Long non‑coding RNA OIP5‑AS1 facilitates the progression of ovarian cancer via the miR‑128‑3p/CCNG1 axis

Abstract

Long non‑coding RNA (LncRNA) o‑phthalaldehyde-interacting protein 5 antisense transcript 1 (OIP5‑AS1) serves major roles in the progression of various types of cancer. The present study investigated its biological function in ovarian cancer (OC) and its mechanisms. The levels of OIP5‑AS1, microRNA‑128‑3p (miR‑128‑3p) and cyclin G1 (CCNG1) were examined by reverse transcription‑quantitative PCR. Cell viability, apoptosis, migration and invasion were detected to analyze cellular progression. Glycolytic metabolism was assessed by detecting the levels of glucose consumption and lactate production. CCNG1 and hexokinase 2 protein levels were measured by western blotting. Dual‑luciferase reporter assay, RNA immunoprecipitation and RNA pull‑down assays were performed to affirm the interaction between two molecules. OIP5‑AS1 was found to be upregulated in OC tissues and cells. Knockdown of OIP5‑AS1 suppressed cell viability, migration, invasion and glycolysis while promoting apoptosis in OC cells. OIP5‑AS1 interacted with miR‑128‑3p and functioned as an oncogene by sequestering miR‑128‑3p. In addition, CCNG1 was a target gene for miR‑128‑3p and miR‑128‑3p regulated the CCNG1‑induced effects on OC cells by downregulating CCNG1. OIP5‑AS1 upregulated the expression of CCNG1 via targeting miR‑128‑3p. OIP5‑AS1 knockdown also inhibited tumor growth of OC in vivo by modulating the expression of miR‑128‑3p and CCNG1. Collectively, these data illustrated that the oncogenic role of OIP5‑AS1 in OC was associated with the miR‑128‑3p/CCNG1 axis at least in part. OIP5‑AS1 might be a probable diagnostic and therapeutic biomarker for the treatment of OC patients.

Keywords: long non‑coding RNA o‑phthalaldehyde‑interacting protein 5 antisense transcript 1; ovarian cancer; microRNA‑128‑3p; cyclin G1.

Figure 1.

OIP5-AS1 was upregulated in OC tissues and cells. Reverse transcription-quantitative PCR was performed to analyze OIP5-AS1 expression in (A) OC tissues or adjacent normal tissues, (B) OC tissues at I+II or III+IV stages and (C) OC cell lines or normal cells. *P<0.05. OIP5-AS1, o-phthalaldehyde-interacting protein 5 antisense transcript 1; OC, ovarian cancer.

Figure 2.

Knockdown of OIP5-AS1 represses cell viability, migration, invasion and glycolysis while promoting apoptosis in OC cells. (A) The knockdown efficiency of si-OIP5-AS1 was detected by reverse transcription-quantitative PCR. (B) MTT assay was used to examine cell viability. (C) Flow cytometry was exploited to determine cell apoptosis rate. Transwell assay was used to measure cell (D) migration and (E) invasion abilities. (F) Glucose consumption and (G) lactate production were evaluated by glucose detection and lactic acid detection kits. (H) Western blotting was employed to detect the protein expression of HK2. *P<0.05. OIP5-AS1, o-phthalaldehyde-interacting protein 5 antisense transcript 1; OC, ovarian cancer; si-, small interfering; HK2, hexokinase 2.

Figure 3.

OIP5-AS1 interacts with miR-128-3p. (A) StarBase software was used to predict the potential miRNA target of OIP5-AS1. The combination between OIP5-AS1 and miR-128-3p was verified by (B) dual-luciferase reporter assay, (C) RNA immunoprecipitation and (D) RNA pull-down assay. (E) The expression of miR-128-3p was detected by RT-qPCR in OC cells. RT-qPCR was performed to determine the overexpression effects of (F) miR-128-3p and (G) OIP5-AS1. (H) Following transfection of OIP5-AS1, OIP5-AS1 + miR-128-3p or relative controls in OC cells, miR-128-3p level was determined by RT-qPCR. *P<0.05. OIP5-AS1, o-phthalaldehyde-interacting protein 5 antisense transcript 1; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; OC, ovarian cancer.

Figure 4.

Overexpression of OIP5-AS1 ameliorates the suppressive effects of miR-128-3p on the progression of OC cells. MTT assay was performed to analyze cell viability in (A) OVCAR-3 and (B) SKOV3 cells transfected with miR-128-3p, miR-128-3p + OIP5-AS1 or matched controls. (C and D) The assessment of apoptosis was performed using Annexin V/PI flow cytometry. The evaluation of cell migration (E and F) and (G and H) invasion was performed via Transwell assay. Glucose detection and lactic acid detection kits were administrated for measuring (I and J) glucose consumption and (K and L) lactate production. (M and N) The detection of HK2 was conducted by western blotting. *P<0.05. OIP5-AS1, o-phthalaldehyde-interacting protein 5 antisense transcript 1; miR, microRNA; OC, ovarian cancer; HK2, hexokinase 2.

Figure 5.

CCNG1 is a target of miR-128-3p. (A) The potential target of miR-128-3p was predicted by TargetScan software. (B) The relationship between miR-128-3p and CCNG1 was explored by the dual-luciferase reporter assay. The mRNA and protein expression levels of CCNG1 were determined by (C) RT-qPCR and (D) western blotting assays in OC cells. (E) The inhibitory effect of anti-miR-128-3p on miR-128-3p level was assayed by RT-qPCR. The determination of CCNG1 was performed by (F) RT-qPCR and (G) western blotting in OVCAR-3 and SKOV3 cells transfected with miR-128-3p, anti-miR-128-3p or the respective controls. *P<0.05. CCNG1, cyclin G1; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type; MUT, mutant-type.

Figure 6.

Downregulation of miR-128-3p restored the si-CCNG1-induced effect on OC cells. (A and B) Cell viability was examined by MTT assay after transfection of si-CCNG1, si-CCNG1+anti-miR-128-3p or the corresponding controls. (C and D) Cell apoptosis was detected by flow cytometry. (E and F) Cell migration and (G and H) invasion were measured by Transwell assay. The examination of (I and J) glucose consumption and (K and L) lactate production was performed via glucose detection and lactic acid detection kits. (M and N) The protein expression of HK2 was determined by western blotting assay. *P<0.05. miR, microRNA; si-, small interfering; CCNG1, cyclin G1; OC, ovarian cancer; HK2, hexokinase 2.

Figure 7.

OIP5-AS1 upregulates CCNG1 expression via sponging miR-128-3p in OC cells. (A) mRNA and (B) protein expression of CCNG1 was measured by reverse transcription-quantitative PCR and western blotting in OVCAR-3 and SKOV3 cells transfected with miR-128-3p, miR-128-3p + OIP5-AS1 or their relative controls. *P<0.05. OIP5-AS1, o-phthalaldehyde-interacting protein 5 antisense transcript 1; CCNG1, cyclin G1; miR, microRNA; OC, ovarian cancer.

Figure 8.

OIP5-AS1 depression inhibited tumor growth of OC by miR-128-3p/CCNG1 axis in vivo. (A) Tumor volume and (B) weight were measured in sh-OIP5-AS1 and sh-control groups. (C) The levels of OIP5-AS1 and (D) miR-128-3p were examined using reverse transcription-quantitative PCR. (E) CCNG1 protein expression was measured using western blotting. *P<0.05. OIP5-AS1, o-phthalaldehyde-interacting protein 5 antisense transcript 1; OC, ovarian cancer; miR, microRNA; sh-, short hairpin; CCNG1, cyclin G1.