Hsa_circ_0008945 promoted breast cancer progression by targeting miR-338-3p
Authors Zhang L, Ding F
Received 30 April 2019
Accepted for publication 22 July 2019
Published 16 August 2019 Volume 2019:12 Pages 6577—6589
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Yao Dai
Li Zhang, Fengping Ding
Department of Pathology, People’s Hospital of Xinchang County, Xinchang Affiliated Hospital of Wenzhou Medical University, Xinchang, Zhejiang, People’s Republic of China
Correspondence: Li Zhang
Department of Pathology, People’s Hospital of Xinchang County, Xinchang Affiliated Hospital of Wenzhou Medical University, 117 Gushan Middle Road, Nanming Street, Xinchang 312500, People’s Republic of China
Tel +86 1 895 756 8155
Purpose: To detect the expression and function of circ_0008945 in breast cancer (BC) and to explore its potential molecular mechanisms in BC tumorigenesis.
Materials and methods: We measured expression levels of circ_0008945, miR-338-3p and homeobox A3 (HOXA3) in BC tissue specimens and cells using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). We examined the effects of all three genes on BC cell proliferation using Cell Counting Kit-8 (CCK-8) and colony formation assays. We also performed a Transwell assay to assess the migratory and invasive ability of treated BC cells. BC cell apoptosis was assessed using flow cytometric (FCM) analysis; interaction between miR-338-3p and circ_0008945 or HOXA3 was verified by dual-luciferase reporter assay as well as by ribonucleic-acid (RNA) pulldown. Finally, we used an in vivo tumor growth assay to assess the role of circ_0008945 overexpression in BC tumor growth.
Results: We found that circ_0008945 expression was significantly increased in both BC tissue specimens and cells. This increase was correlated with poor prognosis in BC patients. Knockdown of circ_0008945 inhibited BC cell proliferation, migration and invasion while promoting BC cell apoptosis in vitro. Overexpression of circ_0008945 remarkably promoted BC tumor growth in vivo. Mechanistically, circ_0008945 acted as a miRNA sponge for miR-338-3p and inhibited its expression in BC cells. Moreover, miR-338-3p targeted and inhibited HOXA3.
Conclusion: We found that circ_0008945 acted as a BC oncogene by physically binding miR-338-3p, which further targeted and regulated HOXA3.
Keywords: apoptosis, breast cancer, circ_0008945, miR-338-3p, proliferation
Breast cancer (BC) is one of the most frequently occurring malignancies, affecting millions of people worldwide.1 It was long considered to occur only in women, but it actually also occurs in men at a low rate of incidence (<1% of all BC cases).2 According to estimates by the American Cancer Society, there will be 271,270 new cases of and 42,260 deaths from BC in the United States in 2019, of which 2670 new cases and 500 deaths will occur in men.3 BC is initiated when the growth of breast cells is out of control.4 These uncontrolled cells form a lump that can frequently be felt and be detected by X-ray.5 BC tumors are considered malignant if the breast cells invade surrounding organs and tissues or metastasize to distant areas of the body.6 Due to the lack of effective measures in BC screening, a considerable percentage of BC patients are diagnosed at an advanced clinical stage, characterized by distant metastases and extremely poor prognosis.7 Combination therapy of surgical removal and chemotherapy is currently the most common and effective treatment measure for BC.8 Nevertheless, therapeutic effects are largely limited not only by the invasive or migratory characters but also by acquired drug resistance.4 Moreover, surgical removal causes considerable damage to the appearance of BC patients, which can affect their confidence. It is therefore a matter of urgent importance to understand the mechanisms underlying the tumorigenesis of BC.
Circular ribonucleic acid (circRNA) is a novel kind of non-coding RNA (ncRNA) that is characterized by circular structures.9 Studies have reported that circRNAs are involved in the pathogeneses of multiple human diseases, such as tumor progression, cardiovascular disease, neurodegenerative disorders and metabolic disorders.10 The biological functions of circRNAs in tumor initiation and development have been studied extensively.11 Several studies have shown the indispensable role that circRNAs play in the proliferation, cell cycle and invasion of cancer cells.12 Recently, numerous circRNAs have been identified as participating in the development of BC, suggesting that circRNAs play an important role in BC.13 We aimed to investigate whether circ_0008945, a novel circRNA with undetermined biological functions, was involved in the tumorigenesis of BC and attempted to understand its underlying molecular mechanisms.
MicroRNAs (miRNAs) are a critical subtype of ncRNA with approximately 22 nucleotides.14 Unlike circRNAs, miRNAs exist as linear structures with a 5′-cap and 3′-poly(A) tail. Moreover, miRNAs have been shown to participate in the modulation of gene expression by degrading target mRNAs.15 Furthermore, research has demonstrated that they are correlated with the development of multiple human tumors, including BC.16 According to the theory of competing endogenous RNAs (ceRNAs), circRNAs might serve as mRNA expression regulators by sponging miRNAs.17 Therefore, the circRNA/miRNA/mRNA pathway is largely considered the most common molecular mechanism underlying the pathogenesis of tumors.17
Herein, we not only detected the expression and function of circ_0008945 in BC but also explored its potential molecular mechanisms that might underlie BC tumorigenesis. Our results indicated that circ_0008945 acted as an important oncogene in BC by modulating miR-338-3p/HOXA3 expression. Therefore, targeting circ_0008945 might offer a promising therapeutic approach for BC treatment.
Materials and methods
Collection of BC tissue samples and cells
We collected BC tissue specimens and adjacent normal samples from BC patients (average age 66.7 years, age range 37–78 years) for use in this study. Written informed consent was obtained from each individual, and approval was obtained from the Ethics Committee of Xinchang County People’s Hospital and First Affiliated Hospital of Wenzhou Medical University (FHWMU) to perform this study. Our research was carried out in strict accordance with the Declaration of Helsinki. We obtained the non-metastatic human mammary epithelial cell line MCF-10A and 4 BC cell lines (MCF-7, MDA-MB-231, HCC1937 and BCAP-37) from the American Type Culture Collection (ATCC; Manassas, Virginia, US). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO [Thermo Fisher Scientific, Waltham, Massachusetts, US]) containing fetal bovine serum (FBS; 10%; Thermo Fisher) at 37 °C and 5% CO2.
Quantitative real-time polymerase chain reaction analysis
We extracted RNA from the tissues and treated cells with TRIzol Reagent (Invitrogen, Carlsbad, California, US). After examining the quality of total RNA with a NanoDrop 2000c spectrophotometer (Thermo Fisher), we used 3 μg total RNA as a template to produce complementary deoxyribonucleic acid (cDNA). We conducted qRT-PCR with BestarTM qPCR MasterMix (DBI Bioscience, China) on an ABI 7500 system (ABI Biosystems, Foster City, California, US). Primer sequences used in this study were as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), F: 5′-TGTTCGTCATGGGTGTGAAC-3′, R: 5′-ATGGCATGGACTGTGGTCAT-3′; U6, F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′; circ_0008945, F: 5′-CGGATGAAATCTGACCTACGA-3′, R: 5′-TCAGAGAGATCGGGACATCTG-3′; miR-338-3p, F: 5′-TGCGGTCCAGCATCAGTGAT-3′, R: 5′-CCAGTGCAGGGTCCGAGGT-3′; HOXA3, F: 5′-TCATTTAAGAGCGCCTGGACA-3′, R: 5′-GAGCTGTCGTAGTAGGTCGC-3′. We quantified gene expression using the 2−ΔΔCt method.
Negative control (NC) RNA, the siRNA target circ_0008945 (si-circ_0008945) and mimics and inhibitors of miR-388-3p and HOXA3 were all designed and purchased from GenePharma (Shanghai, China). The sequence for si-NC was 5′-TTCTCCGAACGTGTCACGT-3′; that for si-circ_0008945, 5′-ATGCTGGTGGCAAGCTGCACA-3′; that for miR-388-3p mimics, 5′-UUGUGCUUGAUCUAACCAUGU-3′; that for NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; and that for miR-388-3p inhibitor, 5′-AGCUGGUGUUGUGAAUCAGGCCG-3′. For HOXA3 plasmid construction, we amplified full-length HOXA3 cDNA from MCF-7 cells with PCR using PrimerSTAR Max DNA Polymerase Mix (TaKaRa Bio, Shiga, Japan) and inserted the PCR products into the pcDNA3.0 vector (Invitrogen). For BC cell transfection, we plated cells in 96-well plates and cultured them for 10 hrs, then transfected them with corresponding RNAs using Lipofectamine 3000 reagent (Invitrogen) per manufacturer’s instructions.
Assessment of cell proliferation
We detected the effects of circ_0008945, miR-338-3p and HOXA3 on BC cell viability using a Cell Counting Kit-8 (CCK-8) assay and colony formation assay (CFA). Briefly, for the CCK-8, after transfection with corresponding RNAs we collected and plated BC cells into 96-well plates at a final density of 3×104 cells/well. We cultured the cells at 37 °C for 24 hrs, incubated them with CCK-8 solution (10 µL) for 10 mins and then measured absorbance in each well using a microplate reader at 450 nm. For the CFA, we cultured the treated BC cells in 6-well plates at a density of 2000 cells/well and maintained them at 37 °C for 2 weeks. After cell colonies formed, we fixed them using 4% paraformaldehyde and then applied Giemsa stain for 30 mins. Finally, we counted the visible colonies manually.
Migration and invasion analysis (Transwell assay)
For invasion analysis, we used Transwell chambers (Corning, Inc., Corning, New York, US) coated with Matrigel matrix. We collected and resuspended BC cells in cultured medium (2×105 cells/mL); subsequently, we added BC cell suspension (200 µL) to the upper chamber and cultured medium supplemented with 20% FBS (500 µL) to the lower chamber. After incubation at 37 °C for 24 hrs, the BC cells that invaded the Matrigel were fixed, stained and counted. For migration analysis, we used Transwell chambers without Matrigel matrix.
We estimated the apoptosis of treated BC cells using propidium iodide (PI)/Annexin V–fluorescein isothiocyanate (FITC) staining and flow cytometric (FCM) analysis. In brief, after staining them with PI and Annexin V–FITC for 10 mins, we analyzed the transfected BC cells using an EPICS XL-4 Flow Cytometer (Beckman Coulter, Brea, California, US).
Dual-luciferase reporter assay
We purchased wild-type (WT) and mutant (mut) circ_0008945 plasmids from Geneseed Biotech Co., Ltd. (Guangzhou, China). In brief, we amplified both the WT and mut sequences of circ_0008945 containing the miR-338-3p seed region by specific oligonucleotides (Invitrogen) and sub-cloned them into psi-CHECK2 vector (Promega, Fitchburg, Wisconsin, US) to form circ_0008945-WT and circ_0008945-Mut. Plasmid synthesis was performed using T4 DNA Ligase Master Mix (Thermo Fisher) with NheI and XhoI restriction sites. We tested the physical relationship between circ_0008945 and miR-338-3p using a dual-luciferase reporter assay. After culturing them at 37 °C for at least 8 hrs, we co-transfected BC cells (2×105 cells/well) with circ_0008945-WT and miR-338-3p mimics for 48 hrs. We examined the firefly and renilla luciferase intensities of BC cells by the interaction between miR-338-3p and HOXA3 in the cells per the protocols listed above.
RNA pulldown assay
In brief, we harvested and lysed BC cells stably transfected with biotinylated miR-338-3p (Bio-miR-338-3p) or mutant miR-338-3p (Bio-miR-338-3p-mut) and incubated the lysates for 2 hrs with C-1 magnetic beads (Life Technologies [Thermo Fisher]) at 4 °C. This was followed by purification using an RNeasy Mini Kit (QIAGEN, Düsseldorf, Germany). Finally, we performed qRT-PCR to examine the expression of circ_0008945.
In vivo tumor growth assay
In this study we used BALB/c nude mice (male, 8 weeks old) provided by Xinchang County People’s Hospital and FHWMU. All animal procedures in this study followed the guidelines for animal welfare approved by the Institutional Animal Care and Use Committee of the Ethics Committee of the aforementioned hospitals. BCAP-37 cells stably transfected with circ_0008945 or control circRNA were harvested and resuspended in culture medium. Subsequently, we subcutaneously injected BC cell suspensions (100 μL) into the mice’s left flanks. The BC tumors were allowed to grow for 40 days, and tumor volume was measured every 10 days using the following formula: Volume = (Length × Width2)/2. Forty days later, we sacrificed the mice and excised and weighed the BC tumors.
All data in this study are presented as mean ± standard error of the mean (SEM) for three repeated experiments. We estimated differences between groups with a one-way analysis of variance (ANOVA) using SPSS software version 20 (IBM Corp., Armonk, New York, US). We analyzed the correlation between HOXA3 and miR-338-3p using Pearson’s correlation coefficient and performed a survival analysis using the Kaplan-Meier estimator. P<0.05 was considered statistically significant.
circ_0008945 was highly expressed in BC and associated with poor prognosis
To investigate the role of circ_0008945 in BC, we first detected its expression in BC tissue samples using qRT-PCR. Compared with the NC group, circ_0008945 expression was upregulated in BC tissue samples (Figure 1A). Moreover, we found higher expression of circ_0008945 in BC patients with metastasis than in those without (Figure 1B). In addition, we analyzed the correlations between circ_0008945 expression and the clinicopathological characteristics of BC and found that circ_0008945 expression was related to differentiation grade (P=0.033) and tumor, node and metastasis (TNM) stage (P<0.001; Table 1). Relative circ_0008945 expression was also increased in BC cell lines MCF-7, MDA-MB-231, HCC1937 and BCAP-37 compared with MCF-10A (Figure 1C). In addition, Kaplan-Meier analysis showed that BC patients with high circ_0008945 expression had poor prognoses (Figure 1D).
Table 1 Correlations between hsa_circ_0008945 expression and clinicopathologic characteristics of breast cancer
Knockdown of circ_0008945 inhibited BC cell proliferation in vitro
To test the effect of circ_008945 inhibition on BC cell proliferation in vitro, we transfected BCAP-37 and HCC1937 cells with si-circ_0008945 or its negative control (NC), then performed a CCK-8 assay and a CFA. As shown by qRT-PCR analysis, si-circ_0008945 treatment resulted in significant downregulation of circ_0008945 in BCAP-37 and HCC1937 cells (Figure 2A and B). In our CCK-8 assay, we found that circ_0008945 knockdown significantly inhibited the viability of BCAP-37 and HCC1937 cells (Figure 2C and D). In our CFA, the number of si-circ_0008945–transfected BCAP-37 and HCC1937 cells was reduced, compared with NC (Figure 2E and F). These findings suggested that inhibition of circ_0008945 suppressed BC cell proliferation in vitro.
Knockdown of circ_0008945 in vitro inhibited migration and invasion of BC cells
Next, we evaluated the effect of circ_0008945 knockdown on BC cell migration, invasion and apoptosis using Transwell and FCM assays. The numbers of migratory and invasive BCAP-37 and HCC1937 cells transfected with si-circ_0008945 were significantly reduced compared with NC-transfected cells (Figure 3A and B). FCM analysis indicated that si-circ_0008945 transfection significantly increased the apoptotic rate of BCAP-37 and HCC1937 cells compared with NC transfection (Figure 3C).
miR-338-3p was bound and negatively regulated by circ_0008945
Using bioinformatic analysis, we found that circ_0008945 possessed putative binding sites for miR-338-3p (Figure 4A). To verify whether circ_0008945 could physically interact with miR-338-3p, we conducted a dual-luciferase reporter analysis of the BCAP-37 and HCC1937 cells. Results indicated that miR-338-3p significantly attenuated the circ_0008945-WT–driven luciferase intensity of these cells, but SCRaMbLE did not; on the other hand, neither miR-338-3p nor SCRaMbLE changed the circ_0008945-Mut–driven luciferase intensity of BCAP-37 and HCC1937 cells (Figure 4B). Additionally, we observed higher levels of miR-338-3p in si-circ_0008945–transfected BCAP-37 and HCC1937 cells compared with NC transfected cells (Figure 4C). We used RNA pulldown to verify the relationship between miR-338-3p and circ_0008945. After pulldown, circ_0008945 expression of the Bio-miR-338-3p group was significantly higher than that of either the Bio-NC or Bio-miR-338-3p mutant group (Figure 4D).
Inhibition of miR-338-3p abolished the effects of si-circ_0008945 on BC cells
To explore the biological functions of miR-338-3p in BC cell colony formation, we performed Transwell and FCM assays on BCAP-37 and HCC1937 cells transfected with NC, si-circ_0008945 or si-circ_0008945 + miR-338-3p inhibitor. Our CFA revealed that miR-338-3p inhibitor could reverse the reduction in the number of BCAP-37 and HCC1937 cell colonies caused by si-circ_0008945 treatment (Figure 5A). Our Transwell assay showed that miR-228-3p inhibitor reversed circ_0008945 knockdown–induced downregulation of invasive-cell numbers (Figure 5B). Moreover, our FCM analysis revealed that co-transfection with si-circ_0008945 and miR-338-3p inhibitor blocked the promotive effects of si-circ_0008945 on cell apoptosis (Figure 5C).
miR-338-3p negatively regulated HOXA3 in BC cells
We observed decreased HOXA3 expression in BCAP-37 and HCC1937 cells transfected with miR-338-3p mimics, compared with NC-transfected cells (Figure 6A). Our qRT-PCR analysis of HOXA3 in BC and corresponding normal tissue samples showed that HOXA3 expression was higher in BC samples than in normal ones (Figure 6B, left panel). Moreover, we observed a negative correlation between miR-338-3p expression and HOXA3 expression in BC samples (Figure 6B, right panel). Additionally, we found that miR-338-3p expression was correlated with differentiation grade (P=0.032), distal metastasis (P=0.002) and TNM stage (P<0.001; Table 2); meanwhile, HOXA3 expression was correlated with distal metastasis (P=0.003) and TNM stage (P=0.005; Table 3). Next, we predicted the binding relationship between miR-338-3p and HOXA3 using bioinformatics software (Figure 6C). Our dual-luciferase reporter assay indicated that the luciferase intensity of BCAP-37 and HCC1937 cells co-transfected with HOXA3-WT and miR-338-3p mimics was significantly attenuated (Figure 6D). These findings suggested that miR-338-3p targeted and negatively regulated HOXA3 in BC cells.
Table 2 Correlations between miR-338-3p expression and clinicopathologic characteristics of breast cancer
Table 3 Correlations between HOXA3 expression and clinicopathologic characteristics of breast cancer
HOXA3 overexpression blocked the effects of miR-338-3p mimics in BC cells
To examine the relationship between miR-38-3p and HOXA3 in BC, we transfected BCAP-37 and HCC1937 cells with NC, miR-338-3p mimics or miR-338-3p mimics + HOXA3; afterward, we assessed cell proliferation, invasion and apoptosis. CFA results indicated that miR-338-3p treatment reduced the number of BCAP-37 and HCC1937 cell colonies compared with NC; however, co-transfection with miR-338-3p mimics + HOXA3 abrogated this effect (Figure 7A). In our Transwell analysis of cell invasive ability, miR-338-3p mimics remarkably reduced the number of invasive BCAP-37 and HCC1937 cells, which was abrogated by co-transfection with miR-338-3p + HOXA3 (Figure 7B). Moreover, in our FCM assessment of cell apoptosis, the apoptosis rate of miR-338-3p–treated BCAP-37 and HCC1937 cells was higher than that of NC-treated cells, suggesting that miR-338-3p promoted BC cell apoptosis (Figure 7C). However, co-transfection with miR-338-3p mimics + HOXA3 blocked miR-338-3p–induced apoptosis of BCAP-37 and HCC1937 cells (Figure 7C).
Overexpression of circ_0008945 promoted BC progression in vivo
We performed an in vivo tumor growth assay to evaluate the effects of circ_0008945 overexpression on BC tumor progression. BCAP-37 cells stably transfected with circ_0008945 or control were subcutaneously injected into the left flanks of mice. The circ_0008945 group showed significantly greater tumor growth and weight compared with the control group (Figure 8A and B). Using qRT-PCR, we then detected the expression of circ_0008945, miR-338-3p and HOXA3 in xenografts formed by BCAP-37 cells stably transfected with circ_0008945 or control. Compared with the control group, expression levels of circ_0008945 and HOXA3 were increased and that of miR-338-3p was reduced in the circ_0008945 group (Figure 8C).
In this study, we showed that circ_0008945 was highly expressed in BC and its expression was negatively correlated with prognosis in BC patients. Knockdown of circ_0008945 in vitro suppressed BC cell proliferation, migration and apoptosis while promoting BC cell apoptosis. Overexpression of circ_0008945 in vivo promoted BC tumor growth. We also found that circ_0008945 served as a miRNA sponge of miR-338-3p and indirectly regulated HOXA3, a target gene of miR-338-3p. Inhibition of miR-338-3p and overexpression of HOXA3 abolished the suppressive effects of si-circ_0008945 and miR-338-3p mimics on BC cell growth. These results suggested that the circ_0008945/miR-338-3p/HOXA3 axis played a critical role in the tumorigenesis of BC.
The roles of circRNAs in tumorigenesis have been well documented; however, the molecular mechanisms remain largely unclear. Numerous studies have demonstrated that stable transcripts possess miRNA-binding sequences or miRNA response elements that might act as potential miRNA sponges.18 Since circRNAs are frequently shown to be enriched in functional miRNA-binding sites in human tumor tissues, circRNAs are typically considered to exhibit their promotive or inhibitory effects on tumors by binding and regulating miRNAs.19 For example, circHIPK3 was reported to suppress bladder cancer cell migration and angiogenesis by modulating miR-558.20 In addition, Gong C et al demonstrated that circ_001783 regulated BC progression by acting as an endogenous miRNA sponge for miR-200c-3p.21 These conclusions are in agreement with our findings, indicating that circ_0008945 might act as a miRNA sponge for miR-338-3p.
Previously, miR-338-3p was reported to be involved in multiple human tumors, such as colorectal, bladder, lung, and prostate cancers;22–25 it was recently also revealed to play a role in BC.26 Yingchun Liang et al have reported that miR-338-3p was downregulated in BC,27 which is consistent with our findings. To further investigate how circRNAs regulate tumor-associated genes through miRNAs, studies have focused on miRNA targets. For instance, circ_MYLK was demonstrated to function as a ceRNA of miR-29a, thus increasing the expression of vascular endothelial growth factor (VEGF) and activating the Ras/extracellular signal-regulated kinase (ERK) pathway in bladder cancer.28 Zhang GJ et al have reported that circTADA2A-E6 preferentially served as a miRNA sponge for miR-203a-3p to restore the expression of SOCS3 (a target gene of miR-203a-3p), resulting in a less aggressive oncogenic phenotype of BC.29 Similarly, in this study we demonstrated that circ_0008945 could indirectly regulate the expression of the miR-338-3p–targeted gene HOXA3.
HOXA3 is one of the HOX transcription factors that plays a critical role in the expression of genes associated with embryonic development.30 Abnormal HOXA3 expression has been reported in multiple human tumors, including leukemia, thyroid cancer and glioma.31–33 HOXA3 has been shown to promote invasive growth and progression of colon cancer cells by activating the epidermal growth factor receptor (EGFR)/Ras/Raf/methyl ethyl ketone (MEK)/ERK signaling pathway.34 However, whether HOXA3 is involved in BC remains largely unclear. In this study, we showed that HOXA3 was upregulated in both BC tissue samples and xenografts formed by BCAP-37 cells that overexpressed circ_0008945. HOXA3 was targeted and negatively regulated by miR-338-3p in BC cells, while overexpression of HOXA3 reversed the effects of miR-338-3p on BC.
In conclusion, our findings suggested that circ_0008945 was persistently upregulated during BC progression and that it promoted BC cell proliferation, migration and invasion by sponging miR-338-3p to release HOXA3. Therefore, inhibition of circ_0008945 might be used as a potential novel therapeutic strategy for BC patients.
The authors report no conflicts of interest in this work.
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