hsa_circ_0061140 Knockdown Reverses FOXM1-Mediated Cell Growth and Metastasis in Ovarian Cancer through miR-370 Sponge Activity.

Circular RNAs (circRNAs) are a class of noncoding RNAs that regulate gene expression at the posttranscriptional level. The specific functions of circRNAs in ovarian cancer are yet to be established. Previous sequencing analyses have revealed an abnormal expression of hsa_circ_0061140 in ovarian cancer. The main aim of the present study is to establish the specific role of hsa_circ_0061140 in ovarian cancer. circRNA expression in ovarian cancer cells was detected via real-time qPCR. The effects on specific cellular characteristics (proliferation, migration, and the EMT) and subcellular localization of hsa_circ_0061140 were assessed via RNA fluorescence in situ hybridization, knockdown, and luciferase reporter assays in the SKOV3 and A2780 cell lines. Tumorigenesis was induced in nude mice to assess the effects of hsa_circ_0061140 on ovarian cancer growth in vivo. Our results showed that hsa_circ_0061140 was upregulated in ovarian cancer cell lines. Knockdown of hsa_circ_0061140 suppressed cell proliferation and migration, both in vivo and in vitro, by inhibiting FOXM1 expression through sponging miR-370. Overexpression of FOXM1 or suppression of miR-370 rescued hsa_circ_0061140 silencing-induced inhibition of cell proliferation, migration, and the EMT. The associations among hsa_circ_0061140, miR-370, and FOXM1 were confirmed via bioinformatic prediction and fluorescein reporter experiments. Thus, hsa_circ_0061140 appeared to function as a competing endogenous RNA of miR-370 that promoted cell growth and metastasis in ovarian cancer through regulation of the miR-370/FOXM1 pathway mediating EMT.

Introduction

An estimated 14.1 million new cancer cases and 8.2 million cancer deaths occurred in 2012 worldwide. Ovarian cancer is the seventh most common lethal gynecological malignancy, causing an estimated 125,000 deaths annually on a global scale. Approximately 90% of ovarian cancers are surface epithelial-stromal tumors, also known as ovarian epithelial carcinoma. Accumulating studies have shown that the epithelial-mesenchymal transition (EMT) plays an important role in metastasis of cancer, because acquisition of invasiveness and metastatic ability is accompanied by the loss of epithelial features.3, 4 Identification of factors that inhibit EMT should, therefore, facilitate the development of effective clinical treatments.

Numerous studies have demonstrated that non-coding RNAs (ncRNAs) play important roles in tumorigenesis.5, 6 Circular RNAs (circRNAs) are a class of ncRNAs with a covalently closed continuous loop structure that regulate gene expression via competitive binding to microRNA (miRNA).7, 8, 9 Abnormal expression of circRNAs during ovarian cancer development has been previously reported. We found that hsa_circ_0061140 expression was abnormally elevated. However, the specific role of hsa_circ_0061140 in ovarian cancer remains to be established.

In the present investigation, we examined hsa_circ_0061140 expression and its biological significance in ovarian cancer cell lines. Our results showed that hsa_circ_0061140 was upregulated in ovarian cancer cells, in turn, promoting cell proliferation, migration, and the EMT. The study provided novel evidence that may aid in the development of effective therapeutic strategies against ovarian cancer in the clinic.

Results

Knockdown of hsa_circ_0061140 Suppresses Ovarian Cancer Cell Proliferation and Invasion

Abnormal expression of circRNAs is associated with development of several tumor types, including ovarian cancer.10, 11 An analysis of sequencing results revealed abnormal expression of hsa_circ_0061140. To determine whether hsa_circ_0061140 participates in the development of ovarian cancer, real-time qPCR analysis was performed on primary cultured normal ovarian epithelial cells and ovarian cancer cell lines. Expression of hsa_circ_0061140 was increased in ovarian cancer, compared with normal ovarian epithelial cells (Figure 1A), with the highest levels observed in SKOV3 and A2780 cells, which were consequently selected for further experiments. In situ hybridization studies revealed that hsa_circ_0061140 was mainly distributed in the cytoplasm (Figure 1B).

Figure 1

Expression and Subcellular Localization of hsa_circ_0061140 in Ovarian Cancer Cell Lines

(A) Real-time qPCR showing expression of hsa_circ_0061140 in primary cultured normal ovarian epithelial and ovarian cancer cell lines. Data are presented as means ± SD (***p < 0.001 versus normal). (B) In situ hybridization was applied to determine the subcellular localization of hsa_circ_0061140.

To establish the specific role of hsa_circ_0061140 in ovarian cancer, SKOV3 and A2780 cells were transfected with specific small interfering RNA (siRNA) against hsa_circ_0061140 for 48 hr, and expression was examined via real-time qPCR, which revealed a significant decrease in the hsa_circ_0061140 level, as expected (Figures 2A and 2B). Data from the Cell Counting Kit-8 (CCK8) detection assay suggested that knockdown of hsa_circ_0061140 suppressed proliferation of both SKOV3 and A2780 cells, compared with the negative control (NC) group (Figures 2C and 2D). Wound-healing (Figures 2E and 2F) and Transwell (Figures 2I–2L) analyses demonstrated that knockdown of hsa_circ_0061140 suppressed migration in both cell lines (Figures 2C and 2D), compared with the NC group. In western blot experiments, knockdown of hsa_circ_0061140 was associated with a decrease in FOXM1 and the EMT relative protein Snail and vimentin expression, along with increased E-cadherin expression in both SKOV3 and A2780 cells (Figures 2M and 3N). FOXM1 was previously shown to confer proliferation and invasion advantages to a variety of tumors, including non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma, gastric cancer, and breast cancer. Upregulation of FOXM1 promotes the EMT, invasion, and migration of tongue squamous cell carcinoma, supporting a role in hsa_circ_0061140-mediated ovarian cancer proliferation and invasion.

Figure 2

Knockdown of hsa_circ_0061140 Suppresses Ovarian Cancer Cell Proliferation and Invasion

(A and B) Expression of hsa_circ_0061140 in SKOV3 (A) and A2780 (B) cells detected via real-time qPCR after transfection with hsa_circ_0061140 interference vector (sicircRNA) or negative control (NC) for 48 hr. Data are presented as means ± SD (***p < 0.001 versus control). (C and D) Evaluation of cell proliferation with the CCK8 assay. (C) SKOV3 cells. (D) A2780 cells. Data are presented as means ± SD (***p < 0.001 versus NC). OD450 nm, optical density. (E–H) Wound-healing assay showing the effect of hsa_circ_0061140 on closure of scratch wounds in both SKOV3 (E and F) and A2780 (G and H) cells. Data are presented as means ± SD (***p < 0.001 versus NC). (I–L) Cell migration was assessed in SKOV3 (I and J) and A2780 (K and L) cells using the Transwell assay. Data are presented as means ± SD (***p < 0.001 versus NC). (M and N) Western blot of the expression of FOXM1 and EMT-related proteins Snail, E-cadherin, and vimentin in (M) SKOV3 and (N) A2780 cells. Data are presented as means ± SD (***p < 0.001 versus NC).

Figure 3

Downregulation of miR-370 Suppresses Ovarian Cancer Cell Proliferation and Invasion

(A and B) Expression of miR-370 in SKOV3 (A) and A2780 (B) cells detected via real-time qPCR after transfection with miR-370 inhibitor or sicircRNA for 48 hr. Data are presented as means ± SD (***p < 0.001 versus control). (C and D) Evaluation of proliferation in both SKOV3 (C) and A2780 (D) cells with the CCK8 assay. Data are presented as means ± SD (***p < 0.001 versus control). (E–H) Wound-healing assays showing the effect of miR-370 on closure of scratch wounds in both SKOV3 (E and F) and A2780 (G and H) cells. Data are presented as mean values ± SD (***p < 0.001 versus control; ###p < 0.001 versus sicircRNA). (I–K) Transwell assay assessing cell migration in SKOV3 (I and J) and A2780 (I and K) cells. Data are presented as means ± SD (***p < 0.001 versus control; ###p < 0.001 versus sicircRNA). (L and M) Western blots of the expression of FOXM1 and the EMT-related proteins Snail, E-cadherin, and vimentin in SKOV3 (L) and A2780 (M) cells.

Downregulation of miR-370 Suppresses Ovarian Cancer Cell Proliferation and Invasion

Accumulating evidence indicates that circRNAs act as competing endogenous RNAs or miRNA sponges to regulate gene expression. Bioinformatics analyses led to the identification of miR-370 as a potential target of hsa_circ_0061140. The miR-370 is known to be downregulated in ovarian cancer.18, 19 To further ascertain whether miR-370 played a role in hsa_circ_0061140-mediated regulation of ovarian cancer proliferation and invasion, SKOV3 and A2780 cells were pretreated with a specific inhibitor. The real-time qPCR analyses confirmed significant suppression of miR-370 in both cell types (Figures 3A and 3B). In the CCK8 detection assay, downregulation of miR-370 rescued hsa_circ_0061140 silencing-induced inhibition of proliferation in both SKOV3 and A2780 cells (Figures 3C and 3D). Data from the wound-healing assay (Figures 3E–3H) and Transwell analysis (Figures 3I–3K) further confirmed that suppression of miR-370 rescued hsa_circ_0061140 silencing-induced inhibition of migration in both cell types. Western blot experiments showed that knockdown of miR-370 promoted FOXM1 and EMT-related Snail and vimentin expression but suppressed E-cadherin expression in the two cell lines (Figures 3L and 3M). Moreover, depletion of miR-370 reversed hsa_circ_0061140 silencing-induced inhibitory effects on FOXM1 expression and EMT. The data suggested that miR-370 was located downstream of hsa_circ_0061140 and regulated expression of FOXM1. The luciferase reporter assay confirmed interactions between hsa_circ_0061140 and miR-370 (Figures 4A and 4B). In situ hybridization experiments showed that miR-370 sponge activity was suppressed following downregulation of hsa_circ_0061140 (Figure 4C).

Figure 4

The miR-370 Is a Direct Target of hsa_circ_0061140

(A) Predicted binding sites of miR-370 and hsa_circ_0061140 and mutated hsa_circ_0061140. (B) Relative luciferase activity was determined 48 hr after transfection with miR-370 mimic/NC or hsa_circ_0061140 wild-type (WT)/Mutant (Mut) in 293T cells. Data are presented as means ± SD (***p < 0.001 versus control). (C) In situ hybridization showing that downregulation of hsa_circ_0061140 reverses the sponge effect on miR-370.

FOXM1 Is a Direct Target of miR-370

Bioinformatics analyses revealed that FOXM1 was a potential target of miR-370 (Figure 5A). Data from the luciferase reporter assay confirmed interactions between miR-370 and the 3′ UTR of FOXM1 (Figure 5B). Following transfection of a miR-370 overexpression vector (mimic) into SKOV3 and A2780 cells for 48 hr, miR-370 levels were significantly increased (Figures 5C and 5D). Overexpression of miR-370 led to the suppression of FOXM1 protein, as evident from western blot data (Figures 5E and 5F). The CCK8 detection assay showed that overexpression of miR-370 suppressed proliferation in both SKOV3 and A2780 cells. However, upregulation of FOXM1 rescued miR-370-induced inhibition of proliferation in both cell lines (Figures 5G and 5H). In wound-healing (Figures 5I–5L) and Transwell analyses (Figures 5M–5O), FOXM1 overexpression rescued miR-370-mediated inhibition of migration in cells. Additionally, FOXM1 rescued miR-370-induced EMT inhibition through the promotion of E-cadherin and suppression of Snail and vimentin, as observed on a western blot (Figures 5P and 5Q).

Figure 5

FOXM1 Is a Direct Target of miR-370

(A) Predicted binding sites of miR-370 in the 3′ UTR of FOXM1. The mutated FOXM1 3′ UTR sequence is presented. (B) Relative luciferase activity was determined 48 hr after transfection with miR-370 mimic/NC or the 3′ UTR of FOXM1 wild-type/Mut in 293T cells. Data are presented as means ± SD (***p < 0.001 versus control). (C and D) Real-time qPCR detection of miR-370 expression after transfection with miR-600 mimic or negative control (NC) in SKOV3 (C) and A2780 (D) cells. Data are presented as means ± SD (***p < 0.001 versus control). (E and F) Western blot of FOXM1 protein expression after transfection with miR-370 mimics in both SKOV3 (E) and A2780 (F) cells. Data are presented as means ± SD (***p < 0.001 versus control). (G and H) Assessment of cell proliferation in SKOV3 (G) and A2780 (H) cells with the CCK8 assay. Data are presented as means ± SD (***p < 0.001 versus control). (G) SKOV3 cells. (H) A2780 cells. (I–L) Wound-healing assays showing the effects of FOXM1 on SKOV3 (I and J) and A2780 (K and L) cells on scratch wound closure (***p < 0.001 versus control; ###p < 0.001 versus mimic). (M–O) Cell migration was assessed in SKOV3 (M and N) and A2780 (M and O) cells using the Transwell assay. Data are presented as means ± SD (***p < 0.001 versus control; ###p < 0.001 versus mimic). (P and Q) Western blot of the expression of EMT-related proteins Snail, E-cadherin, and vimentin in SKOV3 (P) and A2780 (Q) cells.

Silencing of hsa_circ_0061140 Suppresses Ovarian Cancer Growth

To further ascertain whether silencing of hsa_circ_0061140 exerted a similar tumor inhibitory effect in vivo, we established xenograft mouse models via subcutaneous injection of equal amounts of SKOV3 cells (n = 6 per group). Notably, silencing of hsa_circ_0061140 significantly suppressed tumor volume, compared with that in the wild-type SKOV3 group (Figures 6A and 6B). The real-time qPCR experiments disclosed increased expression of miR-370 in the hsa_circ_0061140-depleted construct group (Figure 6C). Conversely, FOXM1 was downregulated in the hsa_circ_0061140-silenced group at both the mRNA and protein levels (Figures 6D and 6E). Western blot results further demonstrated that silencing induced inhibition of EMT by promoting E-cadherin and suppressing Snail and vimentin expression (Figure 6E).

Figure 6

Silencing of hsa_circ_0061140 Suppresses Ovarian Cancer Growth

Xenotransplantation studies with SKOV3 were performed in BALB/c nude mice. (A) Representative images of tumor formation in xenografts of nude mice. (B) Tumor volumes were measured every 5 days for 30 days. Data are presented as means ± SD (***p < 0.001 versus NC). (C and D) Real-time qPCR analysis of miR-370 (C) and FOXM1 (D) expression. Data are presented as means ± SD (***p < 0.001 versus control). (E) Western blot detection of FOXM1 and the EMT-related proteins Snail, E-cadherin, and vimentin is presented. Data are presented as means ± SD (***p < 0.001 versus control).

Discussion

In this study, we showed that hsa_circ_0061140 expression was abnormally elevated in the ovarian cancer cell lines SKOV3 and A2780. Notably, knockdown of hsa_circ_0061140 suppressed cell proliferation, migration, and the EMT in both cell lines, clearly suggesting a role in the development of ovarian cancer. Increasing evidence has confirmed the regulatory effects of circRNAs on gene expression via competitive binding to miRNAs, leading to the designation “miRNA sponge.”20, 21, 22 Our bioinformatics analyses showed that hsa_circ_0061140 interacted with miR-370, which was further confirmed with fluorescein reporter and in situ hybridization assays.

The anticancer activity of miR-370 has been demonstrated previously. Overexpression of miR-370 is reported to induce significant suppression of cell proliferation and invasion in various cancer types, including bladder cancer, breast cancer, and hepatocellular carcinoma. Moreover, miR-370 is downregulated in ovarian cancer.18, 19, 26 Data from the present study showed that knockdown of hsa_circ_0061140 promoted miR-370 expression. Conversely, downregulation of miR-370 rescued hsa_circ_0061140 knockdown-induced inhibition of cell proliferation, migration, and the EMT. The data suggested that hsa_circ_0061140 promoted ovarian cancer development by acting as a miR-370 sponge.

Bioinformatics analyses revealed interactions between miR-370 and the 3′ UTR of FOXM1, which were confirmed using a fluorescein reporter experiment. Overexpression of miR-370 suppressed FOXM1 at the posttranscriptional level, while overexpression of FOXM1 rescued miR-370-induced inhibition of cell proliferation, migration, and the EMT. FOXM1, a key member of the FOX transcription factor family, plays a vital role in a series of physiological processes. FOXM1 confers proliferation and invasion advantages to many cancer types, including NSCLC,13, 27 esophageal squamous cell carcinoma, gastric cancer, and breast cancer. Moreover, expression of FOXM1 promotes EMT, invasion, and migration of a variety of tumor types.17, 28, 29, 30 Here, we additionally showed that downregulation of hsa_circ_0061140 suppressed FOXM1 expression but inhibits miR-370-rescued FoxM1 expression inhibition after hsa_circ_0061140 silence. In vivo experiments further confirmed that hsa_circ_0061140 knockdown led to the suppression of ovarian cancer growth by inhibiting FOXM1 expression through sponging miR-370.

In conclusion, we have identified interactions among hsa_circ_0061140, miR-370, and FOXM1 that potentially contributed to ovarian cancer development. Our results collectively suggested that hsa_circ_0061140 positively regulated FOXM1 expression by acting as a competing endogenous RNA for miR-370 binding, supporting its utility as a promising therapeutic target and predictive marker for ovarian cancer.

Materials and Methods

Animals and Ethics Statement

Four-week-old male BALB/c nude mice (21–23 g) were purchased from the Shanghai Laboratory Animal Center (SLAC; Shanghai, China). All experiments and animal procedures were approved by the Animal Care and Utilization Committee of Tongji Hospital and performed following the guidelines of the Tongji Hospital Ethics Committee.

Cell Culture

Primary cultured human normal ovarian epithelial cells were maintained in Ovarian Epithelial Cell Medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). The ovarian cancer cell lines SKOV3, A2780, OV2008, IGROV1, and ES-2 were maintained in RPMI-1640 (GIBCO) supplemented with 10% FBS. All cells were cultured at 37°C in a 5% CO2 chamber with penicillin (100 IU/mL) and streptomycin (100 μg/mL).

Cell Proliferation Assay

Different groups of SKOV3 or A2780 cells (2 × 103 cells) were cultured in 96-well flat-bottomed microtiter plates in RPMI-1640 containing 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 U/mL) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Following removal of medium, cells were cultured for different time periods and washed three times with PBS. RPMI-1640 (90 μL) and CCK8 solution (10 μL) were added to each well and incubated for 1.5 hr at 37°C. A microplate reader was used to measure optical density at 450 nm (OD450 nm).

Western Blotting

Proteins were resolved on an SDS-denaturing polyacrylamide gel and transferred onto nitrocellulose membrane. Membranes were incubated with antibodies against FOXM1, Snail, E-cadherin, vimentin, and GAPDH overnight at 4°C before blocking with 5% nonfat milk for 2 hr. Next, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at 4°C for 2 hr. Protein expression was assessed via enhanced chemiluminescence and band intensities quantified with LabWorks Image Acquisition and Analysis Software (UVP, Upland, CA, USA). All antibodies were purchased from Abcam (Cambridge, MA, USA).

Fluorescence In Situ Hybridization

Specific cy5-labeled probes for the hsa_circ_0061140 sequence were used for in situ hybridization, as described previously. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. All procedures were performed according to the manufacturer’s instructions (GenePharma, Shanghai, China).

Wound-Healing Assay

Ovarian cancer SKOV3 or A2780 cell lines (2 × 105 cells per well) were seeded into 6-well plates. Cell layers were wounded using a sterile 10-μL pipette tip and washed with PBS three times to remove debris after ∼90% confluence was reached. Next, cells were cultured in RPMI-1640 supplemented with 1% FBS. Images of the scrape lines were obtained under a light microscope at the indicated time points. Each experiment was performed in triplicate.

Plasmid Construction and Stable Transfection

For miR-370, hsa_circ_0061140, and FOXM1 expression analyses, miR-370-specific mimic and inhibitor, hsa_circ_0061140 interference vector, FOXM1 overexpression vector, and the corresponding NC vector were obtained from GenePharma. SKOV3 and A2780 cells were transfected with the aforementioned constructs at a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Cells were collected for further experiments after 48 hr of transfection.

Transwell Migration Assay

Cell migration was analyzed using Transwell chambers (Corning, New York, NY, USA), according to the manufacturer’s protocol. After incubation for 24 hr, cells on the upper surfaces of the chambers were removed with cotton swabs and those located on the lower surfaces were fixed with methanol for 10 min, followed by staining with crystal violet. Images of stained cells were obtained and counted in five randomly selected fields.

Luciferase Reporter Assay

The 293T cells were co-transfected with a bioluminescent reporter vector (pMIR-REPORT-3′ UTR-FOXM1-wt/-mut or pMIR-REPORT-circRNA-wt/-mut) and the miR-370 ectopic expression vector. Equivalent amounts of Renilla expression vector pRLTK (Promega, Wisconsin, USA) were co-transfected into every group for normalization. After 48 hr, cells were harvested, and luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega, Fitchburg, WI, USA).

Real-Time qPCR

Total RNA was isolated from ovarian carcinoma cell lines and tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Next, 2 μg RNA was used for reverse transcription into complementary DNA (cDNA) using random primers and cDNA amplified via real-time qPCR with the SYBR Premix Ex Taq II kit (TaKaRa, Shiga, Japan). The relative expression of target genes was determined by comparing the threshold cycle (Ct) of the target genes to that of U6 or GAPDH using the 2−ΔΔCt method (GenePharma, Suzhou, China).

Prediction of miRNA Targets

The TargetScanHuman database (Release 6.2; http://www.targetscan.org) was used to predict the target genes of miR-370 and the interactions between miR-370 and circRNAs were predicted with the Circular RNA Interactome database (https://circinteractome.nia.nih.gov/miRNA_Target_Sites/mirna_target_sites.html).

Xenograft Assays

Four-week-old male BALB/c nude mice were housed in a pathogen-free and temperature-controlled environment. PBS (200 μL) containing approximately 1 × 106 SKOV3 cells transfected with siRNA against hsa_circ_0061140 was injected into the right flanks of mice. Tumor volumes were measured every 5 days from the fifth day following inoculation, and weights were calculated using the formula (length × width2)/2.

Author Contributions

Yanqiu Wang designed the study and wrote the protocols; Qizhen Chen and Jiarong Zhang managed the literature retrieval and performed the experimental work and data analysis; Yinyan He and Yanqiu Wang wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.