immortalized human gastric mucosal epithelial cell line ges-1  (Procell Inc)

Journal: Journal of Translational Medicine

Article Title: Circular RNA 0001789 sponges miR-140-3p and regulates PAK2 to promote the progression of gastric cancer

doi: 10.1186/s12967-022-03853-2

Figure Lengend Snippet: Upregulation of circ_0001789 in GC clinical samples is associated with poor prognosis. A Volcano plot showing differentially expressed circRNA expression between GC samples and normal gastric mucosa samples in the GSE83521 dataset. B Relative expression level (fragments per kilobase million) of circ_0001789 between normal mucosa tissues (negative control) and GC tissues in the GSE83521 dataset. C RT-qPCR analysis of the relative circ_0001789 expression in normal mucosa tissues of healthy controls (n = 50) and GC tumor samples (n = 70). D RT-qPCR analysis of the relative circ_0001789 expression in GC samples between patients with and without metastasis. E The overall survival rate of patients with GC exhibiting high or low circ_0001789 expression levels was analyzed by Kaplan Meier plotter. F Relative expression level of circ_0001789 in GC cell lines (AGS, HGC27, MKN-45 and MKN-74) and in normal gastric epithelial cells (GES-1). G Schematic illustration of circ_0001789, which is formed by exon2 of the RAB11FIP1 gene. H The relative levels of circ_0001789 and RAB11FIP1 mRNA in AGS and HGC27 cells at different time points (0, 6, 12, 18 and 24 h) were examined by RT-qPCR after actinomycin D treatment. I Total RNA was partially digested with RNase R (RNase R + group), while the other part of the sample was used as a control (mock group). The relative level of linear RAB11FIP1 mRNA and circ_0001789 in each sample was detected by RT-qPCR. * P < 0.05, ** P < 0.01, *** P < 0.001. circ, circular RNA; GC, gastric cancer; RT-qPCR, reverse transcription-quantitative PCR; RAB11FIP1, RAB11 family interacting protein 1

Article Snippet: GC cell lines (AGS, HGC27, MKN-45 and MKN-74), primary human umbilical vein endothelial cells (HUVECs) and the human gastric mucosal epithelial cell line (GES-1) were maintained in RPMI-1640, Ham’s F-12 K or MEM media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, penicillin/streptomycin (1:100; Sigma-Aldrich; Merck KGaA), 4 mM l -glutamine and 0.19% HEPES (Sigma-Aldrich; Merck KGaA) in a 37 °C incubator with 5% CO 2 .

Techniques: Expressing, Negative Control, Quantitative RT-PCR, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Circular RNA 0001789 sponges miR-140-3p and regulates PAK2 to promote the progression of gastric cancer

doi: 10.1186/s12967-022-03853-2

Figure Lengend Snippet: circ_0001789 participates in cell-to-cell communication via exosomes in GC cells. A A representative TEM image of exosomes in the serum samples of normal individuals and patients with GC. B The diameter of exosomes was analyzed by TEM. C Relative circ_0001789 expression level in exosome samples of healthy controls (n = 50) and patients with GC (n = 70). D Receiver operating characteristic curve analysis of the specificity and sensitivity of exosomal circ_0001789 as a diagnostic marker for GC. E Western blot detection of exosomal markers (CD63 and tumor susceptibility gene 101) in the exosome samples of GC cell lines (AGS, HGC27 and MKN-45) and normal gastric epithelial cells (GES-1). F The relative circ_0001789 levels in exosomal samples of GC cell lines (AGS, HGC27 and MKN-45) and normal gastric epithelial cells (GES-1) were detected by RT-qPCR. G Relative circ_0001789 expression level in cells and exosomal samples of MKN-45 cells transfected with empty vector or circ_0001789 expression vector. H AGS and HGC27 cells were incubated with exosomes from MKN-45 cells transfected with empty vector (vector-exo) or circ_0001789 expression vector (circ_0001789-exo) for 48 h, and the relative circ_0001789 levels were determined by RT-qPCR. I Cell Counting Kit-8 proliferation assay in AGS and HGC27 cells treated with vector-exo or circ_0001789-exo. J and K Cell migration and invasion assays in AGS and HGC27 cells treated with vector-exo or circ_0001789-exo. L Tube formation assay in human umbilical vein endothelial cells treated with vector-exo or circ_0001789-exo. M Protein levels of E-cadherin, N-cadherin, vimentin and VEGF-A in AGS and HGC27 cells treated with vector-exo or circ_0001789-exo. * P < 0.05, ** P < 0.01, *** P < 0.001. circ, circular RNA; GC, gastric cancer; TEM, transmission electron microscopy; RT-qPCR, reverse transcription-quantitative PCR

Article Snippet: GC cell lines (AGS, HGC27, MKN-45 and MKN-74), primary human umbilical vein endothelial cells (HUVECs) and the human gastric mucosal epithelial cell line (GES-1) were maintained in RPMI-1640, Ham’s F-12 K or MEM media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, penicillin/streptomycin (1:100; Sigma-Aldrich; Merck KGaA), 4 mM l -glutamine and 0.19% HEPES (Sigma-Aldrich; Merck KGaA) in a 37 °C incubator with 5% CO 2 .

Techniques: Expressing, Diagnostic Assay, Marker, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Incubation, Cell Counting, Proliferation Assay, Migration, Tube Formation Assay, Transmission Assay, Electron Microscopy, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Circular RNA 0001789 sponges miR-140-3p and regulates PAK2 to promote the progression of gastric cancer

doi: 10.1186/s12967-022-03853-2

Figure Lengend Snippet: circ_0001789 targets miR-140-3p. A Fluorescence in situ hybridization images showing the subcellular localization of circ_0001789 in AGS and HGC27 cells. Green: circ_0001789; blue: Nucleus. Scale bar, 100 μm. B ‘circBank’ and ‘circInteractome’ database searching identified hsa-miR-1246, hsa-miR-1286, hsa-miR-140-3p, hsa-miR-658 and hsa-miR-890 as potential targets of circ_0001789. C RNA pull-down assay using biotin-labeled control oligo or circ_0001789 probe. RT-qPCR analysis showed significant enrichment of miR-140-3p by the circ_0001789 probe. D Schematics of the binding site of circ_0001789 sequence to miR-140-3p, and the corresponding mutations. E Dual luciferase reporter assay in AGS and HGC27 cells using reporter containing a wild-type or mutated binding site, in the presence of miR-negative control or miR-140-3p mimic. F RNA immunoprecipitation RT-qPCR analysis using anti-argonaute 2 or IgG isotype was performed in AGS and HGC27 cells. G RT-qPCR was used to detect the miR-140-3p expression level in normal gastric mucosa samples of healthy controls (n = 50) and in GC tissues (n = 70). H Kaplan–Meier plot showing the overall survival of patients with GC exhibiting high or low expression of miR-140-3p (n = 35 in each group). I Pearson’s correlation analysis showed a negative correlation between circ_0001789 and miR-140-3p in patients with GC (r > 0.6; P < 0.01). J RT-qPCR was used to detect the expression level of miR-140-3p in GC cell lines (AGS, HGC27, MKN-45 and MKN-74) and normal gastric epithelial cells (GES-1). * P < 0.05, ** P < 0.01, *** P < 0.001. circ, circular RNA; miR, microRNA; hsa, Homo sapiens ; GC, gastric cancer; RT-qPCR, reverse transcription-quantitative PCR

Article Snippet: GC cell lines (AGS, HGC27, MKN-45 and MKN-74), primary human umbilical vein endothelial cells (HUVECs) and the human gastric mucosal epithelial cell line (GES-1) were maintained in RPMI-1640, Ham’s F-12 K or MEM media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, penicillin/streptomycin (1:100; Sigma-Aldrich; Merck KGaA), 4 mM l -glutamine and 0.19% HEPES (Sigma-Aldrich; Merck KGaA) in a 37 °C incubator with 5% CO 2 .

Techniques: Fluorescence, In Situ Hybridization, Pull Down Assay, Labeling, Control, Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Reporter Assay, Negative Control, RNA Immunoprecipitation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Circular RNA 0001789 sponges miR-140-3p and regulates PAK2 to promote the progression of gastric cancer

doi: 10.1186/s12967-022-03853-2

Figure Lengend Snippet: circ_0001789 sponges miR-140-3p to upregulate PAK2 expression. A The ‘Targetscan’, ‘miRDB’, ‘TarBase’ and ‘UP-GEPIA’ databases were employed to predict the downstream target mRNAs of miR-140-3p: ADAM10, PAK2, NFYA, CAND1, MED13 and USP31. B RT-qPCR was used to detect the candidate mRNA levels of ADAM10, PAK2, NFYA, CAND1, MED13 and USP31 in AGS and HGC27 cells transfected with miR-NC or miR-140-3p mimics. C The Cancer Genome Atlas database analysis of GC samples and normal gastric samples showed upregulation of PAK2 in GC. D RT-qPCR revealed the expression levels of PAK2 in normal gastric mucosa samples of healthy controls (n = 50) and GC tissues (n = 70). E Kaplan–Meier plot showing the overall survival of patients with GC exhibiting high or low expression of PAK2 (n = 35 in each group). F and G Pearson’s correlation analysis revealed a positive correlation between PAK2 and circ_0001789, and a negative correlation between PAK2 and miR-140-3p expression (r > 0.6, P < 0.01). H Western blot analysis of the PAK2 level in GC cell lines (AGS, HGC27, MKN-45 and MKN-74) and normal gastric epithelial cells (GES-1). I Schematic diagram of the binding site between miR-140-3p and the 3’ untranslated region of PAK2 mRNA, and the mutated binding sequence. J Dual luciferase reporter assay in AGS and HGC27 cells using a reporter containing a wild-type or a mutated binding site in the presence of miR-NC or miR-140-3p mimic. K PAK2 expression levels in different groups of AGS and HGC27 cells (sh-NC, sh- circ_0001789 or circ_0001789 + miR-140-3p inhibitor) were detected by western blotting. * P < 0.05, ** P < 0.01, *** P < 0.001. circ, circular RNA; miR, microRNA; PAK2, p21 activated kinase 2; ADAM10, a disintegrin and metalloproteinase domain-containing protein 10; NFYA, nuclear transcription factor Y subunit α; CAND1, cullin associated and neddylation dissociated 1; MED13, mediator complex subunit 13; USP31, ubiquitin specific peptidase 31; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; GC, gastric cancer

Article Snippet: GC cell lines (AGS, HGC27, MKN-45 and MKN-74), primary human umbilical vein endothelial cells (HUVECs) and the human gastric mucosal epithelial cell line (GES-1) were maintained in RPMI-1640, Ham’s F-12 K or MEM media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, penicillin/streptomycin (1:100; Sigma-Aldrich; Merck KGaA), 4 mM l -glutamine and 0.19% HEPES (Sigma-Aldrich; Merck KGaA) in a 37 °C incubator with 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Binding Assay, Sequencing, Luciferase, Reporter Assay, Ubiquitin Proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: World Journal of Gastrointestinal Oncology

Article Title: Interleukin-34 promotes the proliferation and epithelial-mesenchymal transition of gastric cancer cells

doi: 10.4251/wjgo.v14.i10.1968

Figure Lengend Snippet: Expression of interleukin-34 in gastric cancer tissues and cell lines, and construction of AGS cell lines with stable knockdown or overexpression of interleukin-34. A: Representative immunohistochemistry (IHC) staining of interleukin (IL)-34 in adjacent normal tissue, gastric cancer (GC) tissues, (scale bar = 25 μm); B: IHC staining scores were used to evaluate IL-34 expression in GC tissues and adjacent normal tissue; C: Western blotting was used to detect IL-34 protein expression in gastric normal epithelial cells (GES-1) and GC cell lines (AGS, HGC-27, and MKN-45); D: The relative densitometric analysis of protein bands was calculated; E: IL-34 mRNA expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR); F and G: Western blotting was used to verify the downregulation of IL-34 in AGS cell lines; H: qRT-PCR was used to verify the downregulation of IL-34 in AGS cell lines; I and J: Western blotting was used to verify the overexpression of IL-34 in AGS cell lines; K: qRT-PCR was used to verify the overexpression of IL-34 in AGS cell lines. b P < 0.01, c P < 0.001.

Article Snippet: The human normal gastric mucosal epithelial cell line (GES-1) and human GC cell lines (AGS, MKN-45 and HGC-27) were provided by Prof. Aman Xu (The First Affiliated Hospital of Anhui Medical University).

Techniques: Expressing, Knockdown, Over Expression, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: World Journal of Gastrointestinal Oncology

Article Title: Interleukin-34 promotes the proliferation and epithelial-mesenchymal transition of gastric cancer cells

doi: 10.4251/wjgo.v14.i10.1968

Figure Lengend Snippet: Interleukin-34 regulates the expression of epithelial-mesenchymal transition-related proteins in AGS cells. A-D: Downregulation of endogenous interleukin (IL)-34 increases E-cadherin expression, and reduces the expression of N-cadherin and vimentin in AGS cells; E-H: Upregulation of endogenous IL-34 reduces the E-cadherin expression, but increases the expression of N-cadherin and vimentin in AGS cells. Data was experiments performed in triplicate and expressed as mean ± standard deviation. b P < 0.01.

Article Snippet: The human normal gastric mucosal epithelial cell line (GES-1) and human GC cell lines (AGS, MKN-45 and HGC-27) were provided by Prof. Aman Xu (The First Affiliated Hospital of Anhui Medical University).

Techniques: Expressing, Standard Deviation