PMC

A-D: Relative expression of the four indicated circRNAs from five cervical cancer cell lines and one normal cervical epithelial cells (NC) by RT-qPCR. *P<0.05 vs. NC group.

Snail-2 is a direct downstream target of miR-506. A: The putative sequences of miR-506 and Snail-2 with two binding sites. B: Luciferase reporter assay was performed to detect the potential binding between miR-506 and Snail-2. MiR-506 significantly inhibited luciferase activity of wild type reporter for Snail-2, however, miR-506 did not inhibit the luciferase activity of reporter vector containing the mutant binding sites of Snail-2 in both cell lines. C: RT-qPCR and Western blot experiments showed that miR-506 suppressed the expression level of Snail-2 at both transcript and protein levels, respectively. *P<0.05 vs. NC group.

CircRNA profile expression in cervical cancer cells. A: Box plot showed the normalized intensities from five cervical cancer cell lines and one normal cervical epithelial cells (NC). B: Volcano plot of the differentially expressed circRNAs. The vertical lines correspond to 2.0-fold up and down, respectively, and the horizontal line represents a p-value of 0.05. The red point in the plot represents the differentially expressed circRNAs with statistical significance. C: Heat map showed the selected 10 upregulated and 10 downregulated circRNAs. Red indicated the upregulated expression with high fold-change and blue indicated the downregulated expression with low fold-change.

Knockdown of circRNA-000284 suppresses cell proliferation and invasion. A: The sketch of structures of si-circRNA, si-NC vector is shown. B: CircRNA-000284 was silenced by transfection of specific silencing vectors in both cervical cell lines. C: CircRNA-000284 was knocked down by specific silencing vectors for 48 h, then, CCK8 assay was performed in cervical cell lines. D: CircRNA-000284 was knocked down by specific silencing vectors for 48 h, then, Immunofluorescence assay was performed to detect the expression change of Ki-67. Blue, DAPI; Green, Ki-67. Merge, the merge of Ki-67 and DAPI. E: CircRNA-000284 was knocked down by specific silencing vectors for 48 h, then, FACS cell-cycle assays were performed and results suggested that knockdown of circRNA-000284 caused cell-cycle arrest in G0/G1 phase. F: Transwell permeable supports were used for the detection of cell invasive, and the results showed that knockdown of circRNA-000284 suppressed the invasive ability of cervical cancer cells. *P<0.05 vs. si-NC group; **P<0.01 vs. si-NC group.

CircRNA-000284 directly binds to miR-506 and suppresses miR-506 activity. A: RNA fluorescence in situ hybridization for circRNA-000284. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). B: The putative sequences of miR-506 and circRNA-000284 with two binding sites. C: MiR-506 was upregulated by transfection of miR-506 mimics in cervical cells. D: Luciferase reporter assay was performed to detect the interaction between miR-506 and circRNA-000284. MiR-506 significantly inhibited luciferase activity of wild type reporter for cirRNA-000284, however, miR-506 did not inhibit the luciferase activity of reporter vector containing the mutant binding sites of cirRNA-000284. E: Anti-AGO2 RIP was performed in HeLa cells transfected with miR-506 mimics or NC, followed by RT-qPCR to detect circRNA-000284. F: RT-qPCR experiment suggested that miR-506 was downregulated in cervical cell lines in contrast to normal epithelial cells. G: Cervical cells were transfected by si-circRNAs, then miR-506 expression was detected via RT-qPCR. *P<0.05 vs. NC group; **P<0.01 vs. NC group; ***P<0.001 vs. Input group.

CircRNA-000284 suppresses cell proliferation and invasion via miR-506/Snail-2 pathway. A: RT-qPCR and Western blot assays suggested that knockdown of circRNA-000284 suppressed Snail-2 expression level at both transcript and protein levels, respectively. B: The sketch of structures of circRNA-000284 is shown. C: RT-qPCR showed that circRNA-000284 expression level was dramatically elevated by the transfection of circRNA-000284 overexpressing vector. D: RIP experiments were performed using the anti-Snail-2 antibody to immunoprecipitate RNA and a primer to detect miR-506, and a significantly decreased enrichment of miR-506 was identified in cells transfected with circRNA-000284 overexpressing vector. E, F: CCK8 assay showed that enhanced expression of circRNA-000284 promoted the cell proliferation rate of both cell lines, however, cotransfection of miR-506 mimics or si-Snail-2 significantly reversed this effect. G: Matrigel invasion assay suggested that the increased cell invasion of cervical cancer cells induced by circRNA-000284 was abrogated by cotransfection with miR-506 mimics or si-Snail-2 vector. *P<0.05; **P<0.01; ***P<0.001 vs. respective control groups.