Circ_0007841 accelerates ovarian cancer development through facilitating MEX3C expression by restraining miR-151-3p activity

Circ_0007841 is highly expressed in OC tissues

The expression of circ_0007841 in OC was firstly detected. Using qRT-PCR, it was found that circ_0007841 level was increased in OC tissues compared to normal tissues (Figure 1A). Circ_0007841 expression was also up-regulated in OC cell lines compared to IOSE80 cells (Figure 1B). To validate circ_0007841 as a circRNA, we treated RNA with RNase R, followed by qRT-PCR. Circ_0007841 was resistant to RNase R digestion (Figure 1C). It is noticed that circ_0007841 was mainly distributed in the cytoplasm of OC cells (Figure 1D). Then, OC samples were divided into two groups based on circ_0007841 median value. We found that circ_0007841 overexpression was associated with a low survival rate (Figure 1E), indicating circ_0007841 may be a prognostic biomarker.

Figure 1. circ_0007841 is highly expressed in OC tissues. (A) Circ_0007841 expression in OC tissues and adjacent normal tissues was detected by qRT-PCR. (B) Circ_0007841 expression in OC cell lines were measured by qRT-PCR. (C) Circ_0007841 expression was analyzed after RNase RNA digestion. (D) Subcellular distribution of circ_0007841 was analyzed by qRT-PCR. (E) Overall survival rate was determined based on circ_0007841 median value in OC tissues. *P<0.05 by Student’s t-test.

Circ_0007841 overexpression promotes OC proliferation, migration and invasion

Next, circ_0007841 was ectopically expressed in SKOV3 and OVCAR3 cells (Figure 2A). CCK8 assay showed that circ_0007841 overexpression enhanced the proliferation rate (Figure 2B). Colony formation assay indicated that circ_0007841 upregulation caused more colony formation (Figure 2C). Transwell migration and invasion assays illustrated that circ_0007841 ectopic expression promoted cell migration and invasion (Figure 2D, 2E).

Figure 2. circ_0007841 overexpression promotes OC proliferation, migration and invasion. (A) Validation of circ_0007841 upregulation in OC cells after transfection. (B, C) CCK8 and colony formation assays used for cell proliferation analysis. (D, E) Transwell assays were performed to determine migration and invasion. *P<0.05 by Student’s t-test.

Circ_0007841 knockdown suppresses malignant biological behaviors

To further validate the roles of circ_0007841, we knocked it down through shRNA in SKOV3 and OVCAR3 cells (Figure 3A). Similarly, CCK8 and colony formation assays showed that circ_0007841 knockdown inhibited the proliferation rate of OC cells (Figure 3B, 3C). Transwell assays also suggested that circ_0007841 downregulation resulted in less migrated or invaded cells (Figure 3D, 3E). Notably, rescue expression of circ_0007841 abrogated the effects of its silencing (Figure 3B–3E). Thus, circ_0007841 promotes OC progression.

Figure 3. circ_0007841 knockdown suppresses malignant biological behaviors. (A) Validation of circ_0007841 silencing by qRT-PCR. (B, C) Cell proliferation was detected by using CCK8 and Transwell assays. (D, E) Transwell assays were carried out to analyze migration and invasion. *P<0.05 by Student’s t-test.

Circ_0007841 is the ceRNA for miR-151-3p

To investigate the molecular mechanism, the potential targets of circ_0007841 were predicted by using circinteractome (https://circinteractome.nia.nih.gov/). The five most potential targets were listed (Figure 4A). Luciferase reporter assay showed that only miR-151-3p mimics inhibited the activity of circ_0007841 reporter (Figure 4B). We then constructed corresponding wide-type and mutant luciferase reporters (Figure 4C). Luciferase reporter assay showed that only circ_0007841-WT reporter activity was suppressed by miR-151-3p mimics (Figure 4D). Besides, miR-151-3p expression was increased after circ_0007841 silencing (Figure 4E). On the contrary, only circ_0007841-WT overexpression repressed miR-151-3p level in OC cells (Figure 4F). qRT-PCR analysis showed that miR-151-3p level was negatively correlated with circ_0007841 in OC tissues (Figure 4G).

Figure 4. circ_0007841 is the ceRNA for miR-151-3p. (A) Predicted targets of circ_0007841 by circinteractome. (B) Luciferase reporter assay was performed to validate the interaction between circ_0007841 and predicted targets. (C) Construction of wild-type (WT) and mutant (MUT) reporter vector. (D) Luciferase reporter assay using constructed luciferase reporters. (E) qRT-PCR analysis of miR-151-3p expression after circ_0007841 silencing. (F) qRT-PCR analysis of miR-151-3p expression after circ_0007841 overexpression. (G) Expression correlation between circ_0007841 and miR-151-3p in OC tissues. *P<0.05 by Student’s t-test.

MiR-151-3p targets MEX3C

Afterwards, the targets of miR-151-3p were predicted through three online tools and MEX3C was identified (Figure 5A). Similarly, the wide-type and mutant luciferase reporters were constructed (Figure 5A). Luciferase reporter assay indicated that MEX3C-WT reporter activity was suppressed by MEX3C mimics (Figure 5B). RIP assay showed that Ago2 could enrich both MEX3C mRNA and miR-151-3p (Figure 5C). In addition, miR-151-3p mimics inhibited the expression of MEX3C (Figure 5D). We noticed that miR-151-3p level was negatively correlated with MEX3C in OC tissues (Figure 5E). However, MEX3C level was positively correlated with circ_0007841 (Figure 5F). Circ_0007841 knockdown suppressed MEX3C expression whereas miR-151-3p inhibitors reversed it (Figure 5G), indicating that circ_0007841 promotes MEX3C expression via acting as the ceRNA for miR-151-3p.

Figure 5. miR-151-3p targets MEX3C. (A) MEX3C was predicted to be the target of miR-151-3p via TargetSan, miRDB and starBase. (B) Luciferase reporter assay. (C) RIP assay was performed to validate the interaction between miR-151-3p and MEX3C. (D) Relative expression of MEX3C was analyzed by qRT-PCR. (E) Expression correlation between MEX3C and miR-151-3p in OC tissues. (F) Expression correlation between MEX3C and circ_0007841 in OC tissues. (G) Relative expression of MEX3C was analyzed by qRT-PCR after transfection. *P<0.05 by Student’s t-test.

Circ_0007841 promotes OC progression through miR-151-3p/MEX3C axis

It was found that MEX3C was upregulated in OC tissues (Figure 6A). To explore whether circ_0007841 promotes OC progression relying on miR-151-3p/MEX3C axis, rescue assays were carried out. CCK8 and colony formation results showed that miR-151-3p inhibition or MEX3C overexpression rescued the inhibitory effects of circ_0007841 knockdown on proliferation (Figure 6B, 6C). Similarly, Transwell assays showed that miR-151-3p inhibition or MEX3C overexpression rescued the inhibitory effects of circ_0007841 knockdown on migration and invasion (Figure 6D, 6E). To confirm the role of circ_0007841/miR-151-3p/MEX3C axis in vivo, animal experiments were performed. Circ_0007841 knockdown suppressed tumor growth while miR-151-3p inhibition or MEX3C overexpression recovered tumor propagation (Figure 6F, 6G).

Figure 6. circ_0007841 promotes OC progression through miR-151-3p/MEX3C axis. (A) MEX3C expression was analyzed in OC tissues by qRT-PCR. (B, C) CCK8 and colony formation assays were performed to analyze proliferation. (D, E) Transwell assay was conducted to detect migration and invasion. (F) Tumor volumes were measured every one week. (G) Tumor size was measured after five weeks. *P<0.05 by Student’s t-test.

Tissues collection

43 OC tissues and adjacent normal tissues were collected from The Second Hospital of Harbin Medical University. Tissues were stored in liquid nitrogen and not treated by chemotherapy or radiotherapy before surgery. Association of circ_0007841 level and clinicopathological parameters in OC patients was presented in Table 1. This study was approved by the Ethics Committee of The Second Hospital of Harbin Medical University. All patients provided written informed consents.

Cell culture

Human OC cell lines and normal ovarian epithelial IOSE80 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured using DMEM medium supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin. All plasmids and oligonucleotides were purchased from RiboBio (Guangzhou, China). Transfection was carried out through Lipofectamine 3000 reagent following the manufacturer’s protocols.

RNA extraction and qRT-PCR

Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) and utilized to synthesize cDNA through igh Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qPCR was performed using SYBR Premix Ex Taq™ kit (Takara). Relative expression was normalized to GAPDH or U6 and calculated through 2^−ΔΔCt method.

Cell proliferation assays

For Cell Counting Kit 8 (CCK8, Beyotime, China) assay, Cells were seeded into 96-well plates and cultured. At indicated time points, CCK8 solution was added and incubated for 2 hours. OD values at 450 nm were detected through a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

For colony formation assay, cells were plated into 6-well plates and cultured for 14 days. Finally, colonies was fixed with 4 % paraformaldehyde, stained with crystal violet and photographed.

Transwell assay

For migration assay, cells were seeded into the upper part with 200 μl serum-free medium. The lower chamber was filled with 600 μl complete medium. After cultured for 48 h, the cells on the bottom chamber was fixed with 4 % paraformaldehyde, stained with crystal violet and photographed. For invasion assay, the upper chamber was pre-coated with Matrigel and other steps were the same as migration assay.

Bioinformatics analysis

The correlation between miR-151-3p and MEX3C was predicted by using TargetScan, miRDB and starBase tools.

Dual-luciferase reporter assay

Reporter vectors were obtained through inserting circ_0007841 or MEX3C sequence into the PGL3 vector (Promega, Madison, WI). For luciferase reporter assay, miR-151-3p and reporter vectors were co-transfected into cells using Lipofectamine 3000. After 48 h, the luciferase activity was measured using the dual-Luciferase Reporter Assay System (Promega, Sunnyvale, CA) based on the manufacturer’s protocols.

RNA-immunoprecipitation (RIP) assay

RIP assay was performed using Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, MA) according to manufacturer’s instructions. Cell lysates were incubated with IgG (as a negative control) and Ago2 antibody. Then precipitated RNA was analyzed by qPCR.

Animal experiments

6-week-old BALB/c nude mice were randomly divided into two groups (n=5 for each group). Then SKOV3 cells were inoculated subcutaneously into the flanks of nude mice. Tumor volumes were measured every one week according to the formula: Tumor volume = (length×width²)/2. Animal experiments were approved by the Ethics Committee of The Second Hospital of Harbin Medical University.

Statistical analyses

Results were analyzed by GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA) and expressed as the mean ± SD. All experiments were repeated at least three times. P values were analyzed using Student’s t-test for two group comparison or one-way ANOVA for multiple group comparison. Kaplan Meier survival analysis was used for analysis of survival rate and P-value was calculated by the log-rank test. P<0.05 was considered as statistically significant.