Journal: Molecular Cancer

Article Title: miR-19a promotes colorectal cancer proliferation and migration by targeting TIA1

doi: 10.1186/s12943-017-0625-8

Figure Lengend Snippet: Identification of TIA1 as a miR-19a target. a Schematic description of the hypothetical duplex formed by the interactions between the binding site in the TIA1 3’-UTR and miR-19a. The miR-19a seed sequence and the seed sequence binding sites in the TIA1 3’-UTR are indicated in red . All nucleotides of the seed sequence of the binding site are conserved in several species, including human, mouse, rat and rabbit. The predicted free energy values of the hybrids are indicated. b Quantitative RT-PCR analysis of miR-19a expression levels in the same 16 pairs of CRC and normal tissue samples. c Pearson’s correlation scatter plot of the fold changes of miR-19a and TIA1 protein in human CRC tissue pairs. *** P < 0.001

Article Snippet: For miRNA pull-down, SW480 cells which were transfected with biotinylated miR-19a (miR-19a probe) or control probe (Genescript, Nanjing, China) were harvested in lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40 and 1 U/ul Recombinant RNAse inhibitor (TaKaRa)), and the total RNA was pretreated with DNaseI and then heated at 65 °C for 5 min, followed by an instant ice bath.

Techniques: Binding Assay, Sequencing, Quantitative RT-PCR, Expressing

Journal: Molecular Cancer

Article Title: miR-19a promotes colorectal cancer proliferation and migration by targeting TIA1

doi: 10.1186/s12943-017-0625-8

Figure Lengend Snippet: miR-19a directly regulates TIA1 expression at the post-transcriptional level. a Quantitative RT-PCR analysis of miR-19 levels in SW480, Caco2 and HT-29 cells. miR-19 level of SW480 was set as control. b Western blot analysis of TIA1 protein levels in SW480, Caco2 and HT-29 cells. TIA1 level of SW480 was set as control. c Pearson’s correlation scatter plot of the level of miR-19 and TIA1 protein in SW480, Caco2 and HT-29 cells. d Quantitative RT-PCR analysis of miR-19a levels in SW480, Caco2 and HT-29 cells transfected with control mimic, miR-19a mimic, control inhibitor or miR-19a inhibitor. e and f Western blot analysis of TIA1 protein levels in SW480, Caco2 and HT-29 cells transfected with control mimic, miR-19a mimic, control inhibitor or miR-19a inhibitor. e : representative images; f : quantitative analysis. g Quantitative RT-PCR analysis of TIA1 and GAPDH mRNA levels in SW480 after pulling down with control probe or miR-19a probe; h Quantitative RT-PCR analysis of miR-19a and miR-22 levels in SW480 after pulling down with control probes or two TIA1 mRNA probes; i The relative luciferase activities in SW480 transfected with wild type or mutant TIA1 3’-UTR. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: For miRNA pull-down, SW480 cells which were transfected with biotinylated miR-19a (miR-19a probe) or control probe (Genescript, Nanjing, China) were harvested in lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40 and 1 U/ul Recombinant RNAse inhibitor (TaKaRa)), and the total RNA was pretreated with DNaseI and then heated at 65 °C for 5 min, followed by an instant ice bath.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Transfection, Luciferase, Mutagenesis

Journal: Molecular Cancer

Article Title: miR-19a promotes colorectal cancer proliferation and migration by targeting TIA1

doi: 10.1186/s12943-017-0625-8

Figure Lengend Snippet: c-MYC, miR-19a, TIA1 and PDCD4 form a regulatory axis in CRC. a and b Western blot analysis of c-Myc and TIA1 protein levels in SW480 cells transfected with control siRNA, si-c-Myc, si-c-Myc plus miR-19a mimic, control plasmid, c-Myc plasmid, or c-Myc plasmid plus miR-19a inhibitor. a : representative images; b : quantitative analysis. c Quantitative RT-PCR analysis of miR-19a levels in SW480 cells transfected with control siRNA, si-c-Myc, si-c-Myc plus miR-19a mimic, control plasmid, c-Myc plasmid, or c-Myc plasmid plus miR-19a inhibitor. d and e Western blot analysis of TIA1 and PDCD4 protein levels in SW480 cells transfected with control mimic, miR-19a mimic, miR-19a mimic plus TIA1 plasmid, control inhibitor, miR-19a inhibitor, or miR-19a inhibitor plus si-TIA1. d : representative images; e : quantitative analysis. f Quantitative RT-PCR analysis of PDCD4 mRNA levels in SW480 cells transfected with control mimic, miR-19a mimic, miR-19a mimic plus TIA1 plasmid, control inhibitor, miR-19a inhibitor, or miR-19a inhibitor plus si-TIA1. ND: Non-Detectable. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: For miRNA pull-down, SW480 cells which were transfected with biotinylated miR-19a (miR-19a probe) or control probe (Genescript, Nanjing, China) were harvested in lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40 and 1 U/ul Recombinant RNAse inhibitor (TaKaRa)), and the total RNA was pretreated with DNaseI and then heated at 65 °C for 5 min, followed by an instant ice bath.

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: miR-19a promotes colorectal cancer proliferation and migration by targeting TIA1

doi: 10.1186/s12943-017-0625-8

Figure Lengend Snippet: miR-19a promotes CRC cell proliferation by targeting TIA1. a Cell proliferation assays (CCK-8) were performed 12, 24, 36, 48 and 60 h after the transfection of SW480 cells with equal doses of control mimic, miR-19a mimic, control inhibitor or miR-19a inhibitor. b and c Cell proliferation assays (EdU) were performed in SW480 cells transfected with equal doses of control mimic, miR-19a mimic, control inhibitor or miR-19a inhibitor. b : representative images; c : quantitative analysis. d Cell proliferation assays (CCK-8) were performed 12, 24, 36 and 48 and 60 h after transfection of SW480 cells with equal doses of control mimic plus control plasmid, miR-19a mimic plus control plasmid, control mimic plus TIA1 overexpression plasmid, or miR-19a mimic plus TIA1 overexpression plasmid. e and f Cell proliferation assays (EdU) were performed in SW480 cells transfected with equal doses of control mimic plus control plasmid, miR-19a mimic plus control plasmid, control mimic plus TIA1 overexpression plasmid, or miR-19a mimic plus TIA1 overexpression plasmid. e : representative images; f : quantitative analysis. ** P < 0.01; *** P < 0.001

Article Snippet: For miRNA pull-down, SW480 cells which were transfected with biotinylated miR-19a (miR-19a probe) or control probe (Genescript, Nanjing, China) were harvested in lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40 and 1 U/ul Recombinant RNAse inhibitor (TaKaRa)), and the total RNA was pretreated with DNaseI and then heated at 65 °C for 5 min, followed by an instant ice bath.

Techniques: CCK-8 Assay, Transfection, Control, Plasmid Preparation, Over Expression

Journal: Molecular Cancer

Article Title: miR-19a promotes colorectal cancer proliferation and migration by targeting TIA1

doi: 10.1186/s12943-017-0625-8

Figure Lengend Snippet: miR-19a promotes CRC cell migration by targeting TIA1. a – c Cell migration assays (Transwell) were performed in SW480 cells that were transfected with equal doses of control mimic, miR-19a mimic, control inhibitor or miR-19a inhibitor (upper panel), or with equal doses of control mimic plus control plasmid, miR-19a mimic plus control plasmid, control mimic plus TIA1 overexpression plasmid, or miR-19a mimic plus TIA1 overexpression plasmid (lower panel). a : representative images; b and c : quantitative analysis of the number of migrated cells. * P < 0.05; ** P < 0.01

Article Snippet: For miRNA pull-down, SW480 cells which were transfected with biotinylated miR-19a (miR-19a probe) or control probe (Genescript, Nanjing, China) were harvested in lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40 and 1 U/ul Recombinant RNAse inhibitor (TaKaRa)), and the total RNA was pretreated with DNaseI and then heated at 65 °C for 5 min, followed by an instant ice bath.

Techniques: Migration, Transfection, Control, Plasmid Preparation, Over Expression

Journal: Molecular Cancer

Article Title: miR-19a promotes colorectal cancer proliferation and migration by targeting TIA1

doi: 10.1186/s12943-017-0625-8

Figure Lengend Snippet: miR-19a promotes CRC growth in vitro and in vivo by targeting TIA1. a and b Colony formation assays of SW480 cells infected with miR-19a lentivirus, transfected with TIA1 vector or both. a : Representative images of colony formation assays; b : quantitative analysis of the numbers of colonies. c Representative images of tumors from the implanted mice. d and e Quantitative analysis of xenografted tumor volume and weight. d : tumor volume; e : tumor weight. f – h H&E-stained sections and immunohistochemical staining for TIA1 and Ki-67 in tumors from implanted mice. f : representative images; g and h : quantitative analysis. i Working model of the c-Myc-miR-19a-TIA1-PDCD4 regulatory axis in CRC. ** P < 0.01; *** P < 0.001

Article Snippet: For miRNA pull-down, SW480 cells which were transfected with biotinylated miR-19a (miR-19a probe) or control probe (Genescript, Nanjing, China) were harvested in lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40 and 1 U/ul Recombinant RNAse inhibitor (TaKaRa)), and the total RNA was pretreated with DNaseI and then heated at 65 °C for 5 min, followed by an instant ice bath.

Techniques: In Vitro, In Vivo, Infection, Transfection, Plasmid Preparation, Staining, Immunohistochemical staining