A Michael Addition–Asymmetric Transfer Hydrogenation One-Pot Enantioselective Tandem Process for Syntheses of Chiral γ‑Secondary Amino Alcohols

An aza-Michael addition–asymmetric transfer hydrogenation tandem process for preparation of chiral γ-secondary amino alcohols has been developed. This one-pot tandem process involves an aza-Michael addition of aryl-substituted enones and amines to form aryl-substituted γ-secondary amino ketones, followed by a Ru-catalyzed asymmetric transfer hydrogenation to form aryl-substituted γ-secondary amino alcohols. An advantageous feature of this tandem reaction is that it provides various γ-secondary amino alcohols in high yields with high enantioselectivities

MicroRNA-142-3p Negatively Regulates Canonical Wnt Signaling Pathway.

Wnt/β-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs) are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/β-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/β-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC) to upregulate Wnt/β-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/β-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding β-catenin) through binding to its 3' untranslated region (3' UTR). Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/β-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/β-catenin signaling, which help to understand the complex regulation of Wnt/β-catenin signaling

Inhibition of miR-155-5p with antagomir-155 rapidly releases DTR expression.

<p>LN cells of CMV-Cre x R26-YFP × R26-DTR-155T mice founder 47 were stimulated with anti-CD3 for 48 hours. The activated cells were then transfected with 200 nM antagomir-155 or 200 nM antagomir-142 as negative control. FACS analysis was performed after 18 hours of transduction for detecting DTR expression. Left panel shows the dot plots of DTR expressing cells transduced with indicated antagomir. Middle panel shows the overlaid histograms of DTR expression between antagomir-155 and antagomir-142 transfected cells. Right panel shows DTR MFI of indicated cells. Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ****P<0.0001.</p

Validation of miR-155-5p-OFF system <i>in vitro</i>.

<p>(A) Validation of miR-155-5p target using luciferase assay, HEK293T cells were transfected with pmirGLO-4xmiR-155-5pT plus pEF-BOS-EX-miR-155, pEF-BOS-EX-miR-146a, and pEF-BOS-EX-empty vector (control), respectively, at a molar ratio of 1:1. (B) Validation of miR-155-5p target by flow cytometry, expression vector for miR-155 (pEF-BOS-EX-miR-155) or a control vector (pEF-BOS-EX) was co-transfected into HEK293T cells with an expression vector for miR-155-5p target reporter sequence (pDTR.BFP-155T) at various molar ratios. The expression of BFP reporter signal was analyzed through flow cytometry after 48 hours of transfection. (C and D) Sensitivity of miR-155-5p-OFF system. Fold change of mean fluorescent intensity of DTR expression in HEK293T cells transfected with pDTR.BFP-155T-N1 plasmid with increasing concentrations (0, 1.25, 2.5, 5, 10, 20, and 40 nM) of synthetic miR-155 or miR control. Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ns: not significant; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.</p

A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity <i>In Vivo</i>

<div><p>MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity <i>in vivo</i> by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.</p></div

Generation and Characterization of miR-155-5p sensor transgenic mice.

<p>(A) Schematic illustration of the BAC transgenic constructs. Left panel-R26-DTR-BAC lacking miR-155-5p target sequence. Right panel-R26-DTR-155T-BAC containing 4×miR-155-5p target sequence in the 3’-UTR of DTR-BFP fusion gene. The fusion cassette gene was then placed downstream of a STOP element flanked by LoxP sites driven by Rosa26 promoter. (B) Schematic illustration of the determination of miR-155-5p activity in single living cell by flow cytometry. The relative activity of miR-155-5p will be inversely proportional to the expression level of DTR or BFP. (C) Phenotype of R26-DTR-155T mice. Figures in the upper panel show FACS profiling of DTR expression in conventional (conv) CD4-gated cells and Treg-gated cells of different mice as shown. Two founders, 47 and 84, of the R26-DTR-155T mice and a R26-DTR mouse were crossed with CMV-Cre mice; lymph node (LN) cells of the littermates were stained with anti-DTR-Biotin and then conjugated with Streptavidin-PE along with anti-CD4 and anti-Foxp3 (intracellular staining) and analyzed through FACS Calibur. Figures in the lower panel represent DTR MFI of indicated cells (shown in the figures of upper panel). Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ns: not significant; *P<0.05; **P<0.01.</p

Among candidate miRNAs, miR-142 negatively regulates Wnt/β-catenin signaling.

<p>(A) Schematic representation of the pGL3-TopFlash reporter. Eight tandem repeats of TCF/LEF response elements were introduced upstream of the SV40 promoter and firefly luciferase gene (Luc) in pGL3 promoter vector. (B) Screening for inhibitors of Wnt/β-catenin signaling. Plasmids pGL3-TopFlash, pRL-TK and miRNA-expressing vectors were cotransfected into HEK293T cells. Cells were treated with 25 mM LiCl 6 h posttransfection, and lysed 24 h posttransfection for dual-luciferase analysis (RLUs, Fluc/Rluc). (C, E) miR-142 inhibits the activated Wnt/β-catenin signaling. Wnt/β-catenin signaling was activated by 25 mM LiCl (C) or increasing doses of Wnt3a (E). TopFlash-mediated firefly luciferase activities (B, C, E) were normalized to the activity of Renilla luciferase (pRL-TK); error bars mark the SEM (n = 3; **P < 0.01, *P < 0.05, t test). (D) miR-142 represses expression of <i>Axin2</i>. miR-142 expressing vector or empty vector (EF) was transfected into HEK293T cells with or without 25 mM LiCl treatment. <i>Axin2</i> mRNA expression was analyzed by quantitative RT-PCR, the data shown were normalized by <i>Actb</i> expression; error bars mark the SEM (n = 2; *P < 0.05, t test).</p

Pre-miR-142 is essential for suppressing Wnt/β-catenin signaling.

<p>A and B, schematic of the MDH1-142 (A) and EF-miR-142, EF-del-miR-142, EF-pre-edited-miR-142 (B) constructs. Pre-miR-142 (59 nt) plus flanking sequences were introduced downstream of the H1 or EF-1a promoter, followed by T5 or polyA termination signal. Whole 59 nucleotides of pre-miR-142 were deleted in the del-miR-142 construction. Four Adenosine residues within pre-miR-142 were substituted by guanosine residues (A➔G) constructed the pre-edited-miR-142 plasmid. (C) HEK293T cells were transfected with MDH1-142 expression vector or control MDH1 vector along with the pGL3-TopFlash or pGL3 empty vector and the pRL-TK vector as a normalization control. Cells were treated with 25 mM LiCl and lysed 24 h later for dual-luciferase analysis. Normalized TopFlash values were further divided by normalized pGL3 control values; error bar mark the SEM (n = 3; *P < 0.05, t test). (D) HEK293T cells were transfected with EF-miR142 expression vector or miR-142-mutant vectors along with the pGL3-TopFlash or pGL3-FopFlash vector and the pRL-TK vector as a normalization control. Normalized TopFlash values were further divided by normalized FopFlash values; error bars mark the SEM (n = 3; ***P < 0.001, **P < 0.01, t test). (E) Schematic diagram showing the structure of pre-miR-142. The part highlighted in green indicates miR-142-5p, and in red shows miR-142-3p. Bases with light-gray background represent the seed sequences of these two distinct miRNAs. The frames above M1 to M5 mark the positions of five structure-changing mutants within pre-miR-142, purple bases within the frames indicate the mutant sequences. (F) TopFlash-mediated reporter assay was performed as described in (D) with M1 ~ M5 mutants; error bars mark the SEM (n = 3; ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05, t test).</p