MiR-124 inhibits invasion and induces apoptosis of ovarian cancer cells by targeting programmed cell death 6

Abstract

Epithelial ovarian cancer remains the most common type of malignant tumor of the female reproductive system worldwide. Routine surgery and chemotherapy are the best treatments available for patients with ovarian cancer; however, almost 40% of ovarian cancer cases are intractable, with poor 5-year survival rates. MicroRNAs (miRNA) are endogenous small non-coding RNA molecules that function in transcriptional and post-transcriptional regulation of gene expression in various cellular processes. Recent studies demonstrated that microRNA (miR)-124 was downregulated in numerous types of tumors; however, the function and mechanism underlying miR-124 in epithelial ovarian cancer remain unclear. The present study revealed that miR-124 may be significantly downregulated in epithelial ovarian cancer. Using prediction algorithms and luciferase reporter gene assays, the present study identified and confirmed programmed cell death 6 (PDCD6) as a novel, direct target of miR-124. Overexpression of miR-124 suppressed PDCD6 expression, inhibited cell proliferation, migration and invasion, and induced apoptosis in SKOV3 and OCVAR3 cells in vitro. In the present study, overexpression of PDCD6 in epithelial ovarian cancer cells co-transfected with miR-124 effectively reversed the miR-124-induced apoptosis. Therefore, the results of the present study suggested that miR-124 is a tumor suppressor miRNA and a potential target for future treatment of ovarian malignant neoplasms.

Keywords: OCVAR3; SKOV3; apoptosis; cell migration; metastasis; miR-124; microRNA; ovarian cancer; programmed cell death 6.

Figure 1.

Expression level of miR-124 in human ovarian tissues and PDCD6 is a target of miR-124. (A) Representative hematoxylin and eosin staining of ovarian tumor tissue specimens (magnification, ×200). (B) Representative hematoxylin and eosin staining of non-neoplastic ovarian tissue specimens (magnification, ×200). (C) Expression of miR-124 was detected in 30 human ovarian cancer tissues by polymerase chain reaction. Data are presented as log2 values of the change in ovarian tumor tissues relative to non-neoplastic ovarian tissues. (D) The expression of miR-124 in ovarian cancer tissues was decreased compared with in non-neoplastic ovarian tissues (***P<0.001). (E) The putative association between PDCD6 and miR-124 was revealed by TargetScan. (F) The dual-luciferase reporter assay revealed regulation of PDCD6 by miR-124, including wild-type PDCD6 or mutant PDCD6 3′-UTR transfected with or without miR-124 mimics or scramble. (G) Diagram of the PDCD6 3′-UTR with potential binding-sites for miR-124. The data from three in vitro independent experiments are presented (**P<0.01). NC, negative control; miR, microRNA; UTR, untranslated region; mut, mutant; PDCD6, programmed cell death 6.

Figure 2.

Overexpression of miR-124 suppressed ovarian cancer cell migration and invasion. (A) OCVAR3 and (B) SKOV3 cells transfected or not with miR-124 mimics or scramble for 24 h. Wounds were made (magnification, ×200). (C) The OCVAR3 cells were transfected or not with miR-124 mimics or scramble for 24 h (magnification, ×200). Results from Transwell invasion assays are presented. (D) The relative invasion of OCVAR3 cells were transfected or not with miR-124 mimics or scramble for 24 h. (E) The SKOV3 cells were transfected or not with miR-124 mimics or scramble for 24 h (magnification, ×200). Results from Transwell invasion assays are presented. (F) The relative invasion of SKOV3 cells were transfected or not with miR-124 mimics or scramble for 24 h. The data from three in vitro independent experiments are presented (**P<0.01). NC, negative control; miR, microRNA.

Figure 3.

Expression of PDCD6 reversed the miR-124-induced suppression of cellular migration and invasion. (A) Wound-healing assay of SKOV3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector (magnification, ×200). (B) Wound-healing assay of OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector (magnification, ×200). (D) Wound-healing assay of representative images are shown at 24 and 48 h. (C) MTT assay of SKOV3 and OCVAR3 cells following co-transfection with NC or miR-124 mimics with either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The data from three in vitro independent experiments are presented (*P<0.05). (E) Transwell assay of SKOV3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector (magnification, ×200). (F) The relative invasion of SKOV3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. (G) Transwell assay of OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector (magnification, ×200). (H) The relative invasion of OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The data from three in vitro independent experiments are shown (**P<0.01). NC, negative control; miR, microRNA; PDCD6, programmed cell death 6.

Figure 4.

Expression of PDCD6 reversed the miR-124-induced cellular apoptosis. (A) The expression level of miR-124 was determined in SKOV3 and OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The expression level of miR-124 markedly increased in cells transfected with miR-124 mimics. Transfection with pcDNA3.1/PDCD6 restored the expression of PDCD6 in SKOV3 and OCVAR3 cells even with co-transfection of miR-124 mimics. (B) The expression levels of PDCD6 were determined in SKOV3 and OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The expression level of PDCD6 was markedly suppressed in cells transfected with miR-124 mimics. Transfection of pcDNA3.1/PDCD6 restored the PDCD6 expression level in SKOV3 and OCVAR3 cells even with co-transfection of miR-124 mimics. (C) The expression level of PDCD6 protein was determined in SKOV3 and OCVAR3 following co-transfection with NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The expression level of PDCD6 protein was suppressed in cells transfected with miR-124. Transfection with pcDNA3.1/PDCD6 restored the expression level of PDCD6 protein in SKOV3 and OCVAR3 cells even with co-transfection of miR-124 mimics. (D) Transfection of miR-124 mimics alone or with pcDNA3.1/vector induced apoptosis of SKOV3 and OCVAR3 cells. (E) The cell cycle status of SKOV3 and OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector was detected by flow cytometry analysis. NC, negative control; miR, microRNA; PDCD6, programmed cell death 6.