Journal: Molecular Oncology

Article Title: Circular RNA circ‐TNRC6B inhibits the proliferation and invasion of esophageal squamous cell carcinoma cells by regulating the miR ‐452‐5p/ DAG1 axis

doi: 10.1002/1878-0261.13432

Figure Lengend Snippet: circ‐TNRC6B serves as a miR‐452‐5p sponge in ESCC cells. (A) CircRNA‐miRNA binding circle diagram showing potential miRNA targets of circ‐TNRC6B predicted by Arraystar's home‐made miRNA target prediction software based on TargetScan and miRanda. (B) Venn diagram showing the intersection of potential miRNA targets of circ‐TNRC6B based on Arraystar's home‐made miRNA target prediction software, ENCORI, and circBank databases. (C) The complementary sequences of circ‐TNRC6B and miR‐452‐5p were predicted by Arraystar's home‐made miRNA target prediction software. (D) The MFE secondary structure of circ‐TNRC6B predicted by RNAfold web server and the part where the secondary structure of circ‐TNRC6B is relevant for the binding of miR‐452‐5p. (E) FISH assay indicated circ‐TNRC6B and miR‐452‐5p were co‐localized in the cytoplasm of KYSE150 cells ( n = 3). Scale bar: 20 μm. (F) RIP analysis in KYSE150 cells co‐transfected with Myc‐AGO2 vector and miR‐452‐5p mimics. Data are represented as mean ± SD ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). (G) RIP analysis in KYSE150 cells co‐transfected with Myc‐AGO2 vector and NC ( n = 2, P ‐values were determined by Mann–Whitney U ‐test). Data are represented as mean ± SD. (H) Dual‐luciferase reporter gene analysis detected the luciferase activity of circ‐TNRC6B when co‐transfected with luc‐empty vector or wild‐type luc‐circ‐TNRC6B vector and NC or miR‐452‐5p mimics in KYSE150 cells. Data are represented as mean ± SD ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). (I) Dual‐luciferase reporter gene analysis detected the luciferase activity of circ‐TNRC6B when co‐transfected with mutant luc‐circ‐TNRC6B vector and NC or miR‐452‐5p mimics in KYSE150 cells. Data are represented as mean ± SD ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). (J) TE1 cells were transfected with pLC5 empty vector or circ‐TNRC6B vector; then, the relative expression of circ‐TNRC6B and miR‐452‐5p were detected by qRT‐PCR assay. Data are represented as mean ± SD ( n = 2, P ‐values were determined by Mann–Whitney U ‐test). (K) KYSE150 cells were transfected with si‐NC or si‐circ‐TNRC6B; then, the relative expression of circ‐TNRC6B and miR‐452‐5p was detected by qRT‐PCR assay. Data are represented as mean ± SD ( n = 2, P ‐values were determined by Mann–Whitney U ‐test). * P < 0.05, ** P < 0.01.

Article Snippet: The results were compared with the miRNAs predicted by home‐made miRNA target prediction software from Arraystar. miR‐452‐5p was selected as a candidate miRNA for further analysis (Fig. ).

Techniques: Binding Assay, Software, Transfection, Plasmid Preparation, MANN-WHITNEY, Luciferase, Activity Assay, Mutagenesis, Expressing, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: Circular RNA circ‐TNRC6B inhibits the proliferation and invasion of esophageal squamous cell carcinoma cells by regulating the miR ‐452‐5p/ DAG1 axis

doi: 10.1002/1878-0261.13432

Figure Lengend Snippet: miR‐452‐5p promotes the proliferation, migration, and invasion abilities of ESCC cells. (A) The relative expression level of miR‐452‐5p in four ESCC cell lines was examined by qRT‐PCR ( n = 2). Data are represented as mean ± SD. (B, C) The proliferative and clonogenic ability of TE1 cells after exogenous miR‐452‐5p mimics transfection was detected by CCK‐8 ( n = 4, P ‐values were determined by two‐way ANOVA) and colony formation assay ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). NC: negative control of miRNA mimics. Data are represented as mean ± SD. (D, E) The migration ability of TE1 cells after exogenous miR‐452‐5p mimics transfection was detected by wound healing and transwell migration assay. Data are represented as mean ± SD ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). Scale bar: 100 μm. (F) The invasion ability of TE1 cells after exogenous miR‐452‐5p mimics transfection was detected by transwell invasion assay. Data are represented as mean ± SD ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). Scale bar: 100 μm. (G, H) The proliferative and clonogenic ability of KYSE170 cells after miR‐452‐5p inhibitor transfection was detected by CCK‐8 ( n = 4, P ‐values were determined by two‐way ANOVA) and colony formation assay ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). Inhibitor NC: negative control of miRNA inhibitor. Data are represented as mean ± SD. (I, J) The migration ability of KYSE170 cells after miR‐452‐5p inhibitor transfection was detected by wound‐healing and transwell migration assay. Data are represented as mean ± SD ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). Scale bar: 100 μm. (K) The invasion ability of KYSE170 cells after miR‐452‐5p inhibitor transfection was detected by transwell invasion assay. Data are represented as mean ± SD ( n = 3, P ‐values were determined by Mann–Whitney U ‐test). Scale bar: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The results were compared with the miRNAs predicted by home‐made miRNA target prediction software from Arraystar. miR‐452‐5p was selected as a candidate miRNA for further analysis (Fig. ).

Techniques: Migration, Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, MANN-WHITNEY, Negative Control, Transwell Migration Assay, Transwell Invasion Assay