circ0000549 knockdown exosome group  (Exosome Diagnostics)

Journal: International Journal of Molecular Medicine

Article Title: Exosomal circ0000549 promotes MNNG-induced gastric cancer through miR-15b-5p/KIF1B

doi: 10.3892/ijmm.2026.5785

Figure Lengend Snippet: Characterization and expression of circ0000549 in TGES-1 cells and exosomes. (A) Coincidence was observed for the differentially expressed circular RNAs in the GEO database. (B) Heat map illustrating the differential expression of circRNAs, with circ0000549 standing out as highly expressed. (C) Diagram of the cyclization mechanism of circ0000549. (D) Verification through agarose gel electrophoresis showing the specificity of circ0000549 amplification from cDNA. (E) RT-qPCR results demonstrating the expression levels of circ0000549 before and after RNase R treatment, confirming its resistance to RNase R and stability compared to linear RNA. (F) The levels of circ0000549 and its parental gene SRSF5 were assessed after treatment with actinomycin D. (G) The expression of circ0000549 in TGES-1 cells and TGES-1-EX by RT-qPCR. (H) Localization of circ0000549 determined by FISH, original magnification, ×60 (oil immersion). (I) The level of circ0000549 in GES-1 cells after TGES-1-EX treatment by RT-qPCR. (J) Serum levels of N-nitrosoproline were determined by LC-MS/MS. (K) The mRNA expression of circ0000549 in serum exosomes, n=26. (L) Diagnostic efficacy of serum exosome circ0000549, n=40. (M) Protein levels of EMT and stemness markers in GES-1 cells after treatment with serum exosomes. (N) The migration ability of GES-1 cells after treatment with serum exosomes, scale bar, 200 μ m. (O) The mRNA expression of EMT and stemness markers in GES-1 cells after treatment with serum exosomes. ** P<0.01, *** P<0.001, **** P<0.0001. GEO, Gene Expression Omnibus; RT-qPCR, reverse transcription-quantitative PCR; EMT, epithelial-mesenchymal transition; GC, gastric cancer; FISH, fluorescence in situ hybridization; Serum ex, serum derived exosomes from patients with gastric cancer; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: Analysis of tumor tissues revealed that circ0000549 expression was markedly decreased in the circ0000549 knockdown exosome group ( ).

Techniques: Expressing, Quantitative Proteomics, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy, Diagnostic Assay, Migration, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Exosomal circ0000549 promotes MNNG-induced gastric cancer through miR-15b-5p/KIF1B

doi: 10.3892/ijmm.2026.5785

Figure Lengend Snippet: Circ0000549 promoted migration, proliferation, invasion and EMT in GES-1 cells. (A) The overexpression of circ0000549 in TGES-1 cells transfection with si1-circ0000549 and si2-circ0000549 was detected by RT-qPCR. (B) The effect of knocking down circ0000549 on the proliferation of TGES-1 cells was determined through a CCK-8 assay. (C) The effect of knocking down circ0000549 on the proliferative capacity of GES-1 cells was observed. (D) The abilities of TGES-1 cells to migrate and invade were evaluated, scale bar, 200 μ m. (E) The protein levels of EMT-related and stemness were analyzed after knocking down circ0000549. (F) The mRNAs expression of EMT-related and stemness markers. (G) The level of circ0000549 in TGES-1 cells was measured 48 h post-transfection with a lentivirus designed to overexpress circ0000549. (H) The expression of circ0000549 after puromycin screening. (I) Assessed the proliferative capacity of TGES-1 cells following overexpression of circ0000549. (J) Changes in the clonogenic ability of TGES-1 cells after overexpression of circ0000549. (K) The effect of overexpression of circ0000549 on the migration and invasion abilities in TGES-1 cells, scale bar, 200 μ m. (L) Changes in the levels of EMT-related and stemness protein after overexpression of circ0000549. (M) The expression of EMT-related and stemness mRNA in TGES-1 cells after overexpression of circ0000549. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. EMT, epithelial-mesenchymal transition; RT-qPCR, reverse transcription-quantitative PCR; si1, siRNA1; si2, siRNA2; siNC, siRNA negative control; LV-OE-NC, lentivirus overexpression negative control; LV-OE, lentivirus overexpression; MOI, multiplicity of infection.

Article Snippet: Analysis of tumor tissues revealed that circ0000549 expression was markedly decreased in the circ0000549 knockdown exosome group ( ).

Techniques: Migration, Over Expression, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Infection

Journal: International Journal of Molecular Medicine

Article Title: Exosomal circ0000549 promotes MNNG-induced gastric cancer through miR-15b-5p/KIF1B

doi: 10.3892/ijmm.2026.5785

Figure Lengend Snippet: Exosomal circ0000549 promotes the proliferation, migration, invasion, EMT and stemness in GES-1 cells. (A) Measure the level of circ0000549 in TGES-1 cells 48 h post-transfection with a lentivirus designed for circ0000549 knockdown. (B) The level of circ0000549 after puromycin screening. (C) The knockdown/overexpression of exosomes was detected by TEM, scale bar, 100 nm. (D) The size of exosomes was determined using NTA. (E) The protein levels of surface markers of exosomes were detected. (F) The levels of circ0000549 in TGES-1-EX with knockdown/overexpression of circ0000549. (G) The level of circ0000549 in GES-1 cells after SH-circ0000549-EX/OE-circ0000549-EX treatment. (H) Assessment of GES-1 cell proliferation post-treatment with SH-circ0000549-EX/OE-circ0000549-EX. (I) Evaluation of the clonality of GES-1 cells after SH-circ0000549-EX/OE-circ0000549-EX treatment. (J) Examination of the migratory and invasive capabilities of GES-1 cells following SH-circ0000549-EX/OE-circ0000549-EX treatment, scale bar, 200 μ m. (K) Determination of EMT and stemness protein expression levels in GES-1 cells treated SH-circ0000549-EX/OE-circ0000549-EX. (L) Quantification of EMT and stemness mRNA levels in GES-1 cells after SH-circ0000549-EX/OE-circ0000549-EX treatment using RT-qPCR. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; EX, exosome; OE-circ549-EX, exosomes from TGES-1 cells treated with circ0000549 lentivirus overexpression; OE-NC-EX, exosomes from TGES-1 cells treated with circ0000549 lentivirus overexpression negative control; SH-circ0000549-EX, exosomes from TGES-1 cells treated with circ0000549 lentivirus knockdown; SH-NC-EX; exosomes from TGES-1 cells treated with circ0000549 lentivirus knockdown negative control; MOI, multiplicity of infection.

Article Snippet: Analysis of tumor tissues revealed that circ0000549 expression was markedly decreased in the circ0000549 knockdown exosome group ( ).

Techniques: Migration, Transfection, Knockdown, Over Expression, Expressing, Quantitative RT-PCR, Transmission Assay, Electron Microscopy, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Infection

Journal: International Journal of Molecular Medicine

Article Title: Exosomal circ0000549 promotes MNNG-induced gastric cancer through miR-15b-5p/KIF1B

doi: 10.3892/ijmm.2026.5785

Figure Lengend Snippet: miR-15b-5p is the target of circ0000549 and weakens the protumor effect of circ0000549. (A) circInteractome database showed that AGO2 had binding ability with the flanking region of circ0000549. (B) The online database predicted the intersection of miRNA bound by circ0000549. (C) The expression of miRNA after knocking down circ0000549 by RT-qPCR. (D) The level of miRNA after overexpression of circ0000549 by RT-qPCR. (E) The expression of miR-15b-5p was assessed. (F) A dual luciferase reporter gene assay was conducted to investigate the interaction between circ0000549 and miR-15b-5p. (G) The level of miR-15b-5p expression in TGES-1 cells was assessed following transfection with miR-15b-5p mimics or an inhibitor. (H) The level of circ0000549 was assessed following transfection with miR-15b-5p mimics or an inhibitor. (I) The proliferation of cells was evaluated after co-transfecting them with OE-circ0000549 and miR-15b-5p mimics, or with si-circ0000549 and miR-15b-5p inhibitor. (J) Evaluation of the clonality of TGES-1 cells after co-transfection of OE-circ0000549 and miR-15b-5p mimics/si-circ0000549 and miR-15b-5p inhibitor. (K) The migration and invasion capabilities of TGES-1 cells were assessed following co-transfection with OE-circ0000549 and miR-15b-5p mimics, or with si-circ0000549 and miR-15b-5p inhibitor, scale bar, 200 μ m. (L) Level of miR-15b-5p following SH-circ0000549-EX/OE-circ0000549-EX treatment by RT-qPCR. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; si1, siRNA1; si2, siRNA2; siNC, siRNA negative control; LV-OE-NC, lentivirus overexpression negative control; LV-OE, lentivirus overexpression; WT, Wild Type; MUT, Mutant; miR-NC, miR-15b-5p mimics negative control; inhibitor Ctrl, miR-15b-5p inhibitor control; OE-circ, lentivirus overexpression of circ0000549; miR mimics, miR-15b-5p mimics; miR-inhibitor, miR-15b-5p inhibitor.

Article Snippet: Analysis of tumor tissues revealed that circ0000549 expression was markedly decreased in the circ0000549 knockdown exosome group ( ).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Over Expression, Luciferase, Reporter Gene Assay, Transfection, Cotransfection, Migration, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Mutagenesis, Control

Journal: International Journal of Molecular Medicine

Article Title: Exosomal circ0000549 promotes MNNG-induced gastric cancer through miR-15b-5p/KIF1B

doi: 10.3892/ijmm.2026.5785

Figure Lengend Snippet: Circ0000549 in exosomes aids in promoting the aggressive evolution of GES-1 cells, mainly through miR-15b-5p/KIF1B/PI3K/AKT axis. (A) Internet databases were used to predict which genes are regulated by miR-15b-5p. (B) The levels of candidate target genes after knockdown/overexpression of circ0000549 or transfection of miR-15b-5p mimics/inhibitor by RT-qPCR. (C) mRNA level of KIF1B was detected. (D) The effect of co transfection with OE circ0000549 and miR-15b-5p mimic/si circ00000549 and miR-15b-5p inhibitor on the expression of KIF1B. (E) A dual luciferase reporter gene assay was employed to elucidate the interaction between KIF1B and miR-15b-5p. (F) GeneCards database was used to predict KIF1B downstream pathways online. (G) The pathway activity of the PI3K/AKT in TGES-1 cells after knocking down/overexpressing circ0000549. (H) Changes in expression levels of KIF1B and PI3K/AKT pathway proteins of GES-1 cells after SH-circ0000549-EX/OE-circ0000549-EX treatment. (I) The effect on KIF1B level in TGES-1 cells subsequent to KIF1B knockdown. (J) The level of KIF1B in TGES-1 cells which was knocked down KIF1B after treatment with OE-circ0000549 exosomes by RT-qPCR. (K) Detection of changes in expression levels of KIF1B and PI3K/AKT pathway proteins in TGES-1 cells which was knocked down KIF1B after treatment with OE-circ0000549 exosomes. (L) Detection of changes in the proliferation ability of TGES-1 cells. (M) Clonality of TGES-1 cells which was knocked down KIF1B after treatment with OE-circ0000549 exosomes. (N) Alterations in the migratory and invasive capabilities of TGES-1 cells treated with OE-circ0000549 exosomes and KIF1B knockdown in combination. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; LV-OE-NC, lentivirus overexpression negative control; LV-OE, lentivirus overexpression; LV-SH-circ0000549, lentivirus knockdown of circ0000549; LV-SH-NC; lentivirus knockdown of circ0000549 negative control; miR-NC, miR-15b-5p mimics negative control; inhibitor Ctrl, miR-15b-5p inhibitor control; OE-circ549, lentivirus overexpression of circ0000549; OE-circ549-EX, exosomes from TGES-1 cells treated with circ0000549 lentivirus overexpression; siKIF1B, siRNA of KIF1B; siNC, siRNA negative control; SH-circ549-EX, exosomes from TGES-1 cells treated with circ0000549 lentivirus knockdown; SH-NC-EX; exosomes from TGES-1 cells treated with circ0000549 lentivirus knockdown negative control.

Article Snippet: Analysis of tumor tissues revealed that circ0000549 expression was markedly decreased in the circ0000549 knockdown exosome group ( ).

Techniques: Knockdown, Over Expression, Transfection, Quantitative RT-PCR, Cotransfection, Expressing, Luciferase, Reporter Gene Assay, Activity Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Control

Journal: International Journal of Molecular Medicine

Article Title: Exosomal circ0000549 promotes MNNG-induced gastric cancer through miR-15b-5p/KIF1B

doi: 10.3892/ijmm.2026.5785

Figure Lengend Snippet: Exosomal circ0000549 tumorigenicity, EMT, proliferation and stemness. (A) The downregulation of circ0000549 impairs the tumorigenic capacity of TGES-1 cells. (B) The effect of SH-circ0000549-EX treatment on tumor weight. (C) The effect of SH-circ0000549-EX treatment on tumor volume. (D) The level of circ0000549 in tumor tissues after SH-circ0000549-EX treatment by RT-qPCR. (E) The levels of stemness, EMT and proliferation proteins in tumor tissues after SH-circ0000549-EX treatment. (F) The mRNA expression of stemness, EMT and proliferation makers mRNA in tumor tissues after SH-circ0000549-EX treatment. (G) Immunohistochemical analysis of tumor tissues treated with SH-circ0000549-EX revealed alterations in the levels of EMT and stemness markers, magnification, ×400. (H) The effect of OE-circ0000549-EX alone or in combination with miR-15b-5p mimic on the tumorigenicity of TGES-1 cells. (I) The effect of OE-circ0000549-EX alone or in combination with miR-15b-5p mimic on tumor weight and volume. (J) The levels of stemness, EMT and proliferation-related markers in tumor tissues following treatment with OE-circ0000549-EX alone or in combination with miR-15b-5p mimic. (K) The expression of stemness, EMT and proliferation mRNA in tumor tissues after treated with OE-circ0000549-EX alone or in combination with miR-15b-5p mimic. (L) The levels of vimentin and Nanog in tumor tissues after SH-circ0000549-EX treatment by Immunohistochemistry, magnification, ×200. * P<0.05, ** P<0.01, *** P<0.001, # P<0.05, ## P<0.01. EMT, epithelial-mesenchymal transition; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; SH-circ549-EX, exosomes from TGES-1 cells treated with circ0000549 lentivirus knockdown; SH-NC-EX; exosomes from TGES-1 cells treated with circ0000549 lentivirus knockdown negative control; OE-circ549-EX, exosomes from TGES-1 cells treated with circ0000549 lentivirus overexpression.

Article Snippet: Analysis of tumor tissues revealed that circ0000549 expression was markedly decreased in the circ0000549 knockdown exosome group ( ).

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemical staining, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Knockdown, Over Expression