Journal: PLoS Biology

Article Title: Long noncoding RNA PM maintains cerebellar synaptic integrity and Cbln1 activation via Pax6/Mll1-mediated H3K4me3

doi: 10.1371/journal.pbio.3001297

Figure Lengend Snippet: (A) Left: illustration of 12 Gm2694 isoforms indicated as different colors. The sh PM -1, sh PM -2, and sh PM -3 used in the study were indicated. Right: the relative expression levels of the 12 isoforms of Gm2694 in the indicated brain regions. Data are shown as means ± SEMs, n = 3. Gm2694-204 is designated as lncRNA-PM (PM). (B) The relative expression levels of Gm2694 (for 201 , 202 , 203 , 205 , 206 , 207 , 209 , and 210 ), Gm2694-201 , PM , Gm2694-205 , Gm2694-207 , and Cbln1 at the indicated cerebellar developmental time points were measured by RT-qPCR. Data are shown as means ± SEMs, n = 3. (C, D) The expression levels of the indicated Gm2694 isoforms (C) and Cbln1 (D) under the indicated treatments. (E) The expression levels of Cbln1 in control or PM shRNAs (sh PM -1/2/3)-treated Neuro2a cells, detected by RT-qPCR. Data are shown as means ± SEMs, n = 3. (F) Left: representative images of Cbln1 mRNA in the control or PM -overexpressed Neuro2a cells, detected by FISH. Right: quantification of the left. Data are shown as means ± SEMs, n = 3. (G) Left: representative immunofluorescence images of Cbln1 protein in the control (Vector) or PM- overexpressed (PM) Neuro2a cells. Right: quantification of the left. Data are shown as means ± SEMs, n = 3. (H) Subcellular distributions of PM , Gapdh , and Neat1 by fractionating assay in Neuro2a cells. Gapdh and Neat1 RNA served as positive controls for RNAs predominantly expressed in cytoplasm and nucleus, respectively. Data are shown as means ± SEMs, n = 3. (I) Representative FISH image of PM RNA (red) in Neuro2a cells. Nuclei were stained with DAPI (blue). (J) Top: schematic illustration of the Cbln1 / PM locus. Bottom left: luciferase assays indicate the activity changes of pGL3- Cbln1 (blue) and pGL3 -Gm2694 (red), compared with pGL3 vector control (black). Bottom right: Luciferase assays indicate the activity changes of pGL3- Cbln1 in the presence of control or PM -expressing plasmid. Data are shown as means ± SEMs, n = 3. Scale bar, 13 um. All RT-qPCR results were normalized to Gapdh . All the data of this figure can be found in the file. * P < 0.05, ** P < 0.01, and *** P < 0.001. Cbln1 , Cerebellin-1 ; Cereb, Cerebellum; FISH, fluorescence in situ hybridization; Hippo, hippocampus; Hypo, hypothalamus; lncRNA-PM , lncRNA-Promoting Methylation ; OB, olfactory bulb; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Antisense RNA oligonucleotides (probes) for lncRNA-PM and LacZ (negative control) were designed by Biosearch Technologies website ( https://www.biosearchtech.com/stellarisdesigner/ ) and synthesized with 3′-biotin-TEG modification (Sangon, China).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Plasmid Preparation, Staining, Luciferase, Activity Assay, Fluorescence, In Situ Hybridization, Methylation, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Long noncoding RNA PM maintains cerebellar synaptic integrity and Cbln1 activation via Pax6/Mll1-mediated H3K4me3

doi: 10.1371/journal.pbio.3001297

Figure Lengend Snippet: (A) Illustrated structure in the investigated cerebellar area. (B) The expression levels of PM (red) and Cbln1 (green) RNA in the cerebellar sections obtained from control (AAV NC) or lncRNA-PM ( PM , red) shRNA-injected mice (AAV sh PM -1 and AAV sh PM -2). Scale bar, 40 um. (C and D) Immunofluorescence images of Cbln1 protein (C) and H3K27ac (D) in the cerebellar sections obtained from AAV NC and AAV sh PM s mice. Scale bar, 40 um. (E) Quantification of the RNA levels of PM and Cbln1 obtained from B. Data are shown as means ± SEMs, n = 3. (F) Quantification of the protein levels of Cbln1 obtained from C. (G) Quantification of the GC numbers. (H) Evaluation of the control and sh PM s-injected mice on cylinder test. Cylinder test was designed to assess limb preference on mice. It showed that compared with the control mice, PM shRNA-injected mice exhibited asymmetric high frequency in forepaw usage. The paw usage was counted and analyzed by normal-impaired/all. Data are shown as means ± SEMs, n = 7 (per group). (I) Evaluation of the control and sh PM s-injected mice on rotarod test. Latency to fall provides the quantification of motor ability on the accelerating rotarod. It showed that compared with the control mice, sh PM s-injected mice exhibited significant reduced duration in latency to fall. Data are shown as means ± SEMs, n = 6 (per group). (J) Representative electron microscope images for the control and sh PM -injected mice. Red asterisk: intact synapse. Yellow triangle: free spine. Scale bar, 500 nm. (K) Quantification of the numbers of intact synapses obtained from J in the indicated treatments. (L) Quantification of the numbers of free spines obtained from (J) in the indicated treatments. All the data of this figure can be found in the file. * P < 0.05, ** P < 0.01, and *** P < 0.001. Cbln1 , Cerebellin-1 ; GCL, granule cell layer; lncRNA-PM , lncRNA-Promoting Methylation ; ML, molecular layer; PCL, Purkinje cell layer; WM, white matter.

Article Snippet: Antisense RNA oligonucleotides (probes) for lncRNA-PM and LacZ (negative control) were designed by Biosearch Technologies website ( https://www.biosearchtech.com/stellarisdesigner/ ) and synthesized with 3′-biotin-TEG modification (Sangon, China).

Techniques: Expressing, shRNA, Injection, Immunofluorescence, Microscopy, Methylation

Journal: PLoS Biology

Article Title: Long noncoding RNA PM maintains cerebellar synaptic integrity and Cbln1 activation via Pax6/Mll1-mediated H3K4me3

doi: 10.1371/journal.pbio.3001297

Figure Lengend Snippet: (A) UV-RIP assay showed PM :Pax6 and PM :Mll1 interactions in mouse cerebellum. Neat1 and lncRNA-lhx1os : negative controls. Data are shown as means ± SEMs, n = 3. (B) RT-qPCR detection following PM RNA pull-down using cerebellum tissue. PM: PM probes; LacZ: control probes. (C) Illustration of H3K4me3 signature on the upstream regulatory region of Cbln1 locus from +300 to −2,000 base pair based on ENCODE data in mouse cerebellum. The 14 pairs of amplicons are indicated. (D) ChIRP-qPCR showed occupancy of PM to the regions amplified by the indicated 14 amplicons and a control region that is 85 kb upstream of Cbln1 transcription start site (Far Region). Data are shown as means ± SEMs, n = 3. (E) Re-ChIP of Pax6 and Mll1. Top: illustrated flowchart of double ChIP experiment. Bottom left: recruitments of Pax6 or IgG to Cbln1 or the negative control region (Far Region) after first ChIP in mouse cerebellum. Bottom right: recruitments of Mll1 or IgG to Cbln1 or Far Region after second ChIP in mouse cerebellum. Data are shown as means ± SEMs, n = 3. (F) Model: LncRNA - PM mediates the transcriptional activation of Cbln1 through Pax6/Mll1-mediated H3K4me3 and maintains mouse motor coordination and motor learning. All the data of this figure can be found in the and Data files. * P < 0.05, ** P < 0.01, and *** P < 0.001. Cbln1 , Cerebellin-1 ; ChIP, chromatin immunoprecipitation; ChIRP, chromatin isolation by RNA purification; IgG, immunoglobulin G; lncRNA-PM , lncRNA-Promoting Methylation ; ns, no significance; qPCR, quantitative PCR; Re-ChIP, Re-chromatin immunoprecipitation; RT-qPCR, reverse transcription-quantitative PCR; UV-RIP, UV-RNA immunoprecipitation.

Article Snippet: Antisense RNA oligonucleotides (probes) for lncRNA-PM and LacZ (negative control) were designed by Biosearch Technologies website ( https://www.biosearchtech.com/stellarisdesigner/ ) and synthesized with 3′-biotin-TEG modification (Sangon, China).

Techniques: Quantitative RT-PCR, Amplification, Negative Control, Activation Assay, Chromatin Immunoprecipitation, Isolation, Purification, Methylation, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: PLoS Biology

Article Title: Long noncoding RNA PM maintains cerebellar synaptic integrity and Cbln1 activation via Pax6/Mll1-mediated H3K4me3

doi: 10.1371/journal.pbio.3001297

Figure Lengend Snippet: (A) Left: heatmap of dysregulated genes (|Fold change|>1.5, FDR < 0.05) in PM knockdown and Pax6 overexpression RNA-seq. Right: GO biological processes of PM and Pax6 coregulated genes. Red: positively regulated genes shared by PM and Pax6 . Blue: negatively regulated genes shared by PM and Pax6 . (B) Modulations of lncRNA-PM and Pax6 impact neuronal associated genes. Expression changes for selected genes are shown as the log2 expression ratio. Red: positively regulated genes shared by PM and Pax6 . Blue: negatively regulated genes shared by PM and Pax6 . (C and D) qPCR detection of the indicated genes upon PM knockdown (C) and Pax6 overexpression (D). All the data of this figure can be found in the file. Data are shown as means ± SEMs, n = 3. * P < 0.05, ** P < 0.01, and *** P < 0.001. FDR, false discovery rate; GO, Gene Ontology; lncRNA-PM , lncRNA-Promoting Methylation ; ns, no significance; qPCR, quantitative PCR; RNA-seq, RNA sequencing.

Article Snippet: Antisense RNA oligonucleotides (probes) for lncRNA-PM and LacZ (negative control) were designed by Biosearch Technologies website ( https://www.biosearchtech.com/stellarisdesigner/ ) and synthesized with 3′-biotin-TEG modification (Sangon, China).

Techniques: Over Expression, RNA Sequencing Assay, Expressing, Methylation, Real-time Polymerase Chain Reaction

Journal: Theranostics

Article Title: Hyperglycemia facilitates EV71 replication: Insights into miR-206-mediated regulation of G3BP2 promoting EV71 IRES activity

doi: 10.7150/thno.93883

Figure Lengend Snippet: miR-206 exhibits significant impacts on EV71 neurovirulence and insulin secretion in hSCARB2-Tg mice. (A) qRT-PCR analysis of viral load in the brainstem of EV71-infected hSCARB2-Tg mice, with or without antagomiR-206 (AM206) treatment (n = 5). Statistical significance was determined using a two-sided T-test, with a significance threshold set at p < 0.05. (B) Survival assessment of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Seven-day-old hSCARB2-Tg mice infected with EV71 were administered LNA miR-206 antagomir (AM206, 1.2 mg/kg, n = 15) or (Ctrl, n = 14). The log-rank test was utilized to determine the statistical significance in survival rates between the treatment groups. (C) Representative micrographs depicting brainstem gliosis in EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Scale bar represents 20 μm. (D) Brainstem expression of proinflammatory cytokines in EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. RNA was extracted from the brainstem tissue of mice at 5 dpi and subjected to qRT-PCR analysis using TNFα-specific, and IL-6-specific primers, normalized to GAPDH (n = 5). (E) Proposed role of miR-206 in targeting GCK-mediated insulin-glucose homeostasis. (F) Representative immunohistochemistry (IF) images displaying GCK levels in the pancreas of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Paraffin-embedded sections were stained using the VP1/2 antibody to detect EV71, while islet staining was performed using an insulin antibody. Scale bar denotes 20 μm. (G) qRT-PCR quantification of GCK levels in the pancreas of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment (n = 5). (H) Plasma insulin levels in EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Insulin levels were measured using the mouse Insulin ELISA assay (n = 5). (I) Plasma glucose levels in EV71-infected mice, with or without AM206 treatment. Glucose levels were measured using a blood glucose assay (n = 5).

Article Snippet: LNA-modified microRNA miR-206 antisense oligonucleotide probes were labeled with Biotin (MDBio Inc.) and the signal was amplified using Tyramide amplification solution (Thermo Fisher Scientific).

Techniques: Quantitative RT-PCR, Infection, Expressing, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, Glucose Assay

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: ChIRP-Seq reveals m-Meg3 enrichment at multiple m-c-Met loci. (A) Representative agarose gel image showing the specificity of the m-Meg3 ChIRP probes. RNA was isolated after m-Meg3 ChIRP from the V3 (vector) and the M5 (stable MIN6-4N cells stably expressing the m-Meg3-3 isoform which lacks exon 4) cell lines. The RNA was then used for RT-PCR. Input corresponds to the RT-PCR product using RNA isolated before m-Meg3 ChIRP-Seq. Odd and even correspond to RT-PCR using RNA after m-Meg3 ChIRP with probes located at odd and even locations on the m-Meg3 RNA. The gel image represents products of the RT-PCR performed with primers 1F/1R (flanking exon 7 and exon 8) that recognize all m-Meg3 isoforms. gapdh served as the negative control. cDNAs from three replicates of V3 and M5 ChIRP-Seq were pooled due to low yields and sequenced. (B) m-Meg3 ChIRP-PCR in a stable cell line expressing full-length m-Meg3. RNA was isolated after m-Meg3 ChIRP from the 9V (vector) and the 14M (stable MIN6-4N cells with the m-Meg3-1 isoform, which encompasses all 10 exons) cell lines. The RNA was then used for RT-PCR. Input corresponds to RT-PCR from RNA isolated before m-Meg3 ChIRP. RT-PCR was performed with primers 1F/1R (flanking exons 7 and 8) that recognize all m-Meg3 isoforms and further confirmed with the ex3F/ex4R primer pair (flanking exons 3 and 4), specific for the m-Meg3-1 isoform. gapdh served as the negative control. (C) m-Meg3 enrichment patterns at discrete m-c-Met genomic regions in different m-Meg3 stable cell lines. DNA was isolated after m-Meg3 ChIRP from two different m-Meg-3 stable MIN6-4N cell lines and their respective vector controls. The DNA was then subjected to whole-genome amplification (WGA) and subsequent purification. The purified WGA DNA was then used to set up qPCRs in duplicate with primers specific for m-c-Met genomic regions identified by m-Meg3 ChIRP-Seq, namely, the m-c-Met upstream region, the m-c-Met exon 18 region, the m-c-Met exon 20 region, and also the previously identified kb +63 enhancer. The qPCR data are represented as percent input of DNA.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Agarose Gel Electrophoresis, Isolation, Plasmid Preparation, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Whole Genome Amplification, Purification

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: m-Meg3 associates with PRC2 components in mouse insulinoma cell line models. (A and B) Association of m-Meg3 RNA with EZH2 and H3K27me3. RNA-ChIP in Men1 cells (Meg3 proficient) was performed using the antibodies directed toward EZH2 (A) and H3K27me3 (B). qPCR was performed on cDNA obtained from RNA isolated after RNA-ChIP. qPCR data were calculated as percent RNA input and are shown as the average from two independent biological replicates and multiple technical replicates (mean ± SD). IgG served as the negative control for the RNA-ChIP assay. (C and D) EZH2 and H3K27me3 enrichment at the m-c-Met regions identified by m-Meg3 ChIRP-Seq. ChIP assay was performed in Men1 cells using the indicated antibodies. DNA isolated after ChIP was used for qPCR, with primers specific for the m-c-Met upstream region, the +63-kb enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. EZH2 (C) and H3K27me3 (D) enrichment was calculated as percent chromatin DNA input and constitutes the average from three independent biological replicates and multiple technical replicates (mean ± SD). (E and F) Enhancer-signature histone modifications at the m-c-Met loci identified by m-Meg3 ChIRP-Seq. MIN6-4N cells were subjected to ChIP assays and qPCR analyses to detect the enrichment of H3K27Ac (E) and H3K4me1 (F) at the m-c-Met upstream region, the kb +63 enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. The data represent an average from two independent biological replicates and multiple technical replicates (mean ± SD).

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Isolation, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: m-Meg3 requires PRC2 components to repress the m-c-Met transcript. (A and B) Effect of the EZH2 inhibitor GSK343 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated from MIN6-4N and Men1 cells after vehicle or GSK343 treatments at the indicated times. The RNA isolated was used in qPCR analyses using primers specific for m-c-Met, m-Meg3-1, and m-EZH2. The qPCR transcript data shown are from representative experiments where technical replicates were set up in triplicate. ***, P ≤ 0.001; *, P ≤ 0.05; N.s., nonsignificant. (C) Depletion of H3K27me3 is a confirmatory readout for the inhibition of EZH2 methyltransferase activity. Representative Western blots for H3K27me3, using whole-cell extracts (WCEs) from MIN6-4N and Men1 cells after 7-day and 6-day treatments, respectively, with 2.5 μM GSK343 or dimethyl sulfoxide (DMSO) vehicle controls are shown. β-Actin served as the loading control.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Expressing, Isolation, Inhibition, Activity Assay, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: m-Meg3 TFO-9 regulates the m-c-Met transcript. (A) Schematic of full-length m-Meg3-1 exon structure showing the TFO-9 coordinates. Full-length m-Meg3-1 is depicted, with exons 1 to 10 numbered (top) and the sequence length in base pairs (bottom). TFO-9, a GA- and GT-rich 16-mer sequence, is shown mapping to the C-terminal portion of exon 10 in m-Meg3-1. TFO-9, spanning bp 1822 to 1838, was predicted by Triplexator to form triplexes with double-stranded DNA. (B) Effect of TFO-9 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated at 48 h and 96 h posttransfection from MIN6-4N cells transiently transfected with TFO RNA oligonucleotides. Purified RNA converted to cDNA was subjected to qPCR analyses with primers specific for m-Meg3-1 or m-c-Met. The data represent an average from three independent experiments and multiple technical replicates (mean ± SD) *, P ≤ 0.05.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Sequencing, Expressing, Isolation, Transfection, Purification

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: Model depicting the mechanisms of Meg3-mediated c-Met regulation in normal pancreatic islets and tumor cells. The model presented here suggests that in the context of intact menin in normal islet beta cells, the presence of Meg3 deters the proto-oncogenic activity of c-Met by multiple mechanisms. c-Met signaling can be repressed by genomic Meg3 binding, perhaps through contact with Meg3 TFO regions and Meg3 interaction with PRC2 components. Meg3 downregulation driven by menin loss and menin-independent Meg3 loss could both favor gene-activating epigenetic changes. These epigenetic alterations in turn potentiate aberrant c-Met signaling, leading to pancreatic islet β-cell tumor formation.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Activity Assay, Binding Assay