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. 2020 Dec 7;19(24):3563–3580. doi: 10.1080/15384101.2020.1852746

The circ_0004463/miR-380-3p/FOXO1 axis modulates mitochondrial respiration and bladder cancer cell apoptosis

Shuiqing Wu 1,*,*, Huanghao Deng 1,*,*, Haiqing He 1, Ran Xu 1,✉, Yinhuai Wang 1, Xuan Zhu 1, Jinhua Zhang 1, Qi Zeng 1, Xiaokun Zhao 1
PMCID: PMC7781606  PMID: 33283616

ABSTRACT

Bladder cancer is one of the most commonly diagnosed and fatal malignancies of the urinary tract. Noncoding RNAs have been reported to be new biomarkers and effective treatment targets for bladder cancer. In the present study, we identified a novel bladder cancer-related circRNA-miRNA-mRNA network, the circ_0004463/miR-380-3p/FOXO1 axis. circ_0004463 is significantly downregulated, whereas miR-380-3p is upregulated in bladder carcinoma tissue samples and cells. circ_0004463 acts as a tumor suppressor by inhibiting bladder cancer cell proliferation. Genes that negatively correlated with miR-380-3p and genes that miR-380-3p might target are enriched in mitochondrial respiration chain-related pathways. miR-380-3p promotes the proliferation of bladder cancer cells and mitochondrial respiration by acting as an oncogenic miRNA. circ_0004463 competes with FOXO1 for miR-380-3p binding to counteract miR-380-3p-mediated repression of FOXO1. Circ_0004463 overexpression inhibits cancer cell proliferation and mitochondrial respiration in bladder cancer cell lines, while miR-380-3p overexpression dramatically reverses the roles of circ_0004463 overexpression. In conclusion, the circ_0004463/miR-380-3p/FOXO1 axis could regulate mitochondrial respiration and bladder cancer cell apoptosis via FOXO1 signaling.

KEYWORDS: Bladder cancer, mitochondrial respiration, circ_0004463/miR-380-3p/FOXO1

Introduction

Bladder cancer is one of the most commonly diagnosed and fatal malignancies of the urinary tract [1,2]. Although surgical resection and chemotherapy have been widely used in the treatment of bladder cancer, the results are not ideal at advanced cancer stages, which are prone to distant metastasis, devastating drug resistance and adverse clinical effects [3]. Due to the lack of diagnostic and specialized treatment methods, many patients have already developed high-grade tumors at the time of initial diagnosis [4]. within the face of these pressing challenges, exploring new biomarkers and identifying effective treatment targets for bladder cancer are essential.

In recent decades, it has been demonstrated that many transcripts associated with the occurrence and development of tumors are dysregulated in bladder cancers, as has been observed for circRNAs (circular RNAs) [5–7], a new class of noncoding RNAs abundant in eukaryotic organisms [8]. CircRNAs, similar to other noncoding RNAs, were originally thought to be products of aberrant RNA splicing [9]. Nevertheless, a growing body of evidence has indicated the multiple effects of circRNAs on cancers and many other biological processes. Reportedly, circRNAs affect the progression and recurrence of bladder cancers [10–12]. More importantly, circRNAs are mainly generated by alternative splicing and back-splicing of pre-mRNAs. Due to their unique loop structure [13], circRNAs show strong resistance to exonucleases and are therefore stable. Currently, many noncoding RNAs could serve as important biomarkers in several pathologies. For example, Donato et al. [14] performed total RNA sequencing and identified lncRNAs differentially expressed in oxidative stress-challenged human retinal pigment epithelium cells; these lncRNAs might participate in biochemical pathways related to a compromised response to oxidative stress, carbohydrate and lipid metabolism impairment, melanin biosynthetic process alteration, deficiency in the cellular response to amino acid starvation, and dysregulation of the cofactor metabolic process. Considering the multiple roles of noncoding RNAs and the stability of circRNAs, circRNAs may be more promising biomarkers and therapeutic targets than other noncoding RNAs.

Mechanistically, circRNAs can act as ceRNAs (competing endogenous RNAs) of miRNAs (microRNAs) [15]. miRNAs posttranscriptionally regulate the expression of downstream targets by complete or incomplete base pairing to the 3ʹUTR (untranslated region) of target mRNAs. By binding to miRNAs to counteract miRNA-mediated repression on mRNAs, circRNAs could regulate mRNA translation [15]. As recently reported, circRNAs interact with miRNAs in bladder cancers, therefore playing roles in cancer development, diagnosis, prognosis, and metastasis progression [10,16–18]. For example, CDR1as is a circRNA that acts as a sponge for miR-7 and contains over 70 conserved miR-7 response elements (MREs), thus playing biological or pathological roles in many cancers [15,19,20]. However, other unknown circRNA-miRNA-mRNA networks need to be further studied.

Herein, we downloaded microarray datasets containing bladder cancer circRNA and miRNA expression information from the GEO (Gene Expression Omnibus) database, namely, GSE97239, GSE92675, and GSE40355. Next, we found differentially expressed circRNAs, verified circRNA expression in collected tissue samples, and selected circ_0004463 due to its strongest downregulation in bladder cancer tissues. Second, we compared miRNAs that might be sponged by circ_0004463 and deregulated miRNAs that might be correlated with bladder cancer patient survival based on data from TCGA-BLCA (The Cancer Genome Atlas Urothelial Bladder Carcinoma), and miR-380-3p was selected. circ_0004463 was characterized, and its specific effects on bladder cancer cell viability and apoptosis were examined. Next, mRNAs related to miR-380-3p were also identified based on data from TCGA-BLCA, and these mRNAs were applied to a Gene Ontology (GO) term enrichment analysis. In addition, mRNAs that could be targeted by miR-380-3p were used in KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling enrichment analysis. The results of the two analyses intersected at the mitochondrial respiratory chain-related PI3K/AKT/FOXO signaling pathway. Thus, the specific roles of miR-380-3p in the proliferation of bladder cancer cells, mitochondrial respiration, and factors of the FOXO signaling pathway were examined. The predicted binding of miR-380-3p to circ_0004463 and FOXO1 (Forkhead box protein O1) was verified. Finally, the dynamic effects of circ_0004463 and miR-380-3p on the proliferation of bladder cancer cells, mitochondrial respiration, and the FOXO signaling pathway were evaluated. In summary, we identified a circRNA–miRNA–mRNA axis around circ_0004463 and examined the specific effects of this network on bladder cancer cells.

Materials and methods

Tissue sample collection

A total of 12 paired bladder cancer and adjacent noncancerous tissues were obtained from patients that were clinically and histologically diagnosed and received surgical resection at the Second Xiangya Hospital, Central South University. The sample collection was performed with the approval of the institutional Ethics Committee. Written informed consent was obtained from each enrolled patient.

Cell lines and cell transfection

The human bladder epithelial SV40 immortalized cell line SV-HUC-1 (ATCC® CRL-9520) was obtained from ATCC (Manassas, VA, USA) and cultured in F-12 K medium (Catalog No. 30–2004, ATCC) supplemented with 10% FBS. The bladder cancer cell line T24 (ATCC® HTB-4) was obtained from ATCC and cultured in McCoy’s 5a modified medium (Catalog No. 30–2007, ATCC) supplemented with 10% FBS. The bladder cancer cell line 5637 (ATCC® HTB-9) was obtained from ATCC and cultured in RPMI-1640 medium (Catalog No. 30–2001, ATCC) supplemented with 10% FBS. All cells were cultured at 37°C in 5% CO2.

The overexpression or knockdown of circ_0004463 was conducted in target cells by the transfection of a circ_0004463-overexpressing plasmid (circ_0004463) or small interfering RNA targeting circ_0004463 (si-circ_0004463; GeneTop, Changsha, China), respectively. The overexpression or inhibition of miR-380-3p in target cells was achieved by the transfection of agomir-380-3p or antagomir-380-3p (GeneTop), respectively. All transfections were performed using Lipofectamine™ 3000 (Thermo Fisher Scientific‎, Waltham, MA, USA).

qRT-PCR-based analysis

qRT-PCR-based analysis (polymerase chain reaction (PCR)-based analysis) was performed to examine the expression levels of target factors. Total RNA was extracted from target tissues or cells using TRIzol reagent (Invitrogen). After that, the expression of circRNA, miRNA, and mRNA was determined by real-time PCR using a SYBR Green qPCR assay (Takara, Dalian, China) following the methods described before [21]. The expression of 18S RNA (for circRNA detection), RNU6B (for miRNA detection), or GAPDH (for mRNA detection) was used as an endogenous control to calculate the relative fold-change using the 2−ΔΔCT method. The primers are listed in Table S1.

Agarose gel electrophoresis for circular RNA identification

To characterize circ_0004463, the study performed agarose gel electrophoresis and RNase R treatment following the methods described before [22]. RNA extracted from 5637 cells (1 µg) was treated with 3 U of RNase R (Epicenter, San Antonio, TX, USA) at 37°C for 1 h. Then, RT-PCR assay with divergent or convergent primers tested the existence of circ_0004463. GAPDH was used as a negative control.

Bioinformatic analysis

GSE97239, GSE92675, and GSE40355 from GEO database (https://www.ncbi.nlm.nih.gov/geo) were downloaded for circ_RNA selection. The difference of cicr_RNA expression was analyzed by R language LIMMA package. The online tool lncTar (http://www.cuilab.cn/lnctar) was used to predict hsa_circ_0004463-targeted miRNAs. The correlation of the miRNA expression levels with bladder cancer patient survival data from TCGA-BLCA (The Cancer Genome Atlas Urothelial Bladder Carcinoma) was analysis by online tool Kaplan-Meier Plotter (KMPLOT) (http://kmplot.com/). The online tool LinkedOmics was used to identify the mRNAs which were related to miR-380-3p expression (person correlation analysis) based on TCGA-BLCA data. Then, these mRNAs were applied to a Gene Ontology (GO) term enrichment analysis. Online tool mirDIP (http://ophid.utoronto.ca/mirDIP/) was used to predict the target gene of mR-380-3p (minimum score “very high”, cuttoff >0.38). The predict targeted genes were applied for Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate the key functional pathways involved in bladder cancer using R language ggplot2 package.

Cell viability evaluated by the MTT assay

By following the methods described before [23], cell viability was detected. After transfection or treatment, MTT (20 μl at a density of 5 mg/ml; Sigma-Aldrich) was added, and another 4-h incubation was conducted. At the end of the incubation, DMSO (200 μl) was added to dissolve the formazan. Next, OD values were measured at 490 nm, and the relative cell viability was calculated by taking the viability of nontreated cells (control) as 100%.

Cell apoptosis examined by flow cytometry

Cell apoptosis was determined by flow cytometry. The cells were digested by trypsin and collected. After resuspension in 100 μl binding buffer, the cells were incubated with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) at room temperature in the dark for 15 min. Then, the cells were analyzed by flow cytometry.

Oxygen consumption rate determination

The cellular oxygen consumption rate (OCR) was determined using a Seahorse XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA) on the Seahorse XFe 96 Extracellular Flux Analyzer (Agilent) following the manufacturer’s protocols and the methods described before [24]. For OCR detection, oligomycin, the reversible inhibitor of the oxidative phosphorylation uncoupler FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone), and the mitochondrial complex III inhibitor antimycin A were sequentially applied. Data were assessed by Seahorse XF-96 Wave software. OCR is shown in pmol/min.

Immunoblotting

The protein levels of FOXO1, NDUFA1, SDHB, and ATP5A were determined using immunoblotting. Total proteins were extracted from target cells with radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China), and the protein concentration was determined with the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA). The proteins were transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). The membranes were first blocked with 5% nonfat milk solution for 2 h at room temperature and then incubated with the following primary antibodies: anti-FOXO1 (ab52857; Abcam, Cambridge, CA, USA), anti-NDUFA1 (ab131423, Abcam), anti-SDHB (ab14714, Abcam), and anti-ATP5A (ab14748, Abcam) at 4°C overnight. Then, the blots were incubated with anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (Abcam) for 1 h at 37°C. The blot signals were then visualized using enhanced chemiluminescence systems.

Luciferase reporter assay

To validate the binding between miR-380-3p and circ_0004463 or the FOXO1 3ʹ-UTR, the wild-type or mutated circ_0004463 or FOXO1 3ʹ-UTR was cloned downstream of the Renilla gene in the psiCHECK2 vector (Promega, Madison, WI, USA), and the resulting constructs were named wt-circ_0004463/mut-circ_0004463 or wt-FOXO1 3ʹ-UTR/mut-FOXO1 3ʹ-UTR.

Next, 293 T cells were cotransfected with the two types of luciferase reporter vectors and agomir-380-3p/antagomir-380-3p following a previously described method [25]. For cell cotransfection, serum-free medium was added with a mixture that included 0.2 μL of Lipofectamine 3000 Reagent (Thermo Fisher Scientific) and 0.2 μL of P3000 Reagent (Thermo Fisher Scientific), and then the cells were incubated for 48 h at 37°C in a humidified atmosphere of 5% CO2 in air. After incubation, cells were washed twice with PBS and lysed by Passive Lysis Buffer (Promega), and then the luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega).

Data processing and statistical analysis

The data were analyzed with GraphPad software. The measurement data are expressed as the mean ± standard deviation (SD). All experiments were repeated at least three times. Intergroup and intragroup data comparisons were performed with Student’s t-tests or ANOVA followed by Tukey’s post hoc test. P< 0.05 indicates a statistically significant difference.

Results

Selection of circular RNA and miRNA related to bladder carcinogenesis

To determine circRNAs related to bladder carcinogenesis, the study examined GSE97239 and GSE92675 for differentially expressed circular RNAs in bladder carcinoma and normal control tissues and found that 11 circular RNAs in total were significantly differentially expressed, namely, hsa_circ_0000375, hsa_circ_0004096, hsa_circ_0003505, hsa_circ_0016650, hsa_circ_0004463, hsa_circ_0056310, hsa_circ_0004305, hsa_circ_0001296, hsa_circ_0008628, hsa_circ_0082306, and hsa_circ_0080251. Among these 11 circular RNAs, circ_0004463 was the most downregulated in bladder cancer tissues (Figure 1(a)). Real-time qPCR results supported the data from the online datasets that among the 11 circular RNAs, circ_0004463 was the most downregulated in collected bladder carcinoma tissues in comparison with adjacent normal control tissues (Figure 1(b)). Thus, the study continued to analyze the possible target miRNAs of circ_0004463.

Figure 1.

Figure 1.

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Selection of circular RNA and miRNA related to bladder cancer carcinogenesis

(A) The expression of circ_0004463 in bladder cancer and noncancerous tissue samples, according to GSE97239 and GSE92675. (B) The expression levels of hsa_circ_0000375, hsa_circ_0004096, hsa_circ_0003505, hsa_circ_0016650, hsa_circ_0004463, hsa_circ_0056310, hsa_circ_0004305, hsa_circ_0001296, hsa_circ_0008628, hsa_circ_0082306, and hsa_circ_0080251 in 12 paired bladder cancer and noncancerous tissue samples were examined using real-time qPCR. (C) A schematic diagram showing the process of miRNA selection. The online tool lncTar (http://www.cuilab.cn/lnctar) was used to predict hsa_circ_0004463-targeted miRNAs and obtained 48 miRNAs meeting the criteria 7-mer + A and 8-mer. Using online tool KMPLOT to analyze data from TCGA-BLCA, the correlation of these 48 miRNAs with bladder cancer patient survival was analyzed, and a total of 5 miRNAs were significantly correlated with bladder cancer patient survival. Among the 5 miRNAs, miR-380-3p was significantly upregulated in bladder cancer tissues compared with normal tissues, based on GSE40355. According to the above three steps, miR-380-3p might be a circ_0004463 target and was correlated with bladder cancer patient survival. (D) The expression levels of miR-380 in noncancerous, low-grade bladder cancer and high-grade bladder cancer tissue samples, according to GSE40355. (E) The expression levels of miR-380 were examined in 12 paired bladder cancer and noncancerous tissue samples using real-time qPCR. *P< 0.05, **P< 0.01.

The online tool lncTar (http://www.cuilab.cn/lnctar) was used to predict hsa_circ_0004463-targeted miRNAs, and a total of 48 miRNAs were obtained in the combined form of 7mer + A and 8mer. Next, we divided cases from TCGA-BLCA into a high miRNA group and a low miRNA group using the median expression value of each of the 48 miRNAs as the cutoff and analyzed the correlation of the miRNA expression levels (high or low) with bladder cancer patient survival to investigate whether they were risk factors for bladder cancer patient survival. The survival analysis was performed by online tool Kaplan-Meier Plotter (http://kmplot.com/) and identified the following 5 miRNAs that were significantly correlated with bladder cancer patient survival: hsa-miR-204, hsa-miR-152, hsa-miR-380, hsa-miR-382, and hsa-miR-576 (Figure. S1A-E). Among these 5 miRNAs, miR-380-3p was significantly upregulated in bladder cancer tissues compared with normal tissues according to GSE40355 (analyzed by R language LIMMA package) (Figure 1(d)). Through these three steps, we found that miR-380-3p might be targeted by circ_0004463 and correlated with bladder cancer patient survival (Figure 1(c)). As a further confirmation, we examined miR-380 expression in 12 paired bladder cancer and noncancerous tissue samples. Figure 1(e) shows that miR-380-3p expression was markedly upregulated in bladder carcinoma tissues compared with noncancerous tissues. Thus, miR-380-3p was selected for further experiments.

Specific effects of circ_0004463 on bladder cancer cell phenotypes

Before examining the specific effects of circ_0004463 on bladder cancer cells, the study first performed agarose gel electrophoresis to characterize circ_0004463. Convergent and divergent primers were designed and synthesized to amplify circ_0004463 and GAPDH (internal reference) using real-time qPCR, taking cDNA and genomic DNA (gDNA) as the templates. As revealed by agarose gel electrophoresis, circ_0004463 was only amplified from cDNA but not gDNA from 5637 cells, while GAPDH could be amplified from both cDNA and gDNA (Figure 2(a)). These results indicate that circ_0004463 is a circular RNA.

Figure 2.

Figure 2.

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Specific effects of circ_0004463 on bladder cancer cell phenotypes

(A) Identification of circ_0004463 in 5637 cells by agarose gel electrophoresis. (B) The expression of circ_0004463 in a human bladder epithelial SV40 immortalized cell line, SV-HUC-1, and two bladder cancer cell lines, 5637 and T24, was determined using real-time qPCR. (C) circ_0004463 was overexpressed in 5637 and T24 cells by the transfection of a circ_0004463-overexpressing plasmid (circ_0004463). The transfection efficiency was confirmed using real-time qPCR. Next, 5637 and T24 cells were transfected with circ_0004463 and examined for (D) cell viability by MTT assay and (E) cell apoptosis by flow cytometry assay. *P< 0.05, **P< 0.01.

Second, we examined circ_0004463 expression in cells. Compared to that in SV-HUC-1, a human bladder epithelial SV40 immortalized cell line, the expression level of circ_0004463 was significantly downregulated in both 5637 and T24, two bladder carcinoma cell lines (Figure 2(b)). To evaluate the specific roles of circ_0004463 in bladder carcinoma cell lines, we transfected a circ_0004463-overexpressing plasmid (circ_0004463) to overexpress circ_0004463 in 5637 and T24 cells, as confirmed by real-time qPCR (Figure 2(c)). In both cell lines, circ_0004463 overexpression significantly downregulated cell viability (Figure 2(d)) and upregulated cell apoptosis (Figure 2(e)).

Selection of genes negatively correlated with miR-380-3p in bladder carcinogenesis

Considering the mechanism of miRNA functions, we analyzed the cellular functions and downstream signaling pathways that miR-380-3p may affect, as well as the targets of miR-380-3p. First, Pearson’s correlation analysis was performed to analyze data from TCGA-BLCA to identify genes that were negatively correlated with miR-380-3p by online tool LinkedOmics (http://linkedomics.org/admin.php) (cutoff r > 0.3, p < 0.05). In this step, a total of 321 genes were found to be negatively correlated with miR-380-3p. Genes negatively correlated with miR-380-3p were subjected to GO (Gene Ontology) term enrichment analysis to identify the cellular functions that might be affected by miR-380-3p. GO-CC (cellular component) analysis revealed that the negatively correlated genes were enriched in the mitochondrial complex, respiratory chain, and NADH dehydrogenase synthesis processes (Figure 3(a)). GO-BP (biological process) analysis indicated that the negatively correlated genes were enriched in the ribosome, oxidative phosphorylation, and spliceosome processes (Figure 3(a)). GO-MF (molecular function) analysis revealed that the negatively correlated genes were enriched in the respiratory chain electron transfer oxidoreductase pathway (Figure 3(a)). Thus, the negatively correlated genes are enriched in the mitochondrial respiratory chain (MRC).

Figure 3.

Figure 3.

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Selection of genes negatively correlated with miR-380-3p in bladder carcinogenesis

(A) Pearson’s correlation analysis was performed to identify genes significantly correlated with miR-380-3p according to TCGA-BLCA dataset from LinkedOmics, and a total of 321 genes were found to be negatively correlated with miR-380-3p. Genes negatively correlated with miR-380-3p were subjected to Gene Ontology (GO) term enrichment analysis. The results from cellular component (GO-CC), biological process (GO-BP), and molecular function (GO-MF) analyses are shown. (B) Predicted targets of miR-380-3p were applied to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. (C) The KEGG pathway of PI3K/AKT signal pathway.

Second, the online tool mirDIP (http://ophid.utoronto.ca/mirDIP/) was used to predict the downstream targets of miR-380-3p, and a total of 628 candidates with “very-high” scores (cutoff >0.38) were obtained. These predicted targets of miR-380-3p were applied in KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis to identify the downstream signaling pathways that might be affected by miR-380-3p, and the predicted targets were found to be enriched in mitochondrial apoptosis upstream of the PI3K/Akt/FOXO signaling pathway (Figure 3(b)).The KEGG pathway of PI3K/Akt signaling pathway was shown in Figure 3(c). In this pathway, FOXO 1 and FOXO 3 are both tumor-suppressive genes in bladder cancer that could regulate cell mitochondrial apoptosis [26]. Thus, the mitochondrial respiratory chain-related pathway was further investigated.

Specific roles of miR-380-3p in the phenotype of bladder cancer cells and FOXO1 signaling

Since the bioinformatics analysis indicated that miR-380-3p might be related to the mitochondrial respiratory chain pathway, the study continued to validate the specific roles of miR-380-3p in the bladder cancer cell phenotype and FOXO1 signaling. Consistent with the online database analysis, in both 5637 and T24, two bladder carcinoma cell lines, miR-380-3p expression was significantly upregulated compared to that in SV-HUC-1 cell lines (Figure 4(a)). Next, we transfected antagomir-380-3p to inhibit miR-380-3p in 5637 and T24 cell lines, as confirmed by real-time qPCR (Figure 4(b)). Similar to circ_0004463 overexpression, miR-380-3p inhibition also significantly inhibited 5637 and T24 cell viability (Figure 4(c)) while promoting cell apoptosis (Figure 4(d)). Regarding the mitochondrial respiratory chain, the oxygen consumption rate (OCR) was determined, and as shown in Figure 4(e-f), miR-380-3p inhibition significantly reduced the OCR, suggesting that miR-380-3p inhibition suppressed mitochondrial respiration in bladder cancer cells. In terms of the related pathway, the protein levels of FOXO1, NDUFA1, SDHB, and ATP5A were examined. miR-380-3p inhibition induced a dramatic increase in FOXO1 protein levels while significantly decreasing NDUFA1, SDHB, and ATP5A protein levels (Figure 4(g)), indicating that miR-380-3p could target FOXO1 signaling and affect bladder cancer cell proliferation and mitochondrial respiration.

Figure 4.

Figure 4.

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Specific effects of miR-380-3p on bladder cancer cell phenotypes and FOXO1 signaling

(A) The expression of miR-380-3p in a human bladder epithelial SV40 immortalized cell line, SV-HUC-1, and two bladder cancer cell lines, 5637 and T24, was determined using real-time qPCR. (B) miR-380-3p was inhibited in 5637 and T24 cells by the transfection of antagomir-380-3p. The transfection efficiency was confirmed using real-time qPCR. Next, 5637 and T24 cells were transfected with antagomir-380-3p and examined for (C) cell viability by MTT assay; (D) cell apoptosis by flow cytometry assay; (E-F) oxygen consumption rate (OCR) using a Seahorse XF Cell Mito Stress Test Kit; and (G) the protein levels of FOXO1, NDUFA1, SDHB, and ATP5A by immunoblotting. *P< 0.05, **P< 0.01.

miR-380-3p directly targets circ_0004463 and the 3ʹ-UTR of FOXO1

After confirming the effects of circ_0004463 and miR-380-3p, the study continued to investigate the predicted binding of miR-380-3p to circ_0004463 and the FOXO1 3ʹ-UTR. The knockdown of circ_0004463 was conducted in 5637 and T24 cells by transfecting small interfering RNA targeting circ_0004463 (si-circ_0004463), as confirmed by real-time qPCR (Figure 5(a)). We transfected the 5637 and T24 cell lines with agomir-380-3p/antagomir-380-3p and examined the expression of circ_0004463. Figure 5(b) shows that the overexpression or inhibition of miR-380-3p caused no changes in circ_0004463 expression.

Figure 5.

Figure 5.

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miR-380-3p directly targets circ_0004463 and the 3ʹ-UTR of FOXO1

(A) circ_0004463 was knocked down in 5637 and T24 cells by transfecting small interfering RNA targeting circ_0004463 (si-circ_0004463). The transfection efficiency was determined using real-time qPCR. (B) 5637 and T24 cells were transfected with agomir-380-3p or antagomir-380-3p and examined for the expression of circ_0004463 by real-time qPCR. (C and E) Wild-type and mutant circ_0004463 luciferase reporter plasmids were constructed (wt-circ_0004463/mut-circ_0004463). The predicted miR-380-3p binding site was mutated in mut-circ_0004463. The reporter plasmids were cotransfected into 293 T cells with agomir-380-3p or antagomir-380-3p, and the luciferase activity was determined. (D) 5637 and T24 cells were transfected with agomir-380-3p or antagomir-380-3p and examined for the protein levels of FOXO1 by immunoblotting. Then, wild-type and mutant FOXO1 3ʹ-UTR luciferase reporter plasmids were constructed (wt-FOXO1 3ʹ-UTR/mut-FOXO1 3ʹ-UTR). The predicted miR-380-3p binding site was mutated in mut-FOXO1 3ʹ-UTR. (E) The reporter plasmids were cotransfected into 293 T cells with agomir-380-3p or antagomir-380-3p, and the luciferase activity was determined. (F) The plasmids were cotransfected into 293 T cells with antagomir-380-3p and si-circ_0004463, and the luciferase activity was determined.

Next, we constructed wild-type and mutant circ_0004463 and FOXO1 3ʹ-UTR luciferase reporter plasmids (wt-circ_0004463/mut-circ_0004463 and wt-FOXO1 3ʹ-UTR/mut-FOXO1 3ʹ-UTR) to perform dual-luciferase reporter assays. The predicted miR-380-3p binding site was mutated in the mutant plasmids. Then, we cotransfected the reporter plasmids in 293 T cells with agomir-380-3p or antagomir-380-3p and examined the luciferase activity. Figure 5(c,e) show that wild-type plasmid luciferase activity was dramatically repressed via the overexpression of miR-380-3p but promoted via the inhibition of miR-380-3p. In cells cotransfected with mutant plasmids, the overexpression or inhibition of miR-380-3p caused no changes in luciferase activity. Thus, miR-380-3p might directly target both circ_0004463 and the FOXO1 3ʹ-UTR. To further confirm competitive binding, wt-FOXO1 3ʹ-UTR/mut-FOXO1 3ʹ-UTR were cotransfected into 293 T cells with antagomir-380-3p and si-circ_0004463, and luciferase activity was determined. As shown in Figure 5(f), when the cells were cotransfected with wt-FOXO1 3ʹ-UTR, miR-380-3p inhibition was enhanced, whereas circ_0004463 silencing repressed the luciferase activity of wt-FOXO1 3ʹ-UTR; the effects of miR-380-3p inhibition on the luciferase activity of wt-FOXO1 3ʹ-UTR were partially reversed by circ_0004463 silencing. When the cells were cotransfected with mut-FOXO1 3ʹ-UTR, miR-380-3p inhibition or circ_0004463 silencing failed to alter the luciferase activity (Figure 5(f)). In 5637 and T24 cells, miR-380-3p overexpression significantly decreased, while miR-380-3p inhibition increased the protein levels of FOXO1 (Figure 5(d)), further indicating that miR-380-3p targets the 3ʹ-UTR of FOXO1 to negatively regulate FOXO1 expression.

Dynamic effects of circ_0004463 and miR-380-3p on the phenotype of bladder cancer cells and FOXO1 signaling

After confirming the binding of miR-380-3p to the FOXO1 3ʹ-UTR and circ_0004463, we cotransfected 5637 and T24 cells with circ_0004463 and agomir-380-3p and examined their dynamic effects. Circ_0004463 overexpression significantly downregulated the viability and upregulated the apoptosis of bladder cancer cells, whereas miR-380-3p overexpression exerted the opposite effects; miR-380-3p significantly attenuated the roles of circ_0004463 overexpression (Figure 6(a-b)). Regarding mitochondrial respiration, circ_0004463 overexpression dramatically downregulated, whereas miR-380-3p overexpression upregulated, the OCR; the overexpression of miR-380-3p significantly attenuated the effects of circ_0004463 overexpression (Figure 6(c-d)). Regarding the related FOXO1 signaling pathway, circ_0004463 overexpression significantly increased FOXO1 protein levels and decreased NDUFA1, SDHB, and ATP5A protein levels, whereas miR-380-3p overexpression decreased FOXO1 protein levels and increased NDUFA1, SDHB, and ATP5A protein levels; the overexpression of miR-380-3p significantly attenuated the effects of circ_0004463 overexpression (Figure 6(e)). These data indicate that circ_0004463 inhibits mitochondrial respiration and bladder cancer cell viability and enhances bladder cancer cell apoptosis through the miR-380-3p/FOXO1 axis and FOXO1 signaling.

Figure 6.

Figure 6.

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Dynamic effects of circ_0004463 and miR-380-3p on bladder cancer cell phenotypes and FOXO1 signaling

5637 and T24 cells were transfected with circ_0004463 and agomir-380-3p and examined for (A) cell viability by MTT assay; (B) cell apoptosis by flow cytometry assay; (C-D) oxygen consumption rate (OCR) using a Seahorse XF Cell Mito Stress Test Kit; and (E) the protein levels of FOXO1, NDUFA1, SDHB, and ATP5A by immunoblotting. *P< 0.05, **P< 0.01, compared to the control group; ##P < 0.01, compared to the vector + agomir-380-3p group.

Dynamic effects of miR-380-3p and FOXO1 on bladder cancer cell phenotypes and FOXO1 signaling

To further demonstrate the link between factors in the circ_0004463/miR-380-3p/FOXO1 axis and the functional importance of Foxo1 for the phenotypes observed upon modulation of miR-380-3p, we next determined the dynamic effects of miR-380-3p inhibition and FOXO1 silencing on cancer cells. 5637 and T24 cells were transfected with antagomir-380-3p and si-FOXO1 and examined similar indexes. Regarding FOXO1 signaling, in both cell lines, miR-380-3p inhibition increased the protein levels of FOXO1 but decreased the protein levels of NDUFA1, SDHB, and ATP5A; FOXO1 silencing exerted the opposite effects and significantly reversed the effects of miR-380-5p on these proteins (Figure 7(a)). Regarding cellular functions, miR-380-3p inhibition significantly suppressed cell viability and promoted cell apoptosis in both cell lines; FOXO1 silencing exerted the opposite effects to miR-380-3p on cancer cells and significantly reversed the effects of miR-380-3p on cancer cells (Figure 7(b-c)). These data indicate that FOXO1 could reverse the effects of miR-380-3p on cancer cell phenotypes and FOXO1 signaling.

Figure 7.

Figure 7.

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Dynamic effects of miR-380-3p and FOXO1 on bladder cancer cell phenotypes and FOXO1 signaling

5637 and T24 cells were transfected with antagomir-380-3p and si-FOXO1 and examined for (A) the protein levels of O1, NDUFA1, SDHB, and ATP5A by immunoblotting; (B) cell viability by MTT assay; and (C) cell apoptosis by flow cytometry assay. *P< 0.05, **P< 0.01, compared to the control group; ##P < 0.01, compared to the antagomir-NC + si-FOXO1 group.

Discussion

As high-throughput sequencing and bioinformatics analyses have developed, an increasing number of circRNAs have been demonstrated to modulate gene expression and exert critical effects on the progression of various cancers [27–29]. Herein, we revealed a novel bladder cancer-related circRNA-miRNA-mRNA network, the circ_0004463/miR-380-3p/FOXO1 axis. circ_0004463 was significantly downregulated, whereas miR-380-3p was upregulated, in bladder carcinoma tissue samples and cells. circ_0004463 acted as a tumor suppressor by inhibiting bladder cancer cell proliferation. Genes that negatively correlated with miR-380-3p and genes that miR-380-3p might target were enriched in mitochondrial respiration chain-related pathways, consistent with a previous study showing that miRNA targets could be enriched in mitochondrial respiration chain-related pathways [30]. miR-380-3p promoted the proliferation of bladder cancer cells and mitochondrial respiration by acting as an oncogenic miRNA. circ_0004463 competed with FOXO1 for miR-380-3p binding to counteract miR-380-3p-mediated repression of FOXO1. Circ_0004463 overexpression inhibited cancer cell proliferation and mitochondrial respiration in bladder cancer cell lines, while miR-380-3p overexpression dramatically reversed the roles of circ_0004463 overexpression. Similarly, miR-380-3p inhibition suppressed, whereas FOXO1 silencing promoted, bladder cancer cell proliferation, and the effects of miR-380-3p inhibition were significantly reversed by FOXO1 silencing.

To date, research on the posttranscriptional regulation of circRNAs has concentrated on the effects of miRNA sponges. CircRNA can act as a miRNA sponge that harbors multiple miRNA binding sites or miRNA response elements and contributes to various cancers, such as bladder cancer [31,32]. For instance, circMYLK can act as a ceRNA for miR-29a to promote bladder cancer aggressiveness [16]. In addition, circBCRC3 has been revealed to act as a miR-182-5p sponge and suppresses the capacity of bladder cancer cells to proliferate [33]. Nevertheless, neither the specific effect of circ_0004463 on bladder carcinoma nor the miRNA sponge role of circ_0004463 has been reported previously. Herein, we found that circ_0004463 overexpression significantly inhibited bladder cancer viability and promoted cell apoptosis. Moreover, circ_0004463 might target a total of 48 miRNAs (7-mer+A and 8-mer), among which miR-380-3p was significantly correlated with bladder cancer patient survival. In summary, circ_0004463 exerts a tumor-suppressive effect on bladder cancer, possibly in a miR-380-3p-related manner.

miR-380-3p expression was shown to be limited to adult embryos and brain [34], indicating that miR-380-3p might play a special role in the function, differentiation, and survival of neurons. However, its role in carcinogenesis is still not clear. Herein, this study demonstrated that miR-380-3p was significantly increased in bladder cancer cells and tissues, exhibiting a negative correlation with circ_0004463. As is widely accepted, miRNAs bind to mRNAs in a sequence-specific manner to modulate gene expression, thus resulting in mRNA degradation or translational inhibition [35,36]. To better validate the specific roles of miR-380-3p in bladder carcinoma cell lines, this study first performed bioinformatics analysis to identify downstream signaling pathways related to miR-380-3p and the mitochondrial respiration chain-related FOXO pathway was found. Importantly, miR-380-3p has been predicted to bind to FOXO1, one of the key factors of this signaling pathway.

FOXO1 exerts a tumor-suppressive effect and modulates genes associated with oxidative stress, mitochondrial dysfunction, and apoptosis [37–40]. Since FOXO1 has been recognized as a master regulator of cell responses to oxidative stress [41], deregulation or mutation of FOXO1 might disturb cell mitochondrial respiration or response to oxidative stress [42,43]. Mitochondria generate most of the cell’s supply of adenosine triphosphate (ATP), which is used as a source of chemical energy. Tumor cells consume a substantial quantity of energy supplied as ATP; therefore, disrupting any part of this energy supply can lead to cell apoptosis [44]. Herein, miR-380-3p inhibition significantly suppressed the viability while enhancing the apoptosis of bladder cancer cells. Notably, miR-380-3p inhibition significantly inhibited the OCR in cancer cells, which is consistent with the bioinformatics analysis showing that miR-380-3p-related or targeted genes are enriched in the mitochondrial respiration chain-related FOXO1 pathway.

Regarding the molecular mechanism underlying miR-380-3p inhibition-induced OCR suppression, we also observed that miR-380-3p inhibition increased the FOXO1 protein level while decreasing NDUFA1, SDHB, and ATP5A protein levels. NDUFA1 is an essential component of complex I of the respiratory chain NADH:ubiquinone oxidoreductase subunits and is essential for respiratory activity [45]. SDHB is a component of mitochondrial respiratory chain complex II, succinate dehydrogenase subunits, which is localized in the mitochondrial inner membrane. Sporadic and familial mutations in this gene result in paragangliomas and pheochromocytoma and support a link between mitochondrial dysfunction and tumorigenesis [46]. ATP5A is a component of mitochondrial respiratory chain complex V, ATP synthase subunits. ATP synthase is an enzyme that creates the energy-storing molecule ATP [47,48]. Considering that miR-380-3p inhibition decreases the OCR in bladder cancer cells, it is reasonable to conclude that miR-380-3p modulates bladder cancer cell mitochondrial respiration and apoptosis by targeting FOXO1. Finally, the effects of circ_0004463 overexpression on bladder cancer cells were significantly reversed by miR-380-3p overexpression, indicating that the circ_0004463/miR-380-3p/FOXO1 axis affects bladder cancer cell mitochondrial respiration and apoptosis through FOXO signaling.

To further demonstrate the link between factors in the circ_0004463/miR-380-3p/FOXO1 axis and the functional importance of Foxo1 for the phenotypes observed upon modulation of miR-380-3p, we also determined the dynamic effects of miR-380-3p inhibition and FOXO1 silencing on cancer cells. Similarly, miR-380-3p inhibition repressed, whereas FOXO1 silencing promoted, bladder cancer cell proliferation; FOXO1 silencing partially attenuated the repressive effects of miR-380-3p inhibition. Regarding FOXO1 signaling, FOXO1 silencing also reversed the effects of miR-380-3p inhibition on FOXO1 signaling factors. Thus, FOXO1 could reverse the effects of miR-380-3p on cancer cell phenotypes and FOXO1 signaling. Notably, FOXO1 activity is correlated with its abundance, posttranscriptional modifications, nuclear-cytoplasmic shuttling and subcellular localization. Although miRNAs [49–52] are important regulators of FOXO1 translation that directly interfere with the level of FOXO1 mRNA, leading to a decreased abundance of FOXO1 protein, other posttranscriptional modifications might also be involved, including phosphorylation [53], ubiquitination [54], and acetylation [55]. For example, AKT-dependent phosphorylation of FOXO1 at Thr24, Ser256, or Ser319 tightly regulated the transcriptional activity of FOXO1 [56,57]. In the present study, we focused only on miRNA-mediated suppression of FOXO1 protein levels (abundance); thus, other mechanisms, such as phosphorylation, should be investigated in the future.

In conclusion, we identified the circ_0004463/miR-380-3p/FOXO1 axis modulating mitochondrial respiration and bladder cancer cell apoptosis. In addition to their miRNA sponge function, circRNAs can also contribute to the regulation of gene splicing or transcription, translating proteins, or interacting with RBPs (RNA-binding proteins). Thus, further research is needed to investigate other effects of circ_0004463 on bladder cancer. Inspired by previous studies reporting the possible joint effects of nuclear and mitochondrial variants in the evaluation of molecular regulatory pathways [58,59], similar analyses might be performed in the future to discover new biomarkers.

Supplementary Material

Supplemental Material
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Funding Statement

This study was supported by the grant from Science and Technology Agency of Hunan Province (no. 2020JJ4820).

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.

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