Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: TAF15 Stabilized LINC00665 and MTF1 Played an Oncogenic Role in Glioma Cells (A) Relative expression of LINC00665 in glioma cells treated with TAF15 overexpression (n = 3, each group; ∗p < 0.05 versus TAF15 + -NC group). (B and C) An RNA-IP assay (B) and RNA pull-down assay (C) were used to identify LINC00665 in the TAF15 complex. LINC00665 enrichment was measured using quantitative real-time PCR (n = 3, each group; ∗∗p < 0.01 versus anti-IgG group). (D) Expression level of nascent LINC00665 was measured by quantitative real-time PCR (n = 3, each group; p > 0.05 versus TAF15 + -NC group). (E) The half-life of LINC00665 in the U87 glioma cells (left) and U251 glioma cells (right) treated with TAF15 overexpression. (F) MTF1 expression levels in NBTs, LGGTs, and HGGTs are shown (∗∗p < 0.01 versus NBTs group; ## p < 0.01 versus LGGTs group). (G) MTF1 expression levels in HA, U87, and U251 cell lines are shown (n = 3, each group; ∗∗p < 0.01 versus HA group). (H) A CCK-8 assay was used to measure the effect of MTF1 on the proliferation of glioma cells. (I) The apoptotic percentages of glioma cells were detected with MTF1 upregulation or downregulation. (J) A transwell assay was used to measure the effect of MTF1 on cell migration and invasion of U87 and U251 glioma cells (n = 3, each group; ∗p < 0.05 versus MTF1 + -NC group; #p < 0.05 versus MTF1 − -NC group). Scale bars represent 200 μm.

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Expressing, Over Expression, Pull Down Assay, Real-time Polymerase Chain Reaction, CCK-8 Assay, Transwell Assay, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: LINC00665 Destabilized MTF1 mRNA by Interacting with STAU1, thus Regulating Glioma Cell Malignant Progression (A and B) Quantitative real-time PCR (A) and western blot (B) were applied to test expression levels of MTF1 (∗p < 0.05 versus LINC00665 + -NC group; # p < 0.05 versus STAU1 − -NC group; ▲ p < 0.05 versus LINC00665 + group). (C) LINC00665 and MTF1 could bind to STAU1 protein, respectively. Both relative enrichment levels of LINC00665 and MTF1 were measured using quantitative real-time PCR (n = 3, each group; ∗∗p < 0.01 versus anti-IgG group). (D) The predicted LINC00665 binding site in MTF1 (MTF1-3′ UTR-WT) and the designed mutant sequence (MTF1-3′ UTR-Mut) are shown (left). Luciferase reporter gene assays of HEK293T cells are shown (right) (n = 3, each group; ∗p < 0.05 versus MTF1-3′ UTR-WT + LINC00665-NC group). (E) Remaining MTF1 mRNA (%) at the different actinomycin D treatment times in control group, LINC00665 + -NC group, and LINC00665 + group. (F) Remaining MTF1 mRNA (%) in control group, STAU1 − -NC group, and STAU1 − group. (G–I) CCK-8 (G), flow cytometry (H), and transwell assays (I) were used to assess the proliferation, apoptosis, migration, and invasion capacity of U87 and U251 glioma cells (n = 3, each group; ∗p < 0.05 versus control group; # p < 0.05 versus LINC00665 + + MTF1 + group; ▲ p < 0.05 versus LINC00665 − + MTF1 − group). Scale bars represent 200 μm.

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Control, CCK-8 Assay, Flow Cytometry, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: GTSE1 Was Upregulated in Glioma Cells, and Knockdown of GTSE1 Inhibited Malignant Biological Behaviors (A) GTSE1 expression levels in NBTs, LGGTs, and HGGTs are shown (∗∗p < 0.01 versus NBTs group; ## p < 0.01 versus LGGTs group). (B) GTSE1 expression levels in HA, U87, and U251 cell lines are shown (n = 3, each group; ∗∗p < 0.01 versus HA group). (C) CCK-8 assay was performed to assess the effect of GTSE1 knockdown on the proliferation of glioma cells. (D) The ratio of apoptotic glioma cells were detected with GTSE1 downregulation. (E) Number of migrated and invaded glioma cells was counted to represent the effect of GTSE1 inhibition on cell migration and invasion (n = 3, each group; ∗p < 0.05 versus GTSE1 − -NC group). Scale bars represent 200 μm. (F) The expression level of GTSE1 mRNA in glioma cells treated with MTF1 variation. (G) The expression level of GTSE1 protein in glioma cells are shown (n = 3, each group; ∗p < 0.05 versus MTF1 + -NC group; # p < 0.05 versus MTF1 − -NC group). (H) The expression level of GTSE1 mRNA in glioma cells treated with YY2 variation. (I) The expression level of GTSE1 protein in glioma cells treated with YY2 variation (n = 3, each group; ∗p < 0.05 versus YY2 + -NC group; # p < 0.05 versus YY2 − -NC group).

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Knockdown, Expressing, CCK-8 Assay, Inhibition, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: MTF1 and YY2 Bound to the GTSE1 Promoter and Regulated the Expression Level of GTSE1 (A) Schematic representation of the human GTSE1 promoter region 2,000 bp upstream of the transcription start site (TSS), which was designated as +1. Putative MTF1 binding site was indicated. PCR was conducted with the resulting precipitated DNA. (B) The change of GTSE1 mRNA expression level caused by LINC00665 and MTF1 variation. (C) The change of GTSE1 protein expression level caused by LINC00665 and MTF1 variation (n = 3, each group; ∗p < 0.05 versus control group; # p < 0.05 versus LINC00665 + + MTF1 + group; ▲ p < 0.05 versus LINC00665 − + MTF1 − group). (D) YY2 bound to the promoter of GTSE1 in glioma cells. Three putative YY2 binding sites were indicated. PCR was conducted with the resulting precipitated DNA. (E) The change of GTSE1 mRNA expression level caused by LINC00665 and YY2 variation. (F) The change of GTSE1 protein expression level caused by LINC00665 and YY2 variation (n = 3, each group; ∗p < 0.05 versus control group; # p < 0.05 versus LINC00665 + + YY2 + group; ▲ p < 0.05 versus LINC00665 − + YY2 − group).

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Expressing, Binding Assay, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: In Vivo Study and the Schematic Picture of the Mechanisms in Our Study (A) Nude mice carrying tumors from respective groups are shown. The sample tumors from respective groups are shown. (B) Tumor volume was calculated every 4 days after injection, and tumors were excised after 44 days (∗p < 0.05 versus control group; # p < 0.05 versus TAF15 + group; ▲ p < 0.05 versus LINC00665 + group; ▪ p < 0.05 versus MTF1 + group; ● p < 0.05 versus YY2 + group). (C) The survival curves of nude mice injected into the right striatum. (D) The schematic picture of the mechanism in which TAF15 combined with LINC00665, thus enhancing the degradation of MTF1 and YY2 mRNA via the SMD pathway in glioma cells. Ter, translation termination codon.

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: In Vivo, Injection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: shRNA Sequences

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: shRNA, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: ChIP Primers

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Control, Binding Assay