Journal: International Journal of Oncology

Article Title: Anisomycin inhibits angiogenesis in ovarian cancer by attenuating the molecular sponge effect of the lncRNA-Meg3/miR-421/PDGFRA axis

doi: 10.3892/ijo.2019.4887

Figure Lengend Snippet: Anisomycin attenuates the molecular sponge effect in the lncRNA-Meg3/miR-421/PDGFRA axis. (A) Heatmap showing that the expression of lncRNA-Meg3 in the anisomycin-treated group was significantly lower than that of the control group. (B) Expression levels in clinical tumour samples and (C) in xenograft tumours suggest an inverse trend in expression for lncRNA-Meg3 and miR-421. (D) Expression levels of miR-421 in HuOCSCs following anisomycin treatment. ** P<0.01 vs. non-treated (n=4). (E) Schematic of the complementary sites of mature miR-421 and the 3'UTR of lncRNA-Meg3 and PDGFRA mRNA. (F) Schematic of the structure of luciferase reporter plasmids. (G) The molecular sponge effect of lncRNA-Meg3/miR-421/PDGFRA. (H-J) Results of the luciferase reporter assays. ** P<0.01 vs. blank plasmid (n=3). (K) RIP results revealed that the binding between Meg3 and Ago2 decreased significantly following treatment of HuOCSCs with anisomycin. lncRNA, long non-coding RNA; Meg3, maternally expressed 3; miR, microRNA; PDGFRA, platelet derived growth factor receptor α; HuOCSCs, human ovarian cancer stem cells; RIP, RNA immunoprecipitation; Ago2, argonaute 2; ORF, open reading frame; UTR, untranslated region; mut, mutant; IgG, immunoglobulin G.

Article Snippet: The mouse anti-human Ago2 monoclonal antibody (clone C34C6; cat no. 2897; Cell Signaling Technology, Inc.; 1:100) was added for 90 min, before the re-addition of 20 μ g of protein A agarose beads to capture the immune complexes.

Techniques: Expressing, Control, Luciferase, Plasmid Preparation, Binding Assay, Derivative Assay, RNA Immunoprecipitation, Mutagenesis

Journal: PLoS ONE

Article Title: Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

doi: 10.1371/journal.pone.0007192

Figure Lengend Snippet: A. Hierarchical clustering dendrogram showing Pearson correlation between ESC and NSC stages of four hESC lines: H1, HSF1, HSF6, and RG7. Expression was determined by counting occurrences based on the list of 609 known or 146 predicted microRNAs found to be Ago2 IP enriched. Counts were normalized to the total number of sequences per sample. For both known microRNAs (top) and predicted microRNAs (bottom), the primary distinction between samples is based on cell stage. This demonstrates that each group of microRNAs is uniformly expressed in a group of biologically distinct cell lines. B. Pearson correlation tables of microRNA expression across development. Using the list of 609 known microRNAs (top) or 146 predicted microRNAs (bottom) selected from the Ago2 IP enriched lists, expression levels were determined by counting sequences and correcting for total numbers of sequences per sample. Known microRNA expression levels (top) distinguished all three adult tissues from a pluripotent group (RG7 ESC and iPS cells obtained from WiCell) and also from a neural lineage group (RG7 NSC, RG7 NRP, and iPS-derived NSC). However, predicted microRNAs included the adult brain sample in a cluster containing iPS NSC and, to a lesser extent, RG7 NSC and NRP. C. The same dataset was K-means clustered to isolate expression patterns. Based on minimizing the mean sum of squares fit , k = 11 clusters were selected. Shown here are the cluster means±SEM, color-coded to match the complete plotting of all 755 clustered known and new microRNAs . D. Conservation of predicted microRNAs determined using the novel SiPhy metric ω; substitution rate relative to an ancestral repeat-based neutral evolution model (grey dashed line) . Known microRNAs (black) exhibit two distinct peaks with the larger one (left) having relatively high conservation across 29 mammalian species. The 818 miRDeep-predicted microRNAs (red), in contrast, overlapped with the less conserved fraction of the known microRNAs. A small portion of the 146 Ago2 IP selected microRNAs (blue) peaked as a conserved-sequence group but the majority aligned with the non-conserved microRNAs.

Article Snippet: Lysed cell extracts were immunoprecipitated with Anti-human Ago2 antibody (Ascenion GmbH, Helmholtz Zentrum, München) or rabbit IgG using protein G Sepharose beads.

Techniques: Expressing, Derivative Assay, Sequencing

Journal: Nature Communications

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

doi: 10.1038/ncomms13287

Figure Lengend Snippet: ( a ) A scheme of vectors expressing different GAS5 truncated transcript. The inserted length of each transcript is indicated and the blue square indicates the position of validated GR-binding sequence. 1-651mut represents the GR-binding sequence-mutated construct. The right panel shows the mRNA level of NANOG and NODAL in different treatment groups. ** P <0.01, t -test, n =3. ( b ) The left panel shows the scheme of constructing NODAL promoter reporter. The right panel shows the luciferase activities of NODAL promoter reporter in hESCs with indicated treatment. NC represents NODAL promoter reporter transfected hESCs treated with empty vector, t -test, n =3. ( c ) The nuclear-plasma separation assay of GAS5 overexpressed or knockdown hESCs are shown in the middle panel using qPCR. Respective FISH images are shown in the left panel, and the GAS5 expression of analysed using total RNAs are shown in the right panel ( n =3). NUCL represents nuclear hESC extracts, CYTO represents cytoplasm hESC extracts. Scale bar, 20 μm. All groups were compared with NC or NC-NUCL group respectively. ** P <0.01, t -test, n =3. ( d ) A scheme of MS2-mediated pulldown of GAS5 and its binding protein. ( e ) The silver staining of pull-down products ( n =2), the red triangle indicates the antibody band, whereas black triangle indicates the bands and position that is cutoff for mass spectrometry analysis shown in the right panel. ( f ) Western blot shows the AGO2 expression in different pulldown groups as indicated by the black triangle ( n =2). The red triangle indicates the antibody heavy chain bands, which were used in pull-down experiment and react with the secondary antibody. ( g ) Confocal microscopy of AGO2 and GAS5 transcript. Scale bar, 100 μm. ( h ) RNA immune-precipitation analysis of the RNA levels of precipitated GAS5, linc-ROR, H19 using indicated protein antibodies by qPCR. ** P <0.01, t -test, n =3. ( i ) AGO2 competing assay using AGO2 antibody-mediated RNA-IP. RNA levels of precipitated NODAL, linc-ROR, H19 are tested using qPCR. ** P <0.01, t -test, n =2. Error bars represent s.d. of the indicated experiment replicates. RNA level of β-actin served as internal reference for qPCR. See also .

Article Snippet: For the detection of proteins, samples were blocked with 5% bovine serum albumin, than incubated with rabbit polyclonal anti-Ago2 (1:100, ab32381, Abcam).

Techniques: Expressing, Binding Assay, Sequencing, Construct, Luciferase, Transfection, Plasmid Preparation, Silver Staining, Mass Spectrometry, Western Blot, Confocal Microscopy

Journal: Nature Communications

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

doi: 10.1038/ncomms13287

Figure Lengend Snippet: ( a ) A scheme of miRNA target network showing the relationship of GAS5 downregulated miRNAs and their predicted target mRNAs. ( b ) A list of top 30 downregulated candidate miRNAs on GAS5 overexpression analysed by high-throughput miRNA sequencing. The right panel shows predicted candidate miRNAs that bind to both GAS5 and NODAL. ( c ) The analysis of NODAL and GAS5 expression in Dicer deficient hESCs. ShDicer represents lentiviral-mediated Dicer knockdown. NC represents scramble siRNA control. ** P <0.01, t -test, n =3. ( d ) The effect of candidate miRNAs on the mRNA levels of GAS5 and NODAL in hESCs. MiRNA mimics are transfected into hESCs at the concentration of 20 pmol per ml. ** P <0.01, t -test, n =3. ( e ) A graph illustrating the binding sites of candidate miRNAs to GAS5 and NODAL transcript, and the scheme of constructing wild-type and mutant NODAL luciferase miRNA reporters and GAS5 MS2 constructs. The red cross indicates mutated sites, see also . ( f ) Luciferase activities of relative reporter in e with different miRNAs transfection in HEK-293T cells. * P <0.05, ** P <0.01, t -test, n =3. ( g ) qPCR analysis of MS2-mediated candidate miRNAs pulldown assay. Data were normalized to input, and the enrichment ratios compared with input level were shown. MS2-RL serve as a negative control, and MS2-OCT4 are served as positive control in detecting miR-145-5p. ** P <0.01, t -test, n =2. ( h ) AGO2 competing assay using AGO2 antibody-mediated RNA-IP in the indicated groups. RNA levels of precipitated NODAL, linc-ROR and H19 are tested using qPCR. NS, not significant, ** P <0.01, t -test, n =2. ( i ) MS2-mediated miRNA pulldown in the indicated transcript overexpression groups. MS2-RL serves as control. NS, not significant, ** P <0.01, t -test, n =2. Error bars represent s.d. of the indicated experiment replicates. RNA level of β-actin served as internal reference for qPCR. See also .

Article Snippet: For the detection of proteins, samples were blocked with 5% bovine serum albumin, than incubated with rabbit polyclonal anti-Ago2 (1:100, ab32381, Abcam).

Techniques: Over Expression, High Throughput Screening Assay, Sequencing, Expressing, Transfection, Concentration Assay, Binding Assay, Mutagenesis, Luciferase, Construct, Negative Control, Positive Control

Journal: Experimental and Therapeutic Medicine

Article Title: Long non-coding RNA SNHG7 facilitates pancreatic cancer progression by regulating the miR-146b-5p/Robo1 axis

doi: 10.3892/etm.2021.9829

Figure Lengend Snippet: miR-146b-5p is a direct target of SNHG7. (A) Complementary sequences between miR-146b-5p and SNHG7, and the MUT sequences of SNHG7. Luciferase activity of SNHG7 WT or SNHG7 MUT reporter in (B) SW1990 and (C) AsPC-1 cells transfected with miR-146b-5p or miR-control was assessed via dual-luciferase reporter. (D) Enrichment of SNHG7 in anti-Ago2 or anti-IgG labeled SW1990 cells transfected with miR-146b-5p or miR-control was evaluated by RIP assay. (E) Enrichment of SNHG7 in SW1990 cells transfected with Bio-miR-146b-5p or Bio-NC was analyzed by RNA pull-down assay. (F) Enrichment of SNHG7 in anti-Ago2 or anti-IgG labeled AsPC-1 cells transfected with miR-146b-5p or miR-control was evaluated by RIP assay. (G) Enrichment of SNHG7 in AsPC-1 cells transfected with Bio-miR-146b-5p or Bio-NC was analyzed by RNA pull-down assay. Transfection efficiency of miR-146b-5p probe was examined in (H) SW1990 and (I) AsPC-1 cells transfected with miR-control or miR-146b-5p. miR-146b-5p expression was measured in SW1990 and AsPC-1 cells transfected with (J) miR-control and miR-146b-5p, or (K) inhibitor-control and miR-146b-5p inhibitor. (L) Expression of miR-146b-5p in cells transfected with sh-control, sh-SNHG7, pcDNA-control or pcDNA-SNHG7 were detected via reverse transcription-quantitative PCR. (M) Correlation between SNHG7 and miR-146b-5p was analyzed by Pearson test. * P<0.05 vs. their respective normal groups. WT, wild-type; MUT, mutant; SNHG7, small nucleolar RNA host gene 7; miR, microRNA; sh, short hairpin RNA; NC, negative control; Ago2, argonaute-2.

Article Snippet: Following lysis of the transfected SW1990 and AsPC-1 cells, the lysate samples were incubated with magnetic beads labelled with anti-argonaute-2 (Ago2; BIOSS) or negative control anti-IgG antibody (BIOSS) for 4 h at 4˚C, and a 10 µl aliquot of RIP mixture was saved as input.

Techniques: Luciferase, Activity Assay, Transfection, Labeling, Pull Down Assay, Expressing, Real-time Polymerase Chain Reaction, Mutagenesis, shRNA, Negative Control

Journal: OncoTargets and therapy

Article Title: LncRNA ZNF667-AS1 Promotes ABLIM1 Expression by Adsorbing micro RNA-1290 to Suppress Nasopharyngeal Carcinoma Cell Progression

doi: 10.2147/OTT.S245554

Figure Lengend Snippet: ZNF667-AS1 competitively binds to miR-1290 . ( A ) Comparison of miRNA results with the possible binding of ZNF667-AS1 predicted by RNA22 and miRNAs up-regulated in GSE70970 microarray dataset, there are 2 miRNAs in the intersection. ( B ) The expression of miR-1290 in GSE70970 microarray dataset. ( C ) RNA22 predicts the binding site between ZNF667-AS1 and miR-1290 . ( D ) RT-qPCR utilized to detect the expression of miR-1290 in tumor and adjacent tissues (n = 36), * p < 0.05 vs adjacent tissues. ( E ) Dual-luciferase reporter gene assay to verify the binding relationship between ZNF667-AS1 and miR-1290 . ( F ) FISH for the detection of subcellular localization of ZNF667-AS1 in CNE-1 cells (×200). ( G ) RNA pull-down assay to verify the binding of ZNF667-AS1 with miR-1290 , * p < 0.05 vs the Bio-NC group. ( H ) RIP assay for the detection of whether ZNF667-AS1 interacted directly with AGO2 protein in CNE-1 cells, * p < 0.05 vs the IgG group; # p < 0.05 vs the MUT- miR-1290 group. Data are expressed as mean ± standard deviation. Paired t test is used for comparison of two groups. One-way analysis of variance is used for comparison among multiple groups. Each experiment was run in triplicate. Abbreviations: ZNF667-AS1 , zinc finger protein 667-antisense RNA 1; miR-1290 , microRNA-1290 ; RT-qPCR, reverse transcription quantitative polymerase chain reaction; FISH, fluorescence in situ hybridization; NC, negative control; RIP, RNA immunoprecipitation; AGO2, argonaute 2; IgG, immunoglobulin G; WT, wild type; MUT, mutant; DAPI, 4ʹ,6-diamidino-2-phenylindole.

Article Snippet: LncRNA ZNF667-AS1 bound to AGO2 protein (rabbit anti-AGO2, ab32381, 1:50) and IgG (ab109489, 1:100, both Abcam) was determined with a RIP kit (Millipore, Billerica, MA, USA).

Techniques: Binding Assay, Microarray, Expressing, Quantitative RT-PCR, Luciferase, Reporter Gene Assay, Pull Down Assay, Standard Deviation, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Negative Control, Immunoprecipitation, Mutagenesis