Effect and mechanism of miR-217 on drug resistance, invasion and metastasis of ovarian cancer cells through a regulatory axis of CUL4B gene silencing/inhibited Wnt/β-catenin signaling pathway activation

H.-R. Liu, J. Zhao

Oncology Department, the Second Affiliated Hospital of Dalian Medical University, Dalian, P.R. China. zhaojianbaxia@163.com

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• Oncology

OBJECTIVE: To explore the effect and mechanism of miR-217 in cisplatin resistance, as well as invasion and metastasis of ovarian cancer by inhibiting the expression of Cullin 4B (CUL4B) and the activation of Wnt/β-catenin signaling pathway.

MATERIALS AND METHODS: Human ovarian cancer cell lines COC1 (cisplatin sensitive) and COC1/DDP (cisplatin resistant) were cultured and were used to construct the COC1 group and COC1/DDP group, respectively. COC1/DDP cells were divided into blank group, NC group, miR-217 mimic group, miR-217 mimic NC group, miR-217 inhibitor group, miR-217 inhibitor NC group, si-CUL4B group, si-CUL4B NC group, overexpressed (oe) oe-CUL4B group, oe-CUL4B NC group, and miR-217 mimic +oe-CUL4B group, with the identification of cell transfection simultaneously. Bioinformatics prediction and Dual-Luciferase reporter gene assay of the targeting effect of miR-217 on CUL4B were performed, followed by MTT assay for cell proliferation, associated with the measurement of median inhibitory concentration (IC50). Real-time quantitative PCR (qRT-PCR) detected the mRNA expression of miR-217 and CUL4B, and Western blotting for detecting CUL4B, Wnt1, Wnt3, Wnt3a and β-catenin protein expression. The cell invasion of cells was detected by transwell assay, cell migration by cell scratch assay and cell apoptosis by flow cytometry.

RESULTS: Bioinformatics prediction and Dual-Luciferase reporter gene assay verified that CUL4B was a target gene of miR-217, and the latter could silence the expression of the former gene. Compared with COC1 group, the relative expression of miR-217 was significantly decreased, while CUL4B mRNA and protein expression, as well as Wnt1, Wnt3, Wnt3a and β-catenin protein expression were increased significantly, with evidently increased cell proliferation, IC50, invasion and migration, and decreased apoptosis rate in COC1/DDP group (all p<0.05). Compared with blank group and corresponding NC groups, miR-217 mimic group had increased expression of miR-217 and decreased expression of CUL4B; miR-217 inhibitor group showed decreased miR-217 expression while increased CUL4B expression; si-CUL4B group indicated no significant change of miR-217 expression but decreased CUL4B expression; oe-CUL4B group showed no difference in miR-217 expression but increased CUL4B expression; miR-217 mimic +oe-CUL4B had increased miR-217 expression and no change of CUL4B expression. Meanwhile, miR-217 mimic group and si-CUL4B group exhibited decreased Wnt/β-catenin, Wnt1, Wnt3, Wnt3a and β-catenin expression, decreased cell proliferation, IC50, invasion and migration, and increased apoptosis (all p<0.05). Furthermore, miR-217 inhibitor group and oe-CUL4B group revealed increased Wnt/β-catenin, Wnt1, Wnt3, Wnt3a and β-catenin expression, increased cell proliferation, IC50, invasion and migration, and decreased apoptosis (all p<0.05). NC group and miR-217 mimic +oe-CUL4B group showed no significant difference in the above indexes (all p>0.05). While compared with miR-217 mimic group, miR-217 mimic +oe-CUL4B group showed increased cell proliferation, IC50, invasion and migration, and decreased apoptosis (all p<0.05).

CONCLUSIONS: CUL4B gene is the target gene of miR-217. MiR-217 can silence the expression of this gene, and inhibit the activation of Wnt/β-catenin signaling pathway to enhance the cisplatin sensitivity and reverse drug resistance, inhibit cell invasion and migration, and promote cell apoptosis.

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