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. 2021 Aug 31;20(19):2021–2039. doi: 10.1080/15384101.2021.1965723

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© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

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Figure 3. — CircLNPEP serves as a sponge for miR-876-3p in ovarian cancer cell lines

(a) The abundance of circLNPEP in the cytoplasm and nucleus was detected by qRT-PCR. GAPDH was used as internal control in the cytoplasm and U6 was used as internal control in the nucleus. (b) Lysates from ovarian cell lines with overexpressed circLNPEP were subjected to biotinylated-circLNPEP pull-down assay and the expression level of circLNPEP was measured by qRT-PCR. GAPDH was used as internal control. (c) The expression levels of four candidate miRNAs predicted by CircInteractome were quantified by qRT-PCR after the biotinylated-circLNPEP pull-down assay in SKOV3 and SK-BR-3 cells. U6 was used as internal control. (d) The biotinylated WT/MUT miR-876-3p was transfected into SKOV3 and SK-BR-3 cells with overexpressed circLNPEP. The expression levels of circLNPEP were tested by qRT-PCR after streptavidin capture. GAPDH was used as internal control. (e) The binding site between circLNPEP and miR-876-3p was predicted by CircInteractome. (f) Luciferase activity in SKOV3 and SK-BR-3 cells co-transfected with luciferase reporter containing circLNPEP sequences with WT/MUT miR-876-3p binding sites and miR-876-3p mimics or control. (g-h) qRT-PCR analysis of miR-876-3p expression in SKOV3 and SK-BR-3 cell lines with silent or overexpressed circLNPEP. U6 was used as internal control. (i) The expression of miR-876-3p in ANT and cancer tissues was determined by qRT-PCR. U6 was used as internal control. (j) The correlation between circLNPEP and miR-876-3p was evaluated by Spearman’s analysis (r = −0.446,p = 0.004). Quantified values were expressed as mean ± standard deviation. Experiments were conducted independently in triplicate. ***P < 0.001 vs oligo probe group, ^{^^^}P < 0.001 vs biotin-miR-876-3p-WT group, ^###P < 0.001 vs MC group, ^†††P < 0.001 vs sh-NC group.LNPEP: leucyl and cystinyl aminopeptidase. qRT-PCR: quantitative reverse transcription polymerase chain reaction. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. WT:wild type. MUT: mutant. ANT: adjacent tissues. MC: mimic control. NC: negative control.