https://orcid.org/0000-0002-8875-0484
Figures & data
Figure 1. Circ_0000228 was remarkably up-modulated in CC tissues and cell lines
(a) A volcano plot was employed to analyze the differentially expressed circRNAs in the GEO dataset (GSE113696) that were remarkably up- and down-modulated in CC cell lines, compared with normal cervical epithelial cells; (b) A heat maps was utilized to present the significantly highly expressed circRNAs in CC; (c) Circ_0000228 expression in 40 CC tissues/paired adjacent cervical tissues was examined by qRT-PCR; (d) Circ_0000228 expression in 21 patients with stage I CC and 19 patients with stage II CC was analyzed by qRT-PCR, respectively; (e) ZEB1 mRNA expression in 21 patients with stage I CC and 19 patients with stage II CC was analyzed by qRT-PCR, respectively; (f) Circ_0000228 expression in HUCEC and CC cell lines (HeLa, HCC94, SW756, C33A) was detected by qRT-PCR; (g) Circ_0000228 expression in the nucleus and cytoplasm of HCC94 and C33A cells was detected by qRT-PCR. All of the experiments were performed in triplicate. ***P < 0.001.
Figure 2. Knockdown of circ_0000228 remarkably impeded CC cell multiplication, migration, and invasion
(a) qRT-PCR was executed to detect circ_0000228 and ZEB1 mRNA expression in CC cells transfected with circ_0000228 siRNA; (b) CCK-8 experiment was conducted to detect the multiplication of CC cells after transfection with circ_0000228 siRNA; (c) Transwell experiment was employed to detect migration and invasion of CC cells after transfection with circ_0000228 siRNA. All of the experiments were performed in triplicate. **P < 0.01 and ***P < 0.001.
Figure 3. Circ_0000228 modulated LOXL2 expression through miR-195-5p
(a) A volcano plot was adopted to show the miRNAs that were remarkably up- and down-modulated in CC tissues compared with normal cervical tissue in the GEO dataset (GSE86100); (b) The Venn diagram was used to screen out the differentially expressed miRNAs in GSE86100, which contained complementary binding sites with circ_0000228; (c) Bioinformatics analysis indicated that high LOXL2 expression suggested poor clinical outcome of CC patients. (d) The complementary binding sites of circ_0000228 and miR-195-5p, miR-195-5p and LOXL2 3ʹUTR are presented, and the corresponding luciferase reporter vectors were designed; (E-F) Dual-luciferase reporter gene experiments confirmed that the binding sites between circ_0000228 and miR-195-5p, miR-195-5p and LOXL2 3ʹUTR were functional. (g) MiR-195-5p expression in 40 pairs of CC tissues/adjacent cervical tissues was detected by qRT-PCR; (h) MiR-195-5p expression in stage I and stage II CC patients was detected by qRT-PCR, respectively; (I) MiR-195-5p in HUCEC and CC cell lines (HeLa, HCC94, SW756, C33A) was detected by qRT-PCR.(j) LOXL2 mRNA expression in 40 pairs of CC tissues/adjacent cervical tissues was detected by qRT-PCR; (k) LOXL2 mRNA expression in HUCEC and CC cell lines (HeLa, HCC94, SW756, C33A) was detected by qRT-PCR; (L) LOXL2 mRNA expression in stage 1 and stage 2 CC patients was detected by qRT-PCR; (m-o) Pearson’s correlation analysis of the correlation relationships among circ_0000228 miR-195-5p, and LOXL2 expression in CC tissues; (p) qRT-PCR was employed to detect miR-195-5p expression after co-transfection with circ_0000228 siRNA and miR-195-5pin in CC cell lines; (q) qRT-PCR and Western blot experiments were employed to detect LOXL2 expression after co-transfection with circ_0000228 siRNA and miR-195-5p inhibitors in CC cell lines; All of the experiments were performed in triplicate. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 4. LOXL2 overexpression partially counteracted the inhibitory effect of knockdown circ_0000228 on CC cell multiplication, migration and invasion
(a,b) CCK-8 experiment was utilized to detect the multiplication of CC cells after the CC cells were co-transfected with circ_0000228 siRNA and LOXL2 overexpression plasmid. (c,d) Transwell experiment was executed to detect cell migration and invasion after the CC cells were co-transfected with circ_0000228 siRNA and LOXL2 overexpression plasmid.*P < 0.05 and **P < 0.01.
