a549 cells  (Aviva Systems)

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: Validation of ITGB3 as a direct target of miR-140-3p (A) Predicted binding sites of ITGB3 3’-UTR and miR-140-3p using an online tool TargetScan. The base pairing between the ITGB3 3’UTR binding site and the seed region of miR-140-3p is showed by vertical lines (top). The binding site of miR-140-3p and the mutated form of ITGB3 3’-UTR includes 3 nucleotides (bottom). (B) Relative luciferase activity levels following 24 hours transfection of A549 cells with the Luciferase reporter vector containing the backbone vector only, ITGB3 3’-UTR (ITGB3wt) and the mutated form of the 3’-UTR (ITGB3mut), respectively. Data are presented as fold change of the mean + SEM, three individual experiments were undertaken in triplicate, ANOVA-test was used to assess significance with * p

Article Snippet: The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: Change of miR expression and invasion properties in lung cancer cells after transfection with miR-140-3p mimic (A) Expression of miR-140-3p in A549 cells. (B) Expression of miR-140-3p in SK-MES-1 cells. (C) Transwell invasion property of A549 cells with response to miR-130-3p mimic. (D) Transwell invasion property of SK-MES-1 cells with response to miR-130-3p mimic. (E) Transwell migration property of A549 cells with response to miR-130-3p mimic. (F) Transwell migration property of SK-MES-1 cells with response to miR-130-3p mimic. Data are presented as mean + SEM, three individual experiments were undertaken in triplicate, Student's t -test was used to assess significance.

Article Snippet: The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Expressing, Transfection, Migration

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: The two strands of miR-140 have minor impact upon the metabolite profile of lung cancer cells Lung cancer cells (A549 and SK-MES-1) and BEAS-2B lung epithelial cells were treated with miRNA mimics for 24 hours before cell samples were collected for metabolite analysis using Mass spectrometry (GC-MS). (A) A principle component analysis of the metabolite data set for the three cell lines without treatment (NEG) and with either miRNA mimic. (B) Example GCMS chromatogram of m/z 174 representing glycine levels in the control (NEG, black line) and after treatment with miR-140-3P (red line) and miR-140-5P (blue line). (C) Normalized peak area of metabolites which differed in level in A549 cells dependent on treatment. (D) Normalized peak area of metabolites which differed in level dependent on the cell line studied. Data are presented as mean + SEM, experiments were undertaken in triplicate, T -test and Mann Whitney tests were used to assess significance among multiple groups with * p

Article Snippet: The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Mass Spectrometry, Gas Chromatography-Mass Spectrometry, MANN-WHITNEY

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: Tubule formation ability of the primary endothelial cells HUVECs co-cultured with TCM of lung cancer cells treated with miR-140-3p and 140-5p mimics HUVECs were seeded onto a layer of Matrigel with completed growth medium supplemented with 40% TCM from miR-treated lung cancer cells for 24 and 48 hours, respectively. The tubule formation was monitored in real-time by using the EVOS Cell Imaging System. Images were taken every 30 minutes for 6 hours. (A) Total length of the tubule structure form by HUVECs in the presence of TCM from A549. (B) Number of closed mesh structure formed by HUVECs in the presence of TCM from A549. (C) Total length of the tubule structure form by HUVECs in the presence of TCM from SK-MES-1. (D) Number of closed mesh structure formed by HUVECs in the presence of TCM from SK-MES-1. Data are presented as mean + SEM, three individual experiments were undertaken in triplicate, Kruskal-Wallis-test was used to assess significance with * p

Article Snippet: The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Cell Culture, Imaging

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: Validation of ITGB3 as a direct target of miR-140-3p (A) Predicted binding sites of ITGB3 3’-UTR and miR-140-3p using an online tool TargetScan. The base pairing between the ITGB3 3’UTR binding site and the seed region of miR-140-3p is showed by vertical lines (top). The binding site of miR-140-3p and the mutated form of ITGB3 3’-UTR includes 3 nucleotides (bottom). (B) Relative luciferase activity levels following 24 hours transfection of A549 cells with the Luciferase reporter vector containing the backbone vector only, ITGB3 3’-UTR (ITGB3wt) and the mutated form of the 3’-UTR (ITGB3mut), respectively. Data are presented as fold change of the mean + SEM, three individual experiments were undertaken in triplicate, ANOVA-test was used to assess significance with * p

Article Snippet: Enzyme linked immunosorbent assay (ELISA) The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: Change of miR expression and invasion properties in lung cancer cells after transfection with miR-140-3p mimic (A) Expression of miR-140-3p in A549 cells. (B) Expression of miR-140-3p in SK-MES-1 cells. (C) Transwell invasion property of A549 cells with response to miR-130-3p mimic. (D) Transwell invasion property of SK-MES-1 cells with response to miR-130-3p mimic. (E) Transwell migration property of A549 cells with response to miR-130-3p mimic. (F) Transwell migration property of SK-MES-1 cells with response to miR-130-3p mimic. Data are presented as mean + SEM, three individual experiments were undertaken in triplicate, Student's t -test was used to assess significance.

Article Snippet: Enzyme linked immunosorbent assay (ELISA) The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Expressing, Transfection, Migration

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: The two strands of miR-140 have minor impact upon the metabolite profile of lung cancer cells Lung cancer cells (A549 and SK-MES-1) and BEAS-2B lung epithelial cells were treated with miRNA mimics for 24 hours before cell samples were collected for metabolite analysis using Mass spectrometry (GC-MS). (A) A principle component analysis of the metabolite data set for the three cell lines without treatment (NEG) and with either miRNA mimic. (B) Example GCMS chromatogram of m/z 174 representing glycine levels in the control (NEG, black line) and after treatment with miR-140-3P (red line) and miR-140-5P (blue line). (C) Normalized peak area of metabolites which differed in level in A549 cells dependent on treatment. (D) Normalized peak area of metabolites which differed in level dependent on the cell line studied. Data are presented as mean + SEM, experiments were undertaken in triplicate, T -test and Mann Whitney tests were used to assess significance among multiple groups with * p

Article Snippet: Enzyme linked immunosorbent assay (ELISA) The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Mass Spectrometry, Gas Chromatography-Mass Spectrometry, MANN-WHITNEY

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: Tubule formation ability of the primary endothelial cells HUVECs co-cultured with TCM of lung cancer cells treated with miR-140-3p and 140-5p mimics HUVECs were seeded onto a layer of Matrigel with completed growth medium supplemented with 40% TCM from miR-treated lung cancer cells for 24 and 48 hours, respectively. The tubule formation was monitored in real-time by using the EVOS Cell Imaging System. Images were taken every 30 minutes for 6 hours. (A) Total length of the tubule structure form by HUVECs in the presence of TCM from A549. (B) Number of closed mesh structure formed by HUVECs in the presence of TCM from A549. (C) Total length of the tubule structure form by HUVECs in the presence of TCM from SK-MES-1. (D) Number of closed mesh structure formed by HUVECs in the presence of TCM from SK-MES-1. Data are presented as mean + SEM, three individual experiments were undertaken in triplicate, Kruskal-Wallis-test was used to assess significance with * p

Article Snippet: Enzyme linked immunosorbent assay (ELISA) The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: Cell Culture, Imaging

Journal: Oncotarget

Article Title: Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells

doi: 10.18632/oncotarget.26356

Figure Lengend Snippet: Distinctive gene regulation profiles and cell signalling pathways induced by two strands of miR-140 (A) Heatmap of the RNA-seq data showing the altered upregulated (red) and downregulated (green) genes in A549 treated with miR-140-3p and miR-140-5p, respectively. Global transcriptome analysis of 20816 gene targets was performed using the Ion AmpliSeq™ human gene expression panel. (B) The Venn diagram showing the differential gene expression induced by miR-140-3p and miR-140-5p, respectively, based on the RNA-Seq data. (C) Heatmap of the Kinex antibody array data showing the distinctive cell signalling pathways altered by the two strands of miR-140. The values of Z-score reflected positive or negative shifts in differential protein expression fold-changes after normalization by protein concentration and negative miRNA mimics. (D) Evaluation the expression of some key downstream effectors of miR-140-3p in A549 cells after treatments for 48 hours by Western blotting.

Article Snippet: Enzyme linked immunosorbent assay (ELISA) The human vascular endothelial growth factor A (VEGF-A) levels in the TCM of A549 cells transfected with negative and miR-140-3p mimics were quantified by using 96-well plates pre-coated with VEGF-A primary antibody by using a specific kit purchased from Aviva System Biology, San Diego, USA.

Techniques: RNA Sequencing Assay, Expressing, Ab Array, Protein Concentration, Western Blot