Circ_0002060 enhances doxorubicin resistance in osteosarcoma by regulating miR-198/ABCB1 axis


 Background Osteosarcoma (OS) is a common aggressive primary sarcoma of bone. Drug resistance is a huge obstacle to chemotherapy for cancer. This study aimed to investigate the role and mechanism of circ_0002060 in OS resistance to doxorubicin (DOX). Methods The levels of circ_0002060, miR-198 and ATP binding cassette subfamily B member 1 (ABCB1) were measured by quantitative real-time polymerase chain reaction or western blot assay. Kaplan-Meier analysis was performed to determine the relationship between circ_0002060 expression and overall survival. The half inhibition concentration (IC50) of doxorubicin was calculated by Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was assessed by colony formation assay. Cell apoptosis was monitored by flow cytometry. The levels of apoptosis-related proteins were measured by western blot assay. Xenograft assay was utilized to analyze the effect of circ_0002060 on DOX resistance in vivo . The interaction among circ_0002060, miR-198 and ABCB1 were confirmed by dual-luciferase reporter assay, RNA immunoprecipitation assay or RNA pull-down assay. Results Circ_0002060 and ABCB1 were up-regulated, while miR-198 was down-regulated in OS tissues and DOX-resistant OS cells. Circ_0002060 silence reduced DOX resistance in vitro and in vivo . Moreover, circ_0002060 enhanced DOX resistance via sponging miR-198. Besides, miR-198 decreased DOX resistance by binding to ABCB1. In addition, circ_0002060 sponged miR-198 to up-regulate ABCB1 expression. Conclusion Circ_0002060 enhanced doxorubicin resistance of OS by regulating miR-198/ABCB1 axis, which provides potential therapeutic targets for OS therapy.


Background
Osteosarcoma (OS) is a common malignant bone cancer that occurs in children and adolescents [1].
With the development of treatment technology, OS therapy is not limited to surgical resection. The introduction of chemotherapy improves the prognosis of OS patients, resulting in a 5-year survival rate of 70% [2]. Therefore, the use of chemical drugs to improve the prognosis of OS patients is promising.
Circular RNAs (circRNAs) are a new type of non-coding RNAs that can modulate tumorigenesis through a competing endogenous RNA mechanism (ceRNA) [3]. Increasing studies have unveiled that several circRNAs that are abnormally expressed in OS are closely related to OS pathogenesis [4]. For instance, circ-0000285 was highly expressed in OS and facilitated OS development by targeting microRNA-599 and increasing TGFB2 expression [5]. CircMMP9 sponged microRNA-1265 to upregulate CHI3L1, thus accelerating OS progression [6]. In osteoporosis, circ_0002060, a transcript of dynein cytoplasmic 1 heavy chain 1 (DYNC1H1), was significantly elevated and might be a potential therapeutic target [7]. Therefore, circ_0002060 expression was analyzed in OS, and we first found that circ_0002060 was markedly up-regulated, hinting its potential role in OS development.
Moreover, many microRNAs (miRNAs) have been identified as playing a vital role in OS progression [8]. For example, miR-150 exerted an anti-cancer effect on OS progression and enhanced the sensitivity of OS cells to doxorubicin via regulating RUNX2 [9].  could sensitize OS cells to epirubicin via modulating FEN1-induced autophagy [10]. Furthermore, previous research discovered that miR-198 was an underlying molecular target for OS treatment [11].
Bioinformatics analysis showed that miR-198 and circ_0002060 have complementary binding sites, so it is speculated that circ_0002060 may interact with miR-198 in OS.
In breast cancer, miR-381 targeted ABCB1 to reduce cisplatin resistance [13]. In osteosarcoma, TIPE2 strengthened cisplatin sensitivity by decreasing ABCB1 expression [14]. However, the mechanism of miR-198 and ABCB1 in doxorubicin resistance of OS is still barely reported.
Hence, we further investigated the function and potential mechanism of circ_0002060, miR-198 and ABCB1 in doxorubicin resistance and tumor progression in OS.

Clinical samples
Forty OS tissues and adjacent normal tissues were obtained from OS patients who received surgery at Weifang People's Hospital. This research was ratified by the Ethics Committee of Weifang People's Hospital. All participants signed written informed consent. All tissues were immediately frozen in liquid nitrogen and then stored at -80°C.

Quantitative real-time polymerase chain reaction (qRT-PCR)
After extracting RNA with Trizol (Invitrogen), RNA was reversely transcribed using HiScript II One Step were purchased from GenePharma.

Cell Counting Kit-8 (CCK-8) assay
Cells (3×10 3 ) were plated into 96-well plates and incubated with escalating doses of DOX for indicated time. Subsequently, cells were reacted with 10 μL CCK-8 solution (Solarbio) for 2 h. Next, a Multi-Mode Reader (BioTek, Burlington, VT, USA) was used to measure the optical density at 450 nm to assess cell viability. The half-maximal inhibitory concentration (IC50) of DOX is the concentration at which cell viability is reduced to 50%.

Colony formation assay
The treated cells were seeded into six-well plates and cultured in DMEM medium containing 10% FBS at 37°C for 14 days. After staining with 0.5% crystal violet, colonies were imaged and counted at least three times.

Flow cytometry
The treated cells were inoculated into six-well plates and washed with cold PBS. Then, AnnexinVfluorescein isothiocyanate (AnnexinV-FITC)/Propidium Iodide (PI) Apoptosis Detection kit (Invitrogen) was used to detect cell apoptosis. Final, Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to monitor the apoptosis rate.

Xenograft tumor experiment
U2OS/DOX cells were transfected with the lentivirus carrying circ_0002060 short hairpin RNA (sh-circ_0002060) or the negative control (sh-NC). Subsequently, stably transfected cells (5×10 6 ) were subcutaneously injected into the right-back of BALB/c nude mice (n=6 per group). Then, DOX (15 mg/kg) was intravenously administered to nude mice twice a week. Tumor volume was monitored every week. After five weeks, the mice were sacrificed, and xenografts were weighted. The xenograft assay was ratified by the Animal Research Committee of Weifang People's Hospital.

RNA immunoprecipitation (RIP) assay
RIP analysis was carried out using EZ-Magna RIP kit (Millipore). Briefly, U2OS/DOX and HOS/DOX cells were lysed by RIP lysis buffer. Then, cell lysates were incubated with magnetic beads conjugated with Ago2 antibody or IgG antibody. The enrichment of circ_0002060, miR-198 or ABCB1 was measured by qRT-PCR.

RNA pull-down assay
Biotin-labeled circ_0002060 probe (Bio-circ_0002060) and the control probe (Bio-NC) were purchased from GenePharma. Briefly, biotinylated probes were reacted with M-280 Streptavidin Dynabeads (Invitrogen) at 37°C for 2 h to construct probe-coated beads. Next, the cells were lysed and incubated with probe-coated beads at 4°C for 3 h. Finally, the expression of miR-198 was measured by qRT-PCR.

Statistical analysis
All data were exhibited as mean ± standard deviation using Graphpad Prism 7.0 software (GraphPad, San Diego, CA, USA). Differences were tested by Student's t-test or one-way analysis of variance.
When P <0.05, the difference was considered statistically significant.

Circ_0002060 was up-regulated in OS tissues and DOX-resistant OS cells
First, we detected the expression of circ_0002060 in OS tissues and adjacent normal tissues, and the results exhibited that circ_0002060 expression in OS tissues was strikingly increased compared with non-cancer tissues (Fig. 1A). Besides, Kaplan-Meier analysis was performed to explore the relationship between circ_0002060 expression and overall survival in OS patients. The results showed that high circ_0002060 was closely related to poor prognosis in OS patients (Fig. 1B). Also, the expression of 7 circ_0002060 in U2OS/DOX and HOS/DOX cells was overtly higher than that in U2OS and HOS cells (Fig. 1C). Moreover, circ_0002060 was resistant to RNase R compared to its linear isoform DYNC1H1 ( Fig. 1D and 1E). In addition, DOX-resistant cells were treated with Actinomycin D, and qRT-PCR showed that circ_0002060 had a half-life of more than 24 h, while DYNC1H1 mRNA had a half-life of less than 6 h ( Fig. 1F and 1G). These data revealed that circ_0002060 might be an oncogene in OS.

Circ_0002060 knockdown decreased DOX resistance in vitro and in vivo
In order to explore the function of circ_0002060 in OS cells, circ_0002060 was silenced by transfecting si-circ_0002060#1, si-circ_0002060#2 or si-circ_0002060#3 into U2OS/DOX and HOS/DOX cells. The knockdown efficiency of circ_0002060 was detected by qRT-PCR analysis, and the expression of DYNC1H1 was not affected ( Fig. 2A and 2B). CCK-8 assay showed that down-regulation of circ_0002060 reduced the IC50 value of DOX in U2OS/DOX and HOS/DOX cells ( Fig. 2C and 2D).
Colony formation assay demonstrated that circ_0002060 silencing and DOX treatment significantly decreased the proliferation ability of U2OS/DOX and HOS/DOX cells (Fig. 2E and 2F). Additionally, flow cytometry revealed that circ_0002060 knockdown and DOX stimulation markedly promoted the apoptosis of DOX-resistant OS cells (Fig. 2G-2I). Consistently, circ_0002060 silencing and DOX stimulation resulted in a striking reduction in Bcl-2 expression and a significant increase in c-caspaes 3/caspaes 3 and Bax levels ( Fig. 2J and 2K). Moreover, a xenograft mouse model was established to investigate the effect of circ_0002060 on DOX sensitivity in vivo. As shown in Fig. 2L and 2M, tumor volume and weight were observably reduced in sh-circ_0002060 group compared with sh-NC group, and DOX treatment enhanced this effect. These results unveiled that silencing of circ_0002060 reduced DOX resistance in vitro and in vivo.

Circ_0002060 directly targeted miR-198
Next, the Circular RNA Interactome online database determined that circ_0002060 and miR-198 had complementary sequences (Fig. 3A). Besides, the expression of miR-198 in OS tissues was significantly lower than that in normal tissues (Fig. 3B). In OS tissues, circ_0002060 expression was inversely correlated with miR-198 expression (Fig. 3C). First, dual-luciferase reporter assay suggested that miR-198 mimics inhibited the luciferase activity of circ_0002060-wt reporter ( Fig. 3D and 3E).
8 Then, the relationship between circ_0002060 and miR-198 was verified by RIP and RNA pull-down assays. As shown in Fig. 3F-3G, circ_0002060 and miR-198 were markedly enriched in Ago2 group compared to lgG group. Additionally, RNA pull-down assay revealed that miR-198 could be pulled down by Bio-circ_0002060, but not by Bio-NC (Fig. 3H). Compared with U2OS and HOS cells, miR-198 level in U2OS/DOX and HOS/DOX cells was significantly reduced (Fig. 3I). In addition, circ_0002060 silencing strikingly elevated miR-198 expression, and circ_0002060 overexpression markedly decreased miR-198 expression ( Fig. 3J and 3K). These data indicated that circ_0002060 was a sponge of miR-198.

Circ_0002060 regulated DOX resistance by sponging miR-198
To investigate the roles of circ_0002060 and miR-198 in DOX resistance, U2OS/DOX and HOS/DOX cells were transfected with si-NC, si-circ_0002060#1, si-circ_0002060#1+anti-miR-NC or si-circ_0002060#1+anti-miR-198. The results showed that the addition of anti-miR-198 reversed the increase of miR-198 expression caused by circ_0002060 silencing (Fig. 4A). CCK-8 analysis revealed that inhibition of miR-198 eliminated the reduction in DOX resistance induced by circ_0002060 knockdown (Fig. 4B-4E). Colony formation assay exhibited that down-regulation of miR-198 partially attenuated the reduction in colony number caused by DOX treatment or circ_0002060 silencing ( Fig.   4F and 4G). In addition, transfection with miR-198 partially reversed the effect of DOX stimulation or circ_0002060 depletion on cell apoptosis ( Fig. 4H and 4I). Consistently, DOX treatment and circ_0002060 silencing increased the levels of c-caspase 3/caspase 3 and Bax, and decreased the level of Bcl-2, while miR-198 knockdown partially abolished the effects (Fig. 4J-4M). These data indicated that circ_0002060 modulated DOX resistance by sponging miR-198 in DOX-resistant OS cells.

ABCB1 was a target of miR-198
Further, TargetScan online database predicted that miR-198 and ABCB1 3'UTR had putative binding sites (Fig. 5A). First, qRT-PCR and western blot assays suggested that the mRNA and protein levels of ABCB1 were remarkably increased in OS tissues relative to normal tissues ( Fig. 5B and 5C). Also, miR-9 reporter assay demonstrated that mature miR-198 reduced the luciferase activity of ABCB1 3'UTR-wt reporter ( Fig. 5E and 5F). RIP assay was performed to validate the interaction between miR-198 and ABCB1, and the results showed that miR-198 and ABCB1 were significantly enriched in Ago2 group compared with lgG group (Fig. 5G and 5H). Compared with U2OS and HOS cells, the mRNA and protein levels of ABCB1 in U2OS/DOX and HOS/DOX cells were significantly up-regulated ( Fig. 5I and   5J). Besides, miR-198 mimics suppressed ABCB1 mRNA and protein expression, while miR-198 knockdown has the opposite effect (Fig. 5K-5N). These data unveiled that ABCB1 was a target of miR-198.

MiR-198 reduced DOX resistance via targeting ABCB1
To explore the relationship between miR-198 and ABCB1 in DOX resistance, U2OS/DOX and HOS/DOX cells were introduced with miR-NC, miR-198, miR-198+pcDNA or miR-198+ABCB1. First, transfection with ABCB1 reversed the decrease in ABCB1 expression caused by miR-198 overexpression (Fig. 6A-6C). In addition, up-regulation of miR-198 decreased the IC50 value of DOX, whereas the impact was abolished after transfection with ABCB1 ( Fig. 6D-6G). Colony formation assay revealed that overexpression of ABCB1 partially abrogated the decrease in colony number induced by DOX stimulation or miR-198 up-regulation ( Fig. 6H and 6I). Additionally, introduction of ABCB1 partially rescued the promoting effect of DOX treatment or miR-198 overexpression on cell apoptosis (Fig. 6J and 6K). Similarly, DOX stimulation and miR-198 up-regulation resulted in increased c-caspase 3/caspase 3 and Bax expression, and decreased Bcl-2 expression, while these changes were partially reversed by overexpressing ABCB1 (Fig. 6L-6O). These data evidenced that miR-198 decreased DOX resistance via targeting ABCB1 in U2OS/DOX and HOS/DOX cells.

Discussion
Recently, the occurrence of chemoresistance has become a major stumbling block in OS treatment [15]. Besides, adriamycin has significant medicinal properties in adjuvant chemotherapy of localized high-grade OS [16]. Additionally, large amounts of abnormally expressed non-coding RNAs (ncRNAs) play a crucial role in anti-tumor drug resistance [17]. Therefore, we strengthened the study of circRNA in doxorubicin resistance of OS.
Accumulating evidence demonstrated that circRNAs could mediate the expression of target mRNAs by serving as ceRNAs or miRNA sponges, thereby regulating chemotherapy resistance [18]. For example, circKDM4C potentiated doxorubicin sensitivity and hindered tumorigenesis in breast cancer via sponging miR-548p and up-regulating PBLD [19]. Additionally, Hu et al. presented that circ-LARP4 attenuated cisplatin and doxorubicin resistance in OS by binding to miR-424 [20]. This research demonstrated that circ_0002060 was remarkably up-regulated in OS tissues and DOX-resistant OS cells, and confirmed that circ_0002060 silencing reduced DOX resistance in OS. Further, we disclosed the negative regulatory and binding relationship between circ_0002060 and miR-198.
More and more researches corroborated that miR-198 was an inhibitor of various tumors, including gastric cancer [21], colorectal cancer [22] and breast cancer [23]. Furthermore, the mining of miRNAs provides a new pathway for early diagnosis of OS. Zhang et al. revealed that miR-198 functioned as a potential target for OS treatment by regulating ROCK1 [11]. Also, several studies demonstrated that miRNAs could regulate chemotherapy resistance of OS by mediating multiple potential molecular mechanisms such as DNA damage response, autophagy induction, and signal transduction [24]. Chen et al. found that miR-198 weakened temozolomide resistance via modulating MGMT in glioblastoma [25]. Huang et al. discovered that miR-198 reduced the increase of cisplatin resistance caused by circAKT3 by targeting PIK3R1 in gastric cancer [26]. Nevertheless, the effect of miR-198 on DOX resistance of OS remains unclear. In this study, we evidenced that miR-198 expression was remarkably decreased, and circ_0002060 sponged miR-198 to enhance DOX resistance in OS.
Meanwhile, the existence of multidrug resistance (MDR) has made tumor treatment more difficult [27]. ABCB1 (also known as MDR1) is considered to be the main factor inducing MDR [28]. In hepatocellular carcinoma, miR-122 improved oxaliplatin sensitivity by repressing ABCB1 expression to inactivate Wnt/β-catenin pathway [29]. In colorectal cancer, overexpression of ABCB1 attenuated oxaliplatin sensitivity, which might be regulated by miR-302c-5p [30]. Also, many studies discovered that ABCB1 was remarkably elevated in OS [31], and ABCB1 inhibitor undermined OS resistance to doxorubicin and paclitaxel [32]. We first unveiled that ABCB1 was a target of miR-198 in DOXresistant OS cells.

Conclusion
Circ_0002060 potentiated doxorubicin resistance and facilitated tumor progression in OS via modulating miR-198/ABCB1 axis. Our research found a novel molecular mechanism for improving OS chemotherapy.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Weifang People's Hospital and written informed consents were collected from all patients and hospitals.
The animal experiment was permitted by the Animal Research Committee of Weifang People's Hospital and performed in accordance with the guidelines of the National Animal Care and Ethics Institution.

Availability of data and materials
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no conflicts of interest.

Authors' contributions
Jun Liu performed experiments, analyzed data, and wrote the manuscript. Wenshuai Zhu designed research, performed experiments, and analyzed data. Jianqin Ji conceived and designed research.