circFMN2 Sponges miR-1238 to Promote the Expression of LIM-Homeobox Gene 2 in Prostate Cancer Cells.

Circular RNAs (circRNAs) regulate gene expression in different malignancies. However, the molecular mechanisms that link circRNAs with the tumorigenesis of prostate cancer (PCa) are not well understood. In the present study, we attempted to provide a novel basis for targeted therapy for PCa from the aspect of circRNA-microRNA (miRNA)-mRNA interaction. We investigated the expression of circRNAs in 5 paired PCa tissues and adjacent non-tumor tissues by microarray analysis. We focused on hsa_circ_0005100, which is located on chromosome 1 and derived from FMN2, and thus we named it circFMN2. The qRT-PCR was used to detect circFMN2 and target miRNA expression in PCa tissues and cell lines. Biological functional experiments were performed to detect the effects of circFMN2 on the biological behavior of PCa cells in vivo and in vitro. Bioinformatic analysis was utilized to predict potential miRNA target sites on circFMN2. High expression of circFMN2 was associated with PCa progression. Function assays revealed that knockdown of circFMN2 significantly reduced PCa cell growth in vitro and in vivo. Finally, we found that circFMN2 acts as a competing endogenous RNA (ceRNA) for miR-1238 to regulate LIM-homeobox gene 2 (LHX2) expression. circFMN2 regulates the miR-1238/LHX2 axis to promote PCa progression.

Introduction

Prostate cancer (PCa) is the most frequent malignancy accounting for 25% of all newly diagnosed cancers in men and is the second leading cause of death from cancer. A lack of the biomarker in early diagnosis resulted in low overall survival of PCa patients. Through the tremendous advances in diagnosis and treatment, the prognosis of PCa is still not satisfactory. Exploring the molecular invents associated with the tumorigenesis of PCa is of great significance for the early diagnosis or treatment of PCa.

Circular RNA (circRNA) is a novel type of RNA that features a covalently closed loop with no 5′-3′ polarity., circRNAs are stable, abundant, conserved, and usually expressed in particular tissues or at a specific developmental stage., Recent evidence demonstrated that circRNAs broadly participate in the initiation and progression of various diseases, especially in malignant tumors., circRNA can act as a “super sponge” for microRNA (miRNA)., To date, some circRNAs have been defined to be PCa-specific, such as circ_0057558, circ_0062019, and circ_0141940. Therefore, investigating the function and mechanism of novel circRNAs is essential for finding inhibitor or facilitator in tumorigenesis.

Exosomes, with diameters of 30–100 nm, are cell-derived vesicles that are present in many, and perhaps all, eukaryotic fluids, including blood, and urine, and in the culture medium of cells. Exosomes are involved in cell-to-cell signaling and may influence processes in normal cells because they can merge with and release their contents into cells that are distant from their cell of origin. Recent studies have demonstrated that circRNAs are abundant and stable in exosomes; however, the expression of exosomal circRNAs in PCa remain undefined.

In this study, we first compared the expression patterns of circRNAs between PCa tissues and controls. We focused on hsa_circ_0005100, which is located on chromosome 1 and derived from FMN2, and thus we named it circFMN2. We further tested circFMN2 in 90 pairs of PCa samples by qRT-PCR and the results showed that the expression of circFMN2 was markedly elevated in PCa tissues. We examined the functions of circFMN2 in PCa and found that knockdown of circFMN2 inhibited cell proliferation, migration, and invasion. In addition, we found that circFMN2 may function as the sponge of oncogenic miR-1238 to upregulate LIM-homeobox gene 2 (LHX2) expression. Moreover, circFMN2 expression was detectable in extracted serum exosomes derived from PCa patients. Therefore, circFMN2 may serve as a promising biomarker and as a potential therapeutic target for PCa patients.

Results

Profiles of circRNAs in PCa

A total of 7,385 circRNAs were detected in PCa and paired non-tumorous samples by the circRNA microarray analysis. Among them, 988 circRNAs were significantly aberrantly expressed (p < 0.05 and fold change [FC] >2.0) between PCa tissues and paired non-tumorous tissues. Of these circRNAs, 436 were significantly upregulated and 552 were significantly downregulated in PCa tissues compared with paired adjacent normal tissues. The five most upregulated circRNAs (circ_0057553, circ_0056881, circ_0074032, circ_0062019, and circ_0005100) and five most downregulated circRNAs (circ_0075539, circ_0034470, circ_0060328, circ_0078704, and circ_0000096) were shown in Figure 1A. We validated the five most upregulated circRNAs expression by qRT-PCR using 90 PCa and paired non-tumorous tissue samples. As shown in Figures 1B–1F, except for circ_0062019, the circRNAs displayed a consistent expression level between the microarray and qRT-PCR analyses. There was an increasing trend in hsa_circ_0005100 (chr1:240458121–240497529) levels from non-tumorous tissues to PCa tissues, with more than 10-FC from microarray analysis. By browsing the human reference genome (GRCh37/hg19), we identified that hsa_circ_0005100 is derived from FMN2, which is located on chromosome 1, with a spliced mature sequence length of 612 base pairs (bp), and thus we named it circFMN2.

Figure 1

Deregulated circRNAs in PCa Tumor Tissues

(A) The heatmap showed the top ten most increased and decreased circRNAs in PCa tissues as compared to that in the matched non-tumor tissues analyzed by circRNAs Arraystar Chip. (B) The level of circ_0056881 was significantly increased in tumor tissues as compared to that in matched non-tumor tissues of 88 pairs of PCa patients. ∗∗∗p < 0.001. (C) The level of circ_0057553 was significantly increased in tumor tissues as compared to that in matched non-tumor tissues of 88 pairs of PCa patients. ∗∗∗p < 0.001. (D) The level of circ_0074032 was significantly increased in tumor tissues as compared to that in matched non-tumor tissues of 88 pairs of PCa patients. ∗∗∗p < 0.001. (E) The level of circ_0062019 was not significantly increased in tumor tissues as compared to that in matched non-tumor tissues of 88 pairs of PCa patients. p = 0.08. (F) The level of circ_0005100 was significantly increased in tumor tissues as compared to that in matched non-tumor tissues of 88 pairs of PCa patients. ∗∗∗p < 0.001.

To further investigate the role of circFMN2 in PCa, we analyzed the relationship between circFMN2 expression in PCa tissues and clinicopathological characteristics of PCa patients. Using the median expression level of circFMN2 as cutoff value, patients who expressed circFMN2 equal to or greater than the average level were assigned to the “circFMN2 high” group. As shown in Table 1, high expression of circFMN2 in PCa tissues was significantly correlated with advanced tumor stage (p = 0.005), lymph node metastasis (p = 0.001), and distant metastasis (p = 0.02), but not with other characteristics of PCa. The data provide evidence of the importance of circFMN2 in the growth and tumorigenesis of PCa.

Table 1

Relationship between circFMN2 Expression and the Clinical Pathological Characteristics of PCa Patients

Clinicopathological Features	Overall (n = 88)	circFMN2		p
		Low	High
		(n = 44)	(n = 44)
Age, y
More than median	43	20	23	0.67
Clinical T stage
T3 and T4	38	12	26	0.005
Lymph node metastasis
Yes	40	12	28	0.001
Distant metastasis
Yes	17	4	13	0.02

circFMN2 Promotes Proliferation of PCa Cells by Facilitating DNA Synthesis and Inhibiting Cell Apoptosis

The expression levels of circFMN2 in multiple PCa cell lines were measured. The expression level of circFMN2 in PCa cell lines was generally higher than that in the human normal prostate epithelial cell line (RWPE-1) (Figure 2A; p < 0.01). The expression of circFMN2 was highest in PC3 and DU145 cells and lowest in VCaP cells. These cells were then used to establish cell lines with knockdown or overexpression of circFMN2. To investigate the functional role of circFMN2 in PCa cells, we performed loss- or gain-of-function experiments. First, we knocked down the expression of circFMN2 and FMN2 mRNA. We designed circFMN2-specific small interfering RNAs (siRNAs) targeting the backsplice sequence (respectively si-circFMN2#1, si-circFMN2#2, or si-circFMN2#3) or the sequence only in the linear transcript (si-FMN2). As expected, siRNA directed against the backsplice sequence knocked down only the circular transcript and did not affect the expression of linear species, and siRNA targeting the sequence in the linear transcript knocked down only the linear transcript and did not affect the expression of the circular transcript in PC3 and DU145 cells (Figures 2B–2E; Figures S1A–S1D; p < 0.01). Due to the highest efficiency of interference, si-circFMN2#3 was chosen for the subsequent experiments. Meanwhile, we induced ectopic overexpression of circFMN2 by transfecting VCaP cell lines with pcDNA-circFMN2 expression vector (Figure 2F; p < 0.01).

Figure 2

Transfection of circFMN2 in PCa Cells

(A) The qRT-PCR assay indicated the expression level of circFMN2 in PCa cell lines was higher than that in the human normal prostate epithelial cell line RWPE-1, ∗∗p < 0.01. (B) The qRT-PCR assay indicated the expression level of circFMN2 in PC3 and DU145 cells treated with si-circFMN2. Data are the means ± SD of three experiments, ∗∗p < 0.01. (C) The qRT-PCR assay indicated the expression level of FMN2 in PC3 and DU145 cells treated with si-FMN2. Data are the means ± SD of three experiments, ∗∗p < 0.01. (D) The qRT-PCR assay indicated the expression level of circFMN2 in VCaP cells infected with circFMN2 overexpression plasmid, ∗∗p < 0.01.

Next, we investigated the stability and localization of circFMN2 in PC3 and DU145 cells. Total RNAs from PC3 and DU145 cells were isolated at the indicated time points after treatment with Actinomycin D, an inhibitor of transcription. Then qRT-PCR was performed to measure the level of circFMN2 and FMN2 mRNA. The results showed that the half-life of circFMN2 exceeded 24 h, whereas that of FMN2 mRNA was about 3 h in both PC3 and DU145 cells (Figures 3A and 3B). Furthermore, we found that circFMN2 was resistant to RNase R digestion (Figures 3C and 3D; p < 0.01). We then investigated the localization of circFMN2. qRT-PCR of RNAs from nuclear and cytoplasmic fractions indicated that circFMN2 was predominantly localized in the cytoplasm of PC3 and DU145 cells (Figures 3E and 3F; p < 0.01). Collectively, the above data suggested that circFMN2 harbored a loop structure and was predominantly localized in the cytoplasm.

Figure 3

Characterization of circFMN2 in PCa Cells

(A) The qRT-PCR for the abundance of circFMN2 and FMN2 in PC3 cells treated with Actinomycin D at the indicated time point. (B) The qRT-PCR for the abundance of circFMN2 and FMN2 in DU145 cells treated with Actinomycin D at the indicated time point. (C) circFMN2 was resistant to RNase R digestion in PC3 cells. Data are the means ± SD of three experiments, ∗∗p < 0.01. (D) circFMN2 was resistant to RNase R digestion in DU145 cells. Data are the means ± SD of three experiments, ∗∗p < 0.01. (E) Levels of circFMN2 in the nuclear and cytoplasmic fractions of PC3 cells. Data are the means ± SD of three experiments, ∗∗p < 0.01. (F) Levels of circFMN2 in the nuclear and cytoplasmic fractions of DU145 cells. Data are the means ± SD of three experiments, ∗∗p < 0.01.

A Cell Counting Kit-8 (CCK-8) assay showed that circFMN2 knockdown significantly induced a proliferation-suppressing effect on PC3 and DU145 cells (Figures 4A and 4B, p < 0.01), whereas the results showed that the growth of VCaP cells transfected with pcDNA-circFMN2 was significantly increased compared with that of control cells (Figure 4C, p < 0.01). Consistently, colony formation assays showed that circFMN2 knockdown decreased colony formation ability of PC3 and DU145 cells (Figures 4D and 4E, p < 0.01). Furthermore, colony-formation assays also indicated that clonogenic survival was significantly increased in VCaP cells transfected with pcDNA-circFMN2 (Figure 4F, p < 0.01).

Figure 4

circFMN2 Inhibits PCa Growth In Vitro

(A) CCK-8 assay showed that circFMN2 knockdown significantly repressed cell proliferation of PC3 cells, ∗∗p < 0.01. (B) CCK-8 assay showed that circFMN2 knockdown significantly repressed cell proliferation of DU145 cells, ∗∗p < 0.01. (C) CCK-8 assay showing overexpression of circFMN2 promoted the proliferation of VCaP cells, ∗∗p < 0.01. (D) Colony formation assay showed that the knockdown of circFMN2 significantly restrained the proliferation of PC3 cells. (E) Colony formation assay showed that the knockdown of circFMN2 significantly restrained the proliferation of DU145 cells. (F) Colony formation assay showed that the ectopic expression of circFMN2 significantly promoted the proliferation of VCaP cells.

To assess whether the pro-proliferative effects of circFMN2 on the PCa cells are mediated by promoting cell-cycle progression, we examined cell cycling in PC3 and DU145 cells by flow cytometry. As shown by flow cytometry analysis, circFMN2 knockdown led to an arrest in G1 phase (Figures 5A and 5B, p < 0.01) and increased apoptotic rates of PC3 and DU145 cells compared with those in the NC groups (Figures 5D and 5E). Correspondingly, overexpression of circFMN2 decreased G0/G1 phase percentage and inhibited apoptosis in VCaP cells (Figures 5C–5F, p < 0.01). Therefore, these data suggested that circFMN2 promotes cell proliferation of PCa by facilitating DNA synthesis and inhibiting cell apoptosis.

Figure 5

Knockdown of circFMN2 Inhibits the Proliferation of PCa Cells by Inducing Cell-Cycle Arrest

(A) The knockdown of circFMN2 significantly increased the percent of PC3 cells in G0/G1 phase and decreased the percent of PC3 cells in G2 phase. Data are the means ± SD of three experiments. ∗∗p < 0.01. (B) The knockdown of circFMN2 significantly increased the percent of DU145 cells in G0/G1 phase and decreased the percent of DU145 cells in G2 phase. Data are the means ± SD of three experiments. ∗∗p < 0.01. (C) The overexpression of circFMN2 significantly decreased the percent of VCaP cells in G0/G1 phase and increased the percent of VCaP cells in G2 phase. Data are the means ± SD of three experiments. ∗∗p < 0.01. (D) The knockdown of circFMN2 significantly increased apoptotic rates of PC3 cells. (E) The knockdown of circFMN2 significantly increased apoptotic rates of DU145 cells. (F) circFMN2 overexpression inhibited apoptosis in VCaP cells.

circFMN2 Promotes Migration and Invasion of PCa Cells In Vitro

We next examined the invasion and migration of PCa cells. As shown in transwell assays, inhibition of circFMN2 expression attenuated the invasion and migration of PC3 and DU145 cells (Figures 6A and 6B), whereas overexpression of circFMN2 dramatically promoted cell invasion and migration in the VCaP cells, as indicated by comparisons with the negative control groups (Figure 6C). To measure the effect of silencing circFMN2 expression on EMT of PCa cells, western blot was performed to examine the expression of EMT-related markers in PCa cells after inhibition of circFMN2. As expected, circFMN2 knockdown remarkably increased the expression of E-cadherin and meanwhile greatly decreased the expression of N-cadherin and Vimentin in PC3 and DU145 cells (Figures 6D and 6E). Correspondingly, overexpression of circFMN2 decreased the expression of E-cadherin and meanwhile greatly increased the expression of N-cadherin and Vimentin in VCaP cells, indicating that downregulation of circFMN2 obviously blocked the EMT process (Figure 6F).

Figure 6

circFMN2 Induces PCa Cell Migration and Invasion via EMT

(A) Transwell assays showed that the inhibition of circFMN2 expression attenuated the invasion and migration of PC3 cells. (B) Transwell assays showed that the inhibition of circFMN2 expression attenuated the invasion and migration of DU145 cells. (C) Transwell assays showed that the overexpression of circFMN2 dramatically promoted cell invasion and migration in the VCaP cells. (D) The western blot assays showed that circFMN2 knockdown remarkably increased the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin in PC3 cells. (E) The western blot assays showed that circFMN2 knockdown remarkably increased the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin in DU145 cells. (F) The western blot assays showed that overexpression of circFMN2 decreased the expression of E-cadherin and increased the expression of N-cadherin and Vimentin in VCaP cells.

circFMN2 Has No Effect on Its Linear Transcript

Some circRNAs regulate the expression and function of the corresponding linear transcripts. Therefore, the regulatory relationship between circFMN2 and its linear transcript (FMN2) was investigated in this study. We first analyzed the prostate adenocarcinoma (PRAD) dataset from the TCGA database and found that the level of FMN2 was not significantly higher in 492 PRAD tissues than 152 normal tissues (p < 0.05; Figure 7A). Kaplan-Meier survival analysis from TCGA PRAD datasets suggested that FMN2 expression is not significantly associated with worse overall survival (OS; log-rank test, p = 0.74, Figure 7B), however, FMN2 expression is significantly associated with worse disease-free survival (DFS) of PRAD patients (log-rank test, p = 0.016, Figure 7C). FMN2 did not change in both mRNA and protein levels when the expression of circFMN2 was artificially changed in PCa cells (Figures S1A and S1B; Figure 7D). These data indicated that FMN2 is not the target gene of circFMN2.

Figure 7

circFMN2 Has No Effect on Its Linear Transcript

(A) The level of FMN2 was not significantly higher in 492 prostate adenocarcinoma (PRAD) tissues than 152 normal tissues from the TCGA database. (B) Kaplan-Meier analyses of the correlations between FMN2 expression and overall survival (OS) of PRAD patients from the TCGA PRAD dataset. (C) Kaplan-Meier analyses of the correlations between FMN2 expression and disease-free survival (DFS) of PRAD patients from the TCGA PRAD dataset. (D) The western blot assay indicated circFMN2 knockdown did not change the protein level of FMN2 in PC3 cells.

Knockdown of circFMN2 Inhibits PCa Growth In Vivo

To identify the effect of circFMN2 on tumor growth in vivo, we subcutaneously injected VCaP cells stably transfected with circFMN2-overexpressing vector or control vector into nude mice. The tumor volumes were monitored from the 14 days after VCaP cell injection. We found that overexpression of circFMN2 drastically increased tumor growth of VCaP cells. The tumor volumes and weights were significantly accelerated by circFMN2 (Figures 8A and 8B). After harvesting the subcutaneous tumor tissues, immunohistochemistry was performed. Immunohistochemistry (IHC) staining results showed that overexpressed circFMN2 significantly promoted the expression of Ki67 when compared with the control group (Figure 8C). In addition, the impact of circFMN2 knockdown upon tumor growth in vivo was also investigated. A xenograft tumor model of PC3 cells was developed and then treated with intratumoral injection of cholesterol-conjugated si-circFMN2 or si-NC. As shown in Figures 8D and 8E, treatment with si-circFMN2 significantly inhibited growth of PC3 in vivo. Results of IHC revealed that xenograft tumors derived from PC3 cells with circFMN2 knockdown had lower expression of Ki67 than the si-NC group (Figure 8F). Taken together, these findings suggest that circFMN2s may play an oncogenic role in PCa in vivo.

Figure 8

circFMN2 Promotes Tumor Growth In Vivo

(A) The volume of subcutaneous xenograft tumors of VCaP cells isolated from nude mice. ∗∗p < 0.01. (B) The weight of subcutaneous xenograft tumors of VCaP cells isolated from nude mice. ∗p < 0.05. (C) IHC analysis was performed to examine the expression levels of Ki67 in xenograft tumors of VCaP cells isolated from nude mice. (D) The volume of subcutaneous xenograft tumors of PC3 cells isolated from nude mice. ∗∗p < 0.01. (E) The weight of subcutaneous xenograft tumors of PC3 cells isolated from nude mice. ∗p < 0.05. (F) IHC analysis was performed to examine the expression levels of Ki67 in xenograft tumors of PC3 cells isolated from nude mice.

circFMN2 Promoted PCa Progression by Sponging miR-1238

Increasing studies have verified that circRNAs can exert their functions by acting as competing endogenous RNAs (ceRNAs) to sponge miRNAs. Considering the cytoplasmic localization of circFMN2 in PCa cells, we investigated whether circFMN2 exerted function in PCa in a miRNA-mRNA-dependent manner. StarBase v2.0 target prediction tool was used to investigate the potential miRNAs associated with circFMN2, and the most potentially complementary binding miRNAs were presented. Among these target miRNAs, we found that circFMN2 has a binding site for miR-1238 (Figure 9A). Next, luciferase reporter assays were carried out to verify whether there was a direct interaction between circFMN2 and miR-1238. As shown in Figure 9B, miR-1238 mimics could significantly decrease the luciferase activity of the vector containing the wild-type (WT) miR-1238 binding site within circFMN2, while the luciferase activity of the vector containing the mutant binding site was restored. A previous study reported that Argonaute 2 (Ago2) protein binds with both circRNAs and miRNAs to form the RNA-induced silencing complex. Therefore, an RNA immunoprecipitation (RIP) assay was presently performed to pull down RNA transcripts bound to Ago2 in PC3 cells. As expected, both circFMN2 and miR-1238 were efficiently pulled down by anti-Ago2, but not by the non-specific anti-immunoglobulin G (IgG) antibody (Figure 9C). These outcomes indicated that the interaction of circFMN2 and miR-1238 was realized by the putative binding site. The qRT-PCR analysis indicated that miR-1238 was expressed at low level in PCa tissues (Figure 9D; p < 0.01). The expression of miR-1238 was obviously decreased in PC3 and DU145 cells, indicating the opposite result to circFMN2 expression (Figure S2A; p < 0.01). The decreased expression level of miR-1238 in circFMN2-overexpressed PCa cells indicated the negative regulatory effect of circFMN2 on miR-1238 expression (Figure 9E; p < 0.01).

Figure 9

circFMN2 Promoted PCa Progression by Sponging miR-1238

(A) The binding sequence between miR-1238 and circFMN2. (B) Dual luciferase reporter showed significant reduction of luciferase activity of the wild-type and luciferase activity is restored by the mutant sequence. (C) The RIP experiment showed that miR-1238 and circFMN2 simultaneously existed in the production precipitated by anti-AGO2. (D) The qRT-PCR analysis indicated that miR-1238 was expressed at low level in PCa tissues. (E) Overexpression of circFMN2 decreased expression level of miR-1238 in PC3 cells. Data are the means ± SD of three experiments. ∗∗p < 0.01.

To functionally confirm that circFMN2s promote PCa progression by sponging miR-1238, we transfected miR-1238 mimics into circFMN2-overexpressing cells to examine whether the tumor-promoting effect of circFMN2 overexpression could be reversed by miR-1238 mimics. The results showed that miR-1238 could significantly reverse the circFMN2 overexpression-mediated promotion of proliferation (Figure S2B; p < 0.01). These data suggested that circFMN2 might exert its functions by sponging miR-1238 in PCa.

circFMN2 Upregulates LHX2 Expression Via Sponging miR-1238

miRNAs can bind to and inhibit mRNA expression. To further understand the mechanism of miR-1238 in PCa progression, we predicted the binding partner of miR-1238 by TargetScan online software. The results showed that miR-1238 may bind to LHX2 mRNA (Figure 10A). To verify whether the 3′ UTR of LHX2 mRNA was a target of miR-1238 in 293T cells, we used a luciferase reporter gene assay. The WT 3′ UTR sequence or mutant (mu) 3′ UTR sequence of LHX2 was cloned into a luciferase reporter vector. The luciferase activity was significantly inhibited by the miR-1238 mimics in WT 3′ UTR sequence-transfected 293T cells. Conversely, the luciferase activity was not inhibited by the miR-1238 mimics in mu 3′ UTR sequence-transfected cells (Figure 10B; p < 0.01). We determined the expression of LHX2 in 88 PCa patient tissues using qRT-PCR and IHC. The results of IHC showed that LHX2 expression in PCa specimens was significantly upregulated compared with that in the adjacent normal tissues (Figure 10C). LHX2 overexpression was observed in 68 of 88 (77.27%) PCa specimens when compared with adjacent normal tissues (17 of 88, 19.32%), the difference of LHX2 expression was statistically significant (p < 0.0001). We also investigated the LHX2 mRNA expression in 88 pairs of PCa non-tumorous tissue samples and found that LHX2 mRNA expression was higher in PCa tissues compared to their normal counterparts (Figure 10D; p < 0.01). To explore the role of LHX2 inhibition on PCa malignant phenotypes, we silenced endogenous LHX2 levels using two siRNAs in PC3 cells (Figure 10E; p < 0.01) and the effect on PCa cell proliferation was assessed via CCK-8 assays. We found that LHX2 silencing significantly decreased cell proliferation of PC3 cells (Figure 10F; p < 0.01). Then, we found that levels of LHX2 mRNA expression in PC3 cells were negatively regulated by circFMN2 knockdown, and the effect of inhibition of circFMN2 was attenuated by miR-1238 inhibitor (Figure 10G; p < 0.01). These data further demonstrated the regulatory network of circFMN2/miR-1238/LHX2. According to above data, we confirmed that circFMN2 can exert function in PCa by sponging miR-1238 to upregulate LHX2 expression.

Figure 10

circFMN2 Upregulates LHX2 Expression via Sponging miR-1238

(A) Bioinformatics analysis revealed the predicted binding sites between LHX2 and miR-1238. (B) Luciferase reporter assay demonstrated miR-1238 mimics significantly decreased the luciferase activity of LHX2-WT in 293T cells. (C) IHC assay indicated the expression of LHX2 was significantly upregulated in PCa tissues compared with adjacent non-tumorous tissues. (D) The qRT-PCR assay indicated the expression level of LHX2 was significantly upregulated in PCa tissues compared with adjacent non-tumorous tissues. (E) Endogenous LHX2 levels were silenced using two siRNAs in PC3 cells. (F) CCK-8 assays showed that LHX2 silencing significantly decreased cell proliferation of PC3 cells. (G) The effect of knockdown circFMN2 on mRNA levels of LHX2 was attenuated by miR-1238 inhibitor. All tests were at least performed three times. Data were expressed as mean ± SD. ∗∗p < 0.01.

circFMN2 Is Secreted by Exosomes into Serum of PCa Patients

Finally, in our current study, we collected abundant serums from 30 PCa patients and 30 normal people. After isolation of serum exosomes by sequential centrifugation, transmission electron microscopy (TEM) analysis showed that isolated PCa-secreted exosomes had similar morphologies (30–150 nm in diameter) and exhibited a round-shaped appearance (Figure S2C). The nanoparticle tracking analysis (NTA) results demonstrated that isolated PCa-secreted exosomes showed a similar size distribution, and the peak size range was 80–135 nm. Moreover, the presence of the exosome markers CD63, TSG101, and HSP70 were confirmed by western blot (Figure S2D). Our results showed that circFMN2 expression is detectable in extracted serum exosomes derived from 30 PCa patients (Figure S2E; p < 0.01). Furthermore, there was a significant inverse correlation between the expression levels of circFMN2 and miR-1238 in serum exosomes derived from 30 PCa patients (Figure S2F, r = −0.727, p < 0.0001).

Discussion

In recent years, circRNAs as a type of regulatory RNAs have attracted great research interest. circRNAs are characterized by covalently closed loop structures with neither 5′-3′ polarity nor a polyadenylated tail. circRNAs have been shown to have critical regulatory roles in cancer biology. Several scholars have shown that circRNAs are dysregulated in many cancers. Despite the progress researchers have made in the field of cancer function, the function of circRNAs in PCa progression remains largely unknown. In this study, a novel upregulated circRNA in PCa, circFMN2, was identified in circRNA microarray analysis from PCa tissues, as well as in PCa cell lines. Moreover, elevated expression of circFMN2 was positively correlated with larger tumor size and higher TNM stage in PCa patients. Loss-of-function experiments revealed that knockdown of circFMN2 inhibited proliferation and promoted apoptosis of PCa cells. Gain-of-function experiments revealed that ectopic expression of circFMN2 promoted proliferation and inhibited apoptosis of PCa cells. In addition, xenograft experiments showed that circFMN2 promoted PCa xenograft growth in vivo. Moreover, our results showed that circFMN2 could significantly promote PCa cell invasion ability in vitro, and in vivo peritoneal metastasis assays also indicated that circFMN2 promotes cell metastasis of PCa, suggesting that circFMN2 might be a potential novel target for PCa therapy. Specifically, we also showed mechanistically that circFMN2 promotes the progression of PCa by functioning as a ceRNA of miR-1238 to upregulate LHX2. Finally, we showed that overexpressed circFMN2 was secreted by exosomes into the serum of PCa patients, suggesting that circFMN2 exerts oncogenic potential and it may be a candidate in diagnosis and treatment of PCa.

A number of recent papers have revealed that circRNAs are highly dysregulated in many types of cancers and exhibit high tissue and disease specificity. The deregulation of circRNAs may result in increased proliferation, invasion or angiogenesis and decrease the level of apoptosis or dedifferentiation, eventually leading to tumor formation. The cancer-associated circRNAs and investigation of their molecular and biological functions are important to provide new insights into the diagnosis of cancer, including PCa. Our research identified 988 differentially expressed circRNAs, with 436 upregulated and 552 downregulated, in tumor tissues of patients with PCa. We first found that circFMN2 was significantly higher in the PCa tissues and PCa cell lines compared to corresponding adjacent nontumorous tissues and the human normal prostate epithelial cell line (RWPE-1). Gain-of-function and loss-of-function experiments demonstrated that circFMN2 was associated with tumor progression. circFMN2 knockdown decreased cell proliferation and caused a dramatic decrease in colony formation of PCa cells, whereas circFMN2 overexpression has the opposite results. In addition, circFMN2 knockdown promoted significant arrest in the G0/G1-phase and an obvious increase in cell apoptosis of PCa cells. These observations of tumor growth were verified in a mouse xenograft model. Our results may enrich the study of the pathogenesis of PCa and provide a theoretical basis for the in-depth exploration of the function of circRNAs in PCa.

To detect the potential mechanism pattern of circFMN2 in PCa, we detected the cellular localization of circFMN2 in PCa cells and determined that circFMN2 was predominantly located in the cytoplasm of PCa cells, indicating that circFMN2 may regulate gene expression at post-transcriptional level. In term of post-transcriptional regulation, circFMN2s have been acknowledged to be ceRNAs by competitively binding to miRNA response elements to upregulate mRNAs. On this basis, we carried out bioinformatics analysis and mechanism experiments to determine downstream genes of circFMN2. It was found that circFMN2 exerted its function as a ceRNA that competitively bound to miR-1238 and then abolished the endogenous suppressive effect of miR-1238 on the target gene LHX2. We found that the miR-1238 was significantly lower in PCa tissues compared with adjacent normal tissues, and LHX2 expression was significantly higher in PCa tissues. The miR-1238 inhibitor could attenuate the anti-tumor effects mediated by circFMN2 knockdown. On the other hand, our current study unveiled that LHX2 was the direct target of miR-1238. All of the above results suggest that miR-1238 can bind with circFMN2 and LHX2, respectively.

Most Recently, Exosomal circRNAs Have Attracted Increasing Interest

Exosomes are a novel class of extracellular vesicles that have gained enormous attention lately as facilitators of the progression of various tumors. Exosomes are now recognized as critical messengers of intercellular crosstalk by transferring molecular cargo to recipient cells and have potential clinical applications in cancer diagnosis. In particular, cancer-derived exosomal circRNAs play a key role in cell-cell communication to promote tumor progression and exist in body fluids, where they can serve as non-invasive biomarkers. In this study, we found that circulating exosomes from PCa patients contain higher levels of circFMN2 than healthy circulating exosomes. Thus we assume that exosomal circFMN2 in the serum of PCa patients could function as a candidate in diagnosis of PCa.

In this study, the relationship between circFMN2 and disease severity suggests that circFMN2 plays a critical role in the development of PCa. We demonstrate that circFMN2 functions as an oncogene to positively regulate PCa progression. We also discovered that downregulation of circFMN2 could suppress cell proliferation and migration, as well as facilitate tumor growth in vitro and in vivo. Inverse correlation and perfect binding sequences between elevated circFMN2 and reduced miR-1238 indicate that circFMN2 may be involved in pathogenesis of PCa via sponging miR1238. Mechanistically, circFMN2 could function as a sponge by harboring miR-1238 and thereby abolishing the suppressive effect on the target gene LHX2 in PCa development. Thus, our data enhance our understanding of circRNA biology and suggest that circFMN2 could play an important role in the pathogenesis and progression of PCa.

Materials and Methods

Patient Specimens and Cell Culture

The 90 matched PCa and adjacent non-tumor prostate tissues were acquired from PCa patients at Department of Urology, The First Affiliated Hospital of China Medical University from May 2010 to May 2018. After resection, all tissues were snap-frozen in liquid nitrogen and stored at −80°C. For exosome purification, serum samples were collected from PCa patients and healthy donors. The written informed consents were provided by all participants. Our study was approved by the Research Ethics Committee of May and complied with the Declaration of Helsinki.

Human PCa cell line (PC-3, DU145, VCaP, LNCaP) and human normal prostate epithelial cell line (RWPE-1) were bought from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cell culture was conducted in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA), 1% penicillin/streptomycin (HyClone) at 37°C with 5% CO2.

circRNAs Microarray Hybridization and Data Analysis

Total RNAs were digested with RNase R (20 U/μL, Epicenter, USA) to remove linear RNAs and enrich circular RNAs. The enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Super RNA Labeling Kit; Arraystar, Rockville, MD, USA). The labeled cRNAs were hybridized onto the Human circRNA Array (8 × 15 K, Arraystar). The slides were incubated for 17 h at 65°C in a hybridization oven (Agilent, Santa Clara, CA, USA). circRNAs differentially expressed with statistical significance between PCa and paired normal tissues (FC ≥ 2 and p ≤ 0.05) were identified through volcano plot filtering. Hierarchical clustering was performed to show the distinguishable expression pattern of circRNAs among samples.

RNA Extraction and Quantitative Real-Time PCR

Total RNAs from cells and tissues were extracted using Trizol (Invitrogen, Carlsbad, CA, USA). Total RNAs from plasma were extracted using TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA). The RNA extraction was performed according to the manufacturer’s instructions. For circRNA and mRNA, PrimeScript RT reagent Kit (TaKaRa, Dalian, China) was used to generate cDNA; TB GreenPremix Ex Taq II (TaKaRa, Dalian, China) was used to perform real-time PCR; 18S rRNA was utilized as internal control. For miRNA, miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, Beijing, China) was used to generate cDNA; miRcute Plus miRNA qPCR Kit (SYBR Green) was used to perform real-time PCR; U6 small nuclear RNA (snRNA) was utilized as internal control. Real-time PCR was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). 2−ΔΔCt method was used to calculate the relative expression of RNAs. The primers used in this study were synthesized by Sangon Biotech (Shanghai, China). The reverse primer for miRNA was supplied by the miRcute Plus miRNA qPCR Kit (SYBR Green).

TCGA Dataset Analysis

The data and the corresponding clinical information of patients were collected from the Cancer Genome Atlas (TCGA) database (http://cancergenome.nih.gov/). We used the edgeR package of R packages to perform the difference analysis (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) and used the pheatmap package of R packages to perform the cluster analysis (https://cran.r-project.org/web/packages/pheatmap/index.html). Sva R package was used to remove the batch effect. Genes with adjusted p values < 0.05 and absolute FCs > 1.5 were considered differentially expressed genes. Kaplan-Meier survival curves were drawn to analyze the relationships between genes and overall survival in the survival package. The corresponding statistical analysis and graphics were performed in R software (R version 3.3.2).

Cell Transfection

For cell transfection, cell lines at 70%–80% confluence were planted in 6-well plates. siRNA targeting the junction region of the circFMN2 sequence and miR-1238 mimics or inhibitors were designed and synthesized by RiboBio (Guangzhou, China). The sequence of circFMN2 siRNA were circFMN2#1: 5′-AAGAAAGACTTGAAAGCTGTT-3′; circFMN2#2: 5′-AAGACTTGAAAGCTGTTGTGA-3′; and circFMN2#3: 5′-AGACTTGAAAGCTGTTGTGAA-3′. A pcDNA 3.1 circRNA mini vector was used to ectopic express circFMN2 level in PCa cells. The sequence of LHX2 siRNA were siLHX2-1: 5′-GCAACCTCTTACGGCAGGAAA-3′ and siLHX2-2: 5′-CAACTGTGACGTCCGTCTTAA-3′. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for transfection following the standard method. 48 h later, cells were reaped for subsequent experiments.

Actinomycin D and RNase R Treatment

To block transcription, we added 2 mg/mL Actinomycin D or dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) as a negative control into the cell culture medium. For RNase R treatment, total RNA (2 μg) was incubated for 30 min at 37°C with or without 3 U/μg of RNase R (Epicenter Technologies, Madison, WI, USA). After treatment with Actinomycin D and RNase R, qRT-PCR was performed to determine the expression levels of circFMN2 and FMN2 mRNA.

Isolating RNAs from Nucleus and Cytoplasmic Fractions

The nuclear and cytoplasmic fractions were isolated using PARIS Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, cells were collected and lysed with cell fractionation buffer, followed by centrifugation to separate the nuclear and cytoplasmic fractions. The supernatant containing the cytoplasmic fraction was collected and transferred to a fresh RNase-free tube. The nuclear pellet was lysed with Cell Disruption Buffer. The cytoplasmic fraction and nuclear lysate were mixed with 2X Lysis/Binding Solution and then added with 100% ethanol. The sample mixture was drawn through a Filter Cartridge, followed by washing with Wash Solution. The RNAs of nuclear and cytoplasmic fractions were eluted with Elution Solution. U6 snRNA and 18S rRNA were employed as positive control for nuclear and cytoplasmic fractions, respectively.

CCK-8 Assay

Cell proliferation was assessed using the CCK-8 assay (Beyotime Biotechnology, Nantong, China). Cells (2 × 103) were seeded into each well of 96-well plates. 10 μL of CCK-8 solution was added to each well at six time points. After 1.5 h of incubation at 37°C, the absorbance at 450 nM was measured using Spectra Max 250 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Experiments were independently performed in triplicate.

Colony Formation Assays

For the colony formation assays, cells (1 × 103) were suspended and plated into each well of 6-well plates. After 14 days, incubation at 37°C in a chamber with an atmosphere of 5% CO2, colonies were fixed with 500 μL of 4% paraformaldehyde (Solarbio, Beijing, China) for 30 min and were stained with crystal violet (Beyotime Biotechnology, Nantong, China) for 25 min. Colonies were counted after photographing the sample (Nikon, Tokyo, Japan).

Cell Cycle and Apoptosis Flow Cytometry Analyses

At 48 h after transfection, transfected cells were harvested by trypsinization and resuspended in cold phosphate-buffered saline for analysis. For the analysis of cell cycle, cells stained with propidium iodide (PI) according to the manufacturer’s manual. The rate of cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions.

Cell Migration and Invasion Assays

Cell migration and invasion assays were conducted using Transwell Permeable Supports, polycarbonate (PC) membrane (Corning, NY, USA), which was coated with (for invasion assays) or without (for migration assays) the matrigel (Corning, NY, USA) according to the manufacturer’s instructions. 24 h after transfection, cells in 200 μL serum-free medium were seeded into the upper compartment, with 700 μL culture medium containing 20% FBS in the lower compartment. After 24 h incubation, the cells located on the upper surfaces of the transwell chamber were erased with cotton swabs and the cells located on the lower surfaces were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min at room temperature. The stained cells were photographed and counted in five randomly selected fields under a Leica DM4000B microscope (Leica, Wetzlar, Germany).

Western Blot Analysis

Briefly, total protein of tissue samples and cell lines was extracted using protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science) according to its protocol. Equal amount of total protein (20 μg) was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking for nonspecific binding, the membranes were incubated with following primary antibodies: E-cadherin (Abcam, 1:1,000), Vimentin (Abcam, 1:1,000), N-cadherin (Cell Signaling Technology, 1:1,000), FMN2 (Abcam, 1:1,000), TSG101 (1:1,000, Proteintech, Chicago, USA), HSP70 (1:2,000, Proteintech, Chicago, IL, USA), and GAPDH antibody (1:2,000; Cell Signaling Technology) overnight at 4°C. Then PVDF membranes were incubated with the appropriate secondary antibody (Santa Cruz Biotechnology, 1:10,000) for 1 h at room temperature. Bands were detected by a Bio-rad ChemiDoc XRS system.

Xenograft Model

All animal experiments were performed under approval by the Experimental Animal Care Commission of Cancer Hospital of China Medical University. For xenograft model of VCaP cells: 6-week-old male BALB/c nude mice were housed under standard conditions and cared for according to protocols. 2 × 106 VCaP cells with circFMN2 overexpressed vector or control vector were suspended in 200 μL serum-free RPMI-1640 and subcutaneously injected into the right flank of each mouse. The volumes of tumors were measured from 14 days after injecting. After 31 days the mice were sacrificed. For xenograft model of PC3 cells: 2 × 106 PC3 cells were subcutaneously injected into a single flank of each mouse (12 mice in total). 2 weeks later, mice with palpable tumors were randomly divided into two groups (six mice per group), 50 nmol cholesterol-conjugated si-NC or si-circFMN2 was intratumorally injected into the two groups three times per week for 2 weeks. Tumor growth was examined every 4 days. After mice were sacrificed, tumors were weighed and processed for further histological analysis. Tumor volume was calculated as follows: V (volume) = (length × width2)/2.

IHC

IHC analysis was performed under the manufacturer’s instructions. Briefly, the slides were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies at room temperature for 2 h. The expression was evaluated using a composite score obtained by multiplying the values of staining intensities (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the percentage of positive cells (0, 0%; 1, <10%; 2, 10%–50%; 3, >50%).

Dual-Luciferase Assay

Cells were seeded in 96-well plates at a density of 1 × 104 cells per well for 24 h before co-transfection. The cells were co-transfected with dual-luciferase reporter vector and miRNA mimics or inhibitors using the Lipofectomine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). After 48 h of incubation, firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions.

RIP Assay

The RIP assay was carried out using a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Bersinbio, Guangzhou, China) according to the manufacturer’s protocol. PC3 cells (2 × 107) were lysed in complete RIP lysis buffer and the cell lysates were divided into two equal parts and incubated with either 5 μg human anti-Argonaute2 (AGO2) antibody (Millipore, Billerica, MA, USA) or non-specific anti-IgG antibody (Millipore) with rotation at 4°C overnight. Magnetic beads were added to the cell lysates and incubation was continued at 4°C for 1 h. The samples were then incubated with Proteinase K at 55°C for 1 h. The enriched RNA was obtained using RNA Extraction Reagent (Enol: Chloroform: Isoamylol = 125:24:1, pH < 5.0; Solarbio). The purified RNA was used to detect the expression levels of the genes of interest by qRT-PCR.

Plasma Exosome Isolation

First, the samples were centrifuged twice at 3,000 × g and 10,000 × g for 20 min at room temperature to remove cells and other debris in the plasma. The supernatants were then centrifuged at 100,000 × g for 30 min at 4°C to remove microvesicles that were larger than exosomes, harvested, and again centrifuged at 10,000 × g for 70 min at 4°C. Subsequently, the supernatants were gently decanted, and the exosome sediments were re-suspended in phosphate-buffered saline (PBS). Concentration of exosomes was determined using BCA method as recommended by the manufacturer (Thermo Scientific, USA).

TEM

The exosome suspension was diluted to 0.5 mg/mL with PBS, and then spotted onto a glow-discharged copper grid placed on a filter paper and dried for 10 min by exposure to infrared light. Next, the exosome samples were stained with one drop of phosphotungstic acid (1% aqueous solution) for 5 min and dried for 20 min by exposure to infrared light. Finally, the exosomes were visualized under a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan) at 100 keV.

NTA

Briefly, the exosomes were resuspended in PBS and filtered with a syringe filter (Millipore). Then, the samples were diluted until individual nanoparticles could be tracked. The size distribution of the exosomes was evaluated using a NanoSight NS300 instrument (Malvern Instruments, Worcestershire, UK).

Statistical Analyses

All statistical analyses were performed using SPSS version 21.0 (IBM SPSS, Chicago, IL, USA) and Prism version 5.0 (GraphPad Software, La Jolla, CA, USA) software. Categorical variables were expressed as a count or percentage and tested using χ2 or Fisher’s exact test, as appropriate. Continuous data are reported as mean ± standard deviation (SD) and compared using Student’s t test, the one-way analysis of variance (ANOVA) test or Mann-Whitney test as appropriate. Correlations were calculated using Pearson’s correlation analysis. The cut-off value used to stratify patients into high and low expression groups was the median expression of target genes. All tests were 2-sided, and p <0.05 was considered statistically significant.

Author Contributions

G.S. and B.S. performed primers design and experiments. Q.L. and Y.Z. contributed flow cytometry assay and animal experiments. C.F. and A.C. collected and classified the human tissue samples. G.S. contributed to qRT-PCR. Q.C. analyzed the data. Q.C. wrote the paper. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no competing interests.