underlying mechanism remains elusive. Given the potential of RNAa as a molecular tool for studying gene function and as a therapeutic for disease, further research is needed to elucidate fully its molecular mechanism in order to refine the rules for target selection and improve strategies for exploiting it therapeutically.
RNA interference (RNAi) is an evolutionary conserved mechanism by which small double-stranded RNA (dsRNA) – termed small interfering RNA (siRNA) – inhibits translation or degrades complementary mRNA sequences. Identifying features and enzymatic components of the RNAi pathway have led to the design of highly-effective siRNA molecules for laboratory and therapeutic application. RNA activation (RNAa) is a newly discovered mechanism of gene induction also triggered by dsRNAs termed small activating RNA (saRNA). It offers similar benefits as RNA interference (RNAi), while representing a new method of gene overexpression. In the present study, we identify features of RNAa and explore chemical modifications to saRNAs that improve the applicability of RNAa. We evaluate the rate of RNAa activity in order to define an optimal window of gene induction, while comparing the kinetic differences between RNAa and RNAi. We identify Ago2 as a conserved enzymatic component of both RNAa and RNAi implicating that saRNA may tolerate modification based on Ago2 function. As such, we define chemical modifications to saRNAs that manipulate RNAa activity, as well as exploit their effects to design saRNAs with enhanced medicinal properties. These findings reveal functional features of RNAa that may be utilized to augment saRNA function for mechanistic studies or the development of RNAa-based drugs.
Argonaute 2 (Ago2); cancer therapeutics; E-cadherin; gene promoter; p21; RNA activation (RNAa); RNA interference (RNAi); small activating RNA (saRNA); small interfering RNA (siRNA); strand modifications
To investigate the effect of CC chemokine receptor 3 (CCR3) signal on corneal neovascularization (CRNV) induced by alkali burn and to explore its mechanism.
Specific pathogen-free male BALB/C mice (aged 6-8 weeks) were randomly divided into CCR3-antagonist treated group (experimental group) and control group. CRNV was induced by alkali burn in mice. The time kinetic CCR3 expression in injured corneas was examined by reverse transcription polymerase chain reaction (RT-PCR). CCR3-antagonist (SB-328437 at different concentration of 125µg/mL, 250µg/mL, and 500µg/mL) was locally administrated after alkali injury. The formation of CRNV was assessed by CD31 corneal whole mount staining at two weeks after injury. Monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 3 (MCP-3) expressions in the early phase after injury were quantified and compared by RT-PCR. Macrophage intracorneal accumulation in the early phase after injury was evaluated and compared by immunohistochemistry.
Alkali injury induced the time kinetic intracorneal CCR3 expression. 500µg/mL of CCR3-antagonist treatment in the early phase but not the late phase resulted in significant impaired CRNV as compared to control group (P<0.05). CCR3-antagonist treatment in the early phase significantly reduced the intracorneal MCP-1 and MCP-3 enhancement compare to control group at day 2 and day 4 (P<0.05). Moreover, the number of intracorneal macrophage infiltration in the experimental group was reduced than those in control group at day 4 (P<0.05).
CCR3 signal is involved in alkali-induced CRNV. CCR3-antagonist can inhibit alkali-induced CRNV by reducing the intracorneal MCP-1 and MCP-3 mRNA expression and the intracorneal macrophage infiltration.
corneal neovascularization; CCR3; monocyte chemotactic protein 1; monocyte chemotactic protein 3; macrophage
Aberrant regulation of ribosomal RNA (rRNA) synthesis and translation control can facilitate tumorigenesis. The ErbB2 growth factor receptor is overexpressed in many human tumors and has been detected in the nucleus, but the role of nuclear ErbB2 is obscure. In this study, we defined a novel function of nuclear ErbB2 in enhancing rRNA gene transcription by RNA polymerase-I (RNA Pol I). Nuclear ErbB2 physically associates with β-actin and RNA Pol I, coinciding with active RNA Pol I transcription sites in nucleoli. RNAi-mediated knockdown of ErbB2 reduced pre-rRNA and protein synthesis. In contrast, wild-type ErbB2 augmented pre-rRNA level, protein production and cell size/cell growth, but not by an ErbB2 mutant which is defective in nuclear translocation. Chromatin immunoprecipitation assays revealed that ErbB2 enhances binding of RNA Pol I to rDNA. Additionally, ErbB2 associated with rDNA, RNA Pol I and β-actin, suggesting how it could stimulate rRNA production, protein synthesis and increased cell size and cell growth. Lastly, ErbB2-potentiated RNA Pol I transcription could be stimulated by ligand and was not substantially repressed by inhibition of PI3-K and MEK/ERK, the main ErbB2 effector signaling pathways. Together, our findings indicate that nuclear ErbB2 functions as a regulator of rRNA synthesis and cellular translation, which may contribute to tumor development and progression.
ErbB-2; ribosomal RNA; RNA polymerase I; translation
We sequenced the 5′ UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)n microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5′ UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5′ UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5′ UTR region may be a new biomarker for genetic susceptibility to breast cancer.
Microsatellite; Breast cancer; AAAG; Polymorphism; Genetic predisposition
RNAa (RNA activation) is a mechanism by which small dsRNA (double-stranded RNA), termed saRNA (small activating RNA), target promoter sequences to induce gene expression. This technique represents a novel approach to gene overexpression without the use of exogenous DNA. In the present study, we investigated whether RNAa can modulate expression of the development-related gene NANOG and manipulate cell fate. Using a lentivirus-based reporter system as a screening tool, we identified synthetic saRNAs that stimulate NANOG expression in human NCCIT embryonic carcinoma cells. Mismatch mutations to saRNA duplexes define sequence requirement for gene activation. Functional analysis of NANOG induction reveals saRNA treatment predictably modulates the expression of several known downstream target genes, including FOXH1 (forkhead box H1), REST (RE1-silencing transcription factor), OCT4 (octamer-binding protein 4) and REX1 (reduced expression protein 1). Treatment with RA (retinoic acid) triggers NCCIT cell differentiation, reducing NANOG and OCT4 expression and up-regulating several neural markers [i.e. ASCL1 (achaete-scute complex homologue 1), NEUROD1 (neuronal differentiation 1) and PAX6 (paired box 6)]. However, co-treatment with saRNA antagonizes NANOG down-regulation and RA-induced differentiation. Ectopic overexpression of NANOG via lentiviral transduction further recapitulates saRNA results, providing proof-of-concept that RNAa may be utilized to activate development-related genes and manipulate cell fate.
cell fate; differentiation; induced pluripotent stem cell (iPS cell); NANOG; RNA activation (RNAa); ASCL1, achaete-scute complex homologue 1; dsRNA, double-stranded RNA; ES, embryonic stem; FOXH1, forkhead box H1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HEK, human embryonic kidney; Hsp70, heat-shock protein 70; iPS, induced pluripotent stem; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NEUROD1, neuronal differentiation 1; OCT4, octamer-binding protein 4; PAX6, paired box 6; RA, retinoic acid; REST, RE1-silencing transcription factor; REX1, reduced expression protein 1; RNAa, RNA activation; RT, reverse transcription; saRNA, small activating RNA; SSEA, stage-specific embryonic antigen; TSS, transcription start site
Ameloblastoma is a benign odontogenic tumor with an aggressive biological behavior, and the surgical treatment frequently results in failure for the postoperative recurrence. The aim of this article was to investigate whether the proliferative ability and prognosis of ameloblastoma could be evaluated by the radiographic boundary. The ameloblastoma cases treated by the conservative therapy in our hospital between 1981 and 2001 were divided into three groups based on the nature of the radiographic borders of the lesions. The biologic behavior was evaluated by Ki-67 antibody immunohistochemically. Comparisons of prognosis and Ki-67 expression were carried out by statistic methods. There were 24 cases of well-defined edge with sclerosis (group I), 41 cases of well-defined edge without sclerosis (group II) and 32 cases of ill-defined edge (group III). The recurrent rates were 29.2% in group I, 43.9% in group II and 62.5% in group III (P<0.05). The cells in group III expressed the highest Ki-67 level (P<0.05). The radiographic boundary could be used as one of indicators in evaluating the proliferative ability of ameloblastoma and the patient's prognosis, which was consistent with Ki-67 expression.
ameloblastoma; Ki-67; radiography; prognosis
In the title complex, [CuCl2(C10H7N3S)(C2H5OH)], the CuII ion is five-coordinated in a distorted square-pyramidal geometry by two N atoms from a 2-(1,3-thiazol-4-yl)-1H-benzimidazole ligand, one O atom from an ethanol molecule and two Cl atoms. In the crystal, O—H⋯Cl and N—H⋯Cl hydrogen bonds link the complex molecules into a layer parallel to (100). π–π interactions between the thiazole rings are observed [centroid–centroid distance = 3.749 (3) Å].
In the title complex, [Cd2(C17H14O4S)2(C10H8N2)2], which was hydrothermally synthesized, the CdII cation is hexacoordinated in a distorted octahedral geometry by two N atoms from a 2,2′-bipyridine ligand and by four O atoms from two different 2-[3-carboxylatomethyl-4-(phenylsulfanyl)phenyl]propanoate ligands, forming a cyclic dimetallic complex.
microRNAs (miRNAs), defined as 21–24 nucleotide non-coding RNAs, are important regulators of gene expression. Initially, the functions of miRNAs were recognized as post-transcriptional regulators on mRNAs that result in mRNA degradation and/or translational repression. It is becoming evident that miRNAs are not only restricted to function in the cytoplasm, they can also regulate gene expression in other cellular compartments by a spectrum of targeting mechanisms via coding regions, 5′ and 3′untransalated regions (UTRs), promoters, and gene termini. In this point-of-view, we will specifically focus on the nuclear functions of miRNAs and discuss examples of miRNA-directed transcriptional gene regulation identified in recent years.
Argonaute proteins; RNA activation; chromatin remodeling; gene regulation; promoter-targeting miRNAs; transcriptional gene silencing
Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21WAF1/ Cip1 (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-of-principle that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease.
CDKN1A; delivery; gene activation; gene therapy; lipid nanoparticles; prostate cancer; saRNA; siRNA
To explore the effect of SDF-1α on the development of experimental corneal neovascularization (CRNV).
CRNV was induced by alkali injury in mice. The expression of SDF-1α and CXCR4 in burned corneas was examined by Flow Cytometry. Neutralizing anti-mouse SDF-1α antibody was locally administrated after alkali injury and the formation of CRNV 2 weeks after injury was assessed by Immunohistochemistry. The expression of VEGF and C-Kit in burned corneas was detected by RT-PCR.
The number of CRNV peaks at 2 weeks after alkali injury. Compared to control group, SDF-1α neutralizing antibody treatment significantly decreased the number of CRNV. RT-PCR confirmed that SDF-1α neutralizing antibody treatment resulted in decreased intracorneal VEGF and C-Kit expression.
SDF-1α neutralizing antibody treated mice exhibited impaired experimental CRNV through down regulated VEGF and C-Kit expression.
corneal neovascularization; alkali injury; chemokine
Alteration of epidermal growth factor receptor (EGFR) is involved in various human cancers and has been intensively investigated. A plethora of evidence demonstrates that posttranslational modifications of EGFR play a pivotal role in controlling its function and metabolism. Here, we show that EGFR can be acetylated by CREB binding protein (CBP) acetyltransferase. Interestingly, EGFR acetylation affects its tyrosine phosphorylation, which may contribute to cancer cell resistance to histone deacetylase inhibitors (HDACIs). Since there is an increasing interest in using HDACIs to treat various cancers in the clinic, our current study provides insights and rationale for selecting effective therapeutic regimen. Consistent with the previous reports, we also show that HDACI combined with EGFR inhibitors achieves better therapeutic outcomes and provides a molecular rationale for the enhanced effect of combination therapy. Our results unveil a critical role of EGFR acetylation that regulates EGFR function, which may have an important clinical implication.
AIM: To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms.
METHODS: Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 μmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry.
RESULTS: The proliferation of gastric carcinoma cells was inhibited by PBA in a dose- and time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G0/G1 phase, whereas cells treated with high concentrations of PBA were arrested at the G2/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G0/G1 phase, cells treated with high concentrations of PBA were arrested at the S phase.
CONCLUSION: The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and G2/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and S phases.
Histone deacetylase inhibitor; 4-phenyl butyric acid; Gastric carcinoma; Anticancer effect; Cell cycle; MGC-803; SGC-7901
KLF4/GLKF4 is a transcription factor that can have divergent functions in different malignancies. The role of KLF4 in prostate cancer etiology remains unclear. We have recently reported that small double-stranded RNA (dsRNA) can induce gene expression by targeting promoter sequence in a phenomenon referred to as RNA activation (RNAa). In the present study, we examine KLF4 levels in prostate cancer tissue and utilize RNAa as a tool for gene overexpression to investigate its function. Expression analysis indicated that KLF4 is significantly downregulated in prostate cancer cell lines compared to non-tumorigenic prostate cells. Meta-analysis of existing cDNA microarray data also revealed that KLF4 is frequently depleted in prostate cancer tissue with more pronounced reduction in metastases. In support, tissue microarray analysis of tumors and patient-matched controls indicated downregulation of KLF4 in metastatic tumor samples. Logistic regression analysis found that tumors with a KLF4 staining score <5 had a 15-fold higher risk for developing metastatic prostate cancer (P = 0.001, 95% CI: 3.0–79.0). In vitro analysis indicated that RNAa-mediated overexpression of KLF4 inhibited prostate cancer cell proliferation and survival, as well as altered the expression of several downstream cell cycle-related genes. Ectopic expression of KLF4 via viral transduction recapitulated the RNAa results validating its inhibitory effects on cancer growth. Reactivation of KLF4 also suppressed migration and invasion of prostate cancer cells. These results suggest that KLF4 functions as an inhibitor of tumor cell growth and migration in prostate cancer and decreased expression has prognostic value for predicting prostate cancer metastasis.
KLF4; RNAa; tumor suppressor gene; prostate cancer; metastasis
Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).
Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5′ untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.
It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3′UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth.
T-cell development from stem cells has provided a highly accessible and detailed view of the regulatory processes that can go into the choice of a cell fate in a postembryonic, stem cell-based system. But, it has been a view from the outside. The problems in understanding the regulatory basis for this lineage choice begin with the fact that too many transcription factors are needed to provide crucial input: without any one of them, T-cell development fails. Furthermore, almost all the factors known to provide crucial functions during the climax of T-lineage commitment itself are also vital for earlier functions that establish the pool of multilineage precursors that would normally feed into the T-cell specification process. When the regulatory genes that encode them are mutated, the confounding effects on earlier stages make it difficult to dissect T-cell specification genetically. Yet both the positive and the negative regulatory events involved in the choice of a T-cell fate are actually a mosaic of distinct functions. New evidence has emerged recently that finally provides a way to separate the major components that fit together to drive this process. Here, we review insights into T-cell specification and commitment that emerge from a combination of molecular, cellular, and systems biology approaches. The results reveal the regulatory structure underlying this lineage decision.
hematopoietic progenitor cells; T cells; transcription factors; cell differentiation; gene regulation; lineage commitment/specification