Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Meg3 expression is induced by the DNA damage response in a p53 dependent manner. ( A ) qPCR analysis of Meg3 expression in HUVECs treated with 200 nM, 600 nM and 1 μM of doxorubicin for 12 h. ( B ) qPCR analysis of Meg3 expression in HUVECs treated with 10 μM nutlin-3 for 12, 24 and 48 h. ( C ) qPCR analysis of Meg3 expression in HUVECs treated with 100 μM palmitic acid for 12, 24 and 48 h. ( D ) qPCR analysis of Meg3 expression in HUVECs treated with doxorubicin (40 nM for 12 h) or nutlin-3 (10 μM for 12 h) with or without lentiviral knockdown of p53. ( E ) In situ hybridization of Meg3 in HUVECs treated with or without 0.2 μM doxorubicin for 12 h. The number of red dots per nucleus from 13 cells was counted for each condition. Data show mean ± S.E.M., n = 3 (A–D), n = 13 (E); * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Expressing, In Situ Hybridization

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Meg3 knockdown induces DNA damage. HUVECs were transfected with control or Meg3 GapmeRs for neutral comet assay ( A and B ) and western blot analysis ( C ). ( A ) Representative images of DNA tail length and moment under basal condition and in ECs treated with 10 ng/ml TNF-α for 16 h. ( B ) Quantification of DNA tail length and moment under basal conditions and in ECs treated with 10 ng/ml TNF-α for 16 h. ( C ) Western blot analysis of key proteins involved in the DNA damage response under basal conditions. Data show mean ± S.E.M., n ≥ 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, Neutral Comet Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Transcriptome analysis identifies p53 signaling as the most significantly upregulated pathway upon Meg3 knockdown. HUVECs were transfected with control or Meg3 GapmeRs. 24 h after transfection, cells were treated with 10 ng/ml TNF-α for 3 h and collected for microarray gene chip analysis. Chromatin immunoprecipitation was performed using transfected cells with or without 10 ng/ml TNF-α treatment for 1 h. ( A ) Volcano plot shows differentially expressed mRNAs in ECs upon Meg3 knockdown. ( B ) KEGG signaling pathways analysis identified significantly regulated pathways among upregulated genes upon Meg3 knockdown. ( C ) Venn diagram shows p53 target genes that are regulated by Meg3. ( D ) qPCR analysis of a group of p53 target genes that were induced upon Meg3 knockdown. (E) Enrichment of p53 at the promoters of indicated genes was identified by chromatin immunoprecipitation using anti-p53 antibodies followed by qPCR analysis. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, Microarray, Chromatin Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Quantitative proteomics analysis identifies p53 signaling as the most significantly regulated pathway upon Meg3 silencing. HUVECs were transfected with control or Meg3 GapmeRs. 24 h after transfection, cells were treated with or without 10 ng/ml TNF-α for 4 h. After treatment, cells were collected for TMT10-plex labeling and mass spectrometry analysis, western blot analysis, or immunoprecipitation. ( A ) Volcano plot shows differentially expressed proteins in ECs upon Meg3 silencing under TNF-α-treated condition. ( B ) KEGG signaling pathways were identified with significant enrichment among upregulated proteins upon Meg3 silencing under TNF-α-treated condition. ( C ) Venn diagram shows proteins encoded by p53 target genes that are regulated by Meg3 under TNF-α-treated condition. ( D ) Western blot analysis of p53, MDM2, and p21. ( E ) Quantifications for (D). ( F ) Co-immunoprecipitation was performed to examine the interaction between p53 and MDM2 in TNF-α-treated HUVECs. ( G ) The level of phosphorylated p53 at serine 15 was examined in immunoprecipitated total p53 in TNF-α-treated HUVECs transfected with control or Meg3 GapmeRs. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, Labeling, Mass Spectrometry, Western Blot, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Meg3 knockdown promotes apoptosis and inhibits proliferation in ECs. HUVECs were transfected with control or Meg3 GapmeRs, treated with or without 10 ng/ml TNF-α for 12 or 16 h. ( A ) Caspase 3/7 activities reflected by luminescence levels were measured, and fold changes were calculated relative to that in ECs transfected with control GapmeRs without TNF-α treatment. ( B ) The number of TUNEL positive cells per high power view were shown. TUNEL positive cells are in red color due to presence of DNA breaks, and DAPI-stained nuclei appear in blue. ( C ) The percentages of EdU positive cells (red) among Hoechst 33342 (blue) stained cells were calculated in transfected cells with or without 12 h TNF-α treatment. ( D ) Cell cycle distribution revealed by flow cytometry. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, TUNEL Assay, Staining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: LncRNA pull-down assay identifies PTBP3 as a binding partner of Meg3. ( A ) LncRNA pull-down assay was used to identify the proteins associated with Meg3 transcript variant 1 (TV1) by incubating the cell lysates with biotinylated sense or antisense Meg3 RNA. The RNA–protein complexes captured by T-1 beads were subjected to silver stain after separation on SDS-PAGE gel. ( B ) The RNA–protein complexes from lncRNA pulldown using antisense Meg3 (negative control RNA), sense Meg3 (TV1), and Meg3 deletion mutants were subjected to western blot analysis of PTBP3 and GAPDH after separation on SDS-PAGE gel. GAPDH was examined as a negative control protein. ( C ) The interaction of endogenous PTBP3 with Meg3 was detected by RNA immunoprecipitation. EC lysates were immunoprecipitated with anti-PTBP3 antibody or Isotype matched control IgG. Meg3 was examined by qPCR in the immuno-precipitates using primer set 2 as shown in (D). LncRNA Neat1 was used as a positive control RNA that interacts with PTBP3. ( D ) Different sets of Meg3 primers were used to detect Meg3 by qPCR following RNA immunoprecipitation using anti-PTBP3 antibodies. Primer sets 1, 2, 5 detect Meg3 transcript variants 1 and 6 (TV1 and TV6); primer sets 3 and 4 detect Meg3 TV1; and primer sets 6 and 7 detect Meg3 TV6. ( E ) Dual-staining of Meg3 and PTBP3 in HUVECs. Linear trajectories (yellow line) crossing the cells with the intensities of Meg3 and PTBP3 signals were presented at the right side of images. White and black arrows indicate partial colocalization of Meg3 and PTBP3 in the nucleus of HUVECs. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Pull Down Assay, Binding Assay, Variant Assay, Silver Staining, SDS Page, Negative Control, Western Blot, Immunoprecipitation, Positive Control, Staining

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: PTBP3 knockdown phenocopies Meg3′s effects on EC apoptosis and proliferation. HUVECs were transfected with control or PTBP3 siRNAs (A–E), treated with or without TNF-α for 12 or 16 h. HUVECs were transfected with control GapmeRs or Meg3 GapmeRs (F). ( A ) PTBP3 knockdown by siRNA significantly reduces PTBP3 mRNA. ( B ) qPCR analysis of a group of p53 target genes in TNF-α-treated HUVECs. ( C ) Caspase 3/7 activities reflected by luminescence levels were measured, and fold changes were calculated relative to that in ECs transfected with control siRNAs without TNF-α treatment. ( D ) The number of TUNEL positive cells per high power view were shown. TUNEL positive cells are in red color due to presence of DNA breaks, and DAPI-stained nuclei appear in blue. ( E ) The percentages of EdU positive cells (red) among Hoechst 33342 (blue) stained cells were calculated in transfected cells with or without 12 h TNF-α treatment. ( F ) Enrichment of PTBP3 at the promoters of indicated genes was identified by chromatin immunoprecipitation using anti-PTBP3 antibodies followed by qPCR analysis in TNF-α-treated HUVECs transfected with Ctl GapmeRs or Meg3 GapmeRs. ( G ) Enrichment of PTBP3 at the promoters of indicated genes was identified by chromatin immunoprecipitation using anti-PTBP3 antibodies followed by qPCR analysis in TNF-α-treated HUVECs transduced with lentivirus expressing Ctl shRNA or p53 shRNA. N.S., non-significant. ( H ) qPCR analysis of CDKN1A, MDM2 , and GADD45a promoters after chromatin isolation by RNA purification using control probes or Meg3 probes. Data show mean ± S.E.M., n = 3 or 4 (A–G) or = 2 ( H ); * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, TUNEL Assay, Staining, Chromatin Immunoprecipitation, Transduction, Expressing, shRNA, Isolation, Purification

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Schematic summary of this study. Under basal conditions, Meg3 protects DNA from damage independent of PTBP3, while it restrains the expression of p53 target genes in cooperation with PTBP3 likely through interactions with the promoters of p53 target genes. DNA damage induces Meg3 expression via a p53 dependent mechanism. Induced Meg3 is likely involved in restoring the homeostasis of DNA damage response through an unknown mechanism, as indicated by a question mark. Meg3 knockdown leads to DNA damage and ATM activation, which in turn stabilizes p53 by disrupting p53 and MDM2 interaction. Activation of p53 signaling results in the expression of p53 target genes, EC apoptosis, and decreased EC proliferation. PTBP3 knockdown phenocopies Meg3′s effects on the activation of p53 signaling, the expression of p53 target genes, EC apoptosis, and decreased EC proliferation.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Expressing, Activation Assay