Fig 2: The reciprocal role between CHTOP and miR-338-3p in DDP-resistant OvCa cells in vitro. SKOV3/DDP and A2780/DDP cells were severally transfected with miR-NC, miR-338-3p, miR-338-3p along with pEGFP empty vector (vector), miR-338-3p together with pEGFP-CHTOP vector (CHTOP). (A) Western blotting analyzed CHTOP expression. (B and C) CCK-8 assay measured IC50 of DDP by monitoring cell viability (%) after 1–120 µM of DDP treatment. (D and E) CCK-8 assay showed OD values at 450 nm after transfection for 0–72 h. (F–L) After transfection for 24 h, (F–H) FCM method examined (F) apoptosis rate (%) and (G and H) cell distribution percentages in different cell cycle phases, (I and J) transwell assay evaluated numbers of migrated cells and invaded cells, and (K and L) Western blotting detected protein expression of Bcl-2, Bax, MMP2, and MMP9, normalized to GAPDH. *P<0.05. Data analysis was performed using two-way ANOVA followed with Tukey’s post-hoc analysis.

Fig 3: Identification of CHTOP as target gene of miR-338-3p in DDP-resistant OvCa cells. (A) Alignment sequences among CHTOP 3'UTR WT, miR-338-3p and CHTOP 3'UTR MUT. (B and C) Dual-luciferase reporter assay measured luciferase activity of reporter vectors carrying CHTOP 3'UTR WT or MUT in SKOV3/DDP and A2780/DDP cells co-transfected with miR-338-3p or miR-NC. (D) RNA pull-down assay assessed miR-338-3p enrichment by bio-circHIPK2 or bio-NC in SKOV3/DDP and A2780/DDP cells. (E–N) RT-qPCR and Western blotting, respectively, detected CHTOP mRNA expression and CHTOP protein expression in (E and F) OvCa tumor tissues and normal tissues, (G and H) DDP-sensitive tumor tissues and DDP-resistant tumor tissues, and (I and J) IOSE80, SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells, as well as (K–N) SKOV3/DDP and A2780/DDP cells severally transfected with miR-NC, miR-338-3p, si-NC, si-circHIPK2#2, si-circHIPK2#2 along with anti-miR-NC, si-circHIPK2#2 together with anti-miR-338-3p. *P<0.05. Data analysis was performed using unpaired t-test and one-way or two-way ANOVA followed with Tukey’s post-hoc analysis.