Journal: Nucleic Acids Research

Article Title: Nuclear Argonaute:miRNA complexes recognize target sequences within chromatin-associated RNA and silence gene expression

doi: 10.1093/nar/gkaf800

Figure Lengend Snippet: AGO2 and other protein factors involved in miRNA-mediated regulation can associate with chromatin. Western blots showing ( A ) AGO2 localization and purity of cytoplasm (CY), nucleoplasm (NP), and chromatin (CH) in wild-type samples ( N = 3), ( B ) purity [calnexin for endoplasmic reticulum (ER)] and localization of DROSHA, Dicer, TNRC6A, and AGO1–4 ( N = 3), and ( C ) AGO2 localization in the nuclear localization sequence (NLS)-AGO2 knock-in cell line ( N = 3).

Article Snippet: Western blot was visualized using anti-AGO2 primary antibody (50683-RP02, SinoBiological) at a 1:4000 dilution, with TrueBlot anti-rabbit secondary antibody (18-8816-31, Rockland) at 1:8000 dilution.

Techniques: Western Blot, Sequencing, Knock-In

Journal: Nucleic Acids Research

Article Title: Nuclear Argonaute:miRNA complexes recognize target sequences within chromatin-associated RNA and silence gene expression

doi: 10.1093/nar/gkaf800

Figure Lengend Snippet: AGO2 interacts with target RNAs in all cellular compartments. ( A ) Schematic outlining the chimeric eCLIP protocol used for the identification of AGO2-binding sites. Distribution of reproducible AGO2 ( B ) nonchimeric and ( C ) chimeric binding peaks across genomic features in cytoplasm ( N = 2), whole nucleus ( N = 2), and chromatin ( N = 2) fractions. Chimeric binding peaks only include chimeric reads that include miRNAs that map to the miRGeneDB database . ( D ) Percentage of chimeric miRNAs from the top eight miRNA families sharing the same seed sequence.

Article Snippet: Western blot was visualized using anti-AGO2 primary antibody (50683-RP02, SinoBiological) at a 1:4000 dilution, with TrueBlot anti-rabbit secondary antibody (18-8816-31, Rockland) at 1:8000 dilution.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Nuclear Argonaute:miRNA complexes recognize target sequences within chromatin-associated RNA and silence gene expression

doi: 10.1093/nar/gkaf800

Figure Lengend Snippet: HMGA2 RNA is bound by AGO2 in all compartments of the cell. Representative IGV browser images of chimeric eCLIP reads within the 3′-UTR of HMGA2 in the ( A ) cytoplasm, ( B ) nucleus, and ( C ) chromatin. HMGA2 is located on chromosome 12 (chr12). Peak height is defined as read density in reads per million (RPM). All AGO2 peaks are reproducible and meet the following criteria: log2FC > 3 (IP versus input) at least three reads per peak.

Article Snippet: Western blot was visualized using anti-AGO2 primary antibody (50683-RP02, SinoBiological) at a 1:4000 dilution, with TrueBlot anti-rabbit secondary antibody (18-8816-31, Rockland) at 1:8000 dilution.

Techniques:

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

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