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. 2023 Apr 16;17(7):1437–1452. doi: 10.1002/1878-0261.13432

Fig. 4.

Fig. 4

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.