Circ_0000231 Promotes Paclitaxel Resistance in Ovarian Cancer by Regulating miR-140/RAP1B

doi:10.21203/rs.3.rs-1764341/v1

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Circ_0000231 Promotes Paclitaxel Resistance in Ovarian Cancer by Regulating miR-140/RAP1B

https://doi.org/10.21203/rs.3.rs-1764341/v1

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Purpose: Circular RNAs (circRNAs) are identified as vital regulators in a variety of cancers. However, the involvement of circ_0000231 in paclitaxel (PTX) resistant ovarian cancer (OC) remains unclear.

Methods: The levels of circ_0000231, microRNA-140 (miR-140) and RAP1B were determined by quantitative real-time PCR (qRT-PCR) or Western blot assay. Cell proliferation, PTX- resistance, apoptosis, migration and invasion were assessed by Cell Counting Kit-8 (CCK-8), flow cytometry and transwell assay. The levels of resistant-related and epithelial–mesenchymal transition (EMT)-related proteins were measured by Western blot assay. Target association between miR-140 and circ_0000231 or RAP1B was demonstrated by dual[1]luciferase reporter assay or RNA Binding Protein Immunoprecipitation (RIP). Biological implications of circ_0000231 in ovarian cancer were implemented in tumor xenograft models.

Results: Circ_0000231 and RAP1B levels were increased, while miR-140 level was decreased in PTX-resistant OC tissues and cells. Depletion of circ_0000231 could inhibit resistance, proliferation, invasion, migration and EMT and promoted apoptosis in PTX-resistant OC cells, which was opposited by overexpression of circ_0000231. Circ_0000231 acted as a sponge for miR- 140, and RAP1B was revealed to be target gene of miR-140. Circ_0000231 was a key molecule required for growth, migration, and PTX-resistance and was involved in EMT. Circ_0000231 knockdown increased PTX sensitivity of OC in vivo.

Conclusion: Knockdown of circ_000231 suppressed PTX-resistant OC progression via regulating miR-140/RAP1B signaling pathway. Circ_0000231 might play vital roles in the tumorigenesis and chemoresistance of OC.

Ovarian cancer

circ_0000231

RAP1B

miR-140

Chemoresistance

Paclitaxel

Ovarian cancer (OC) is the eighth most common cause of cancer death and the second cause of gynecologic cancer death in women around the world [1]. OC is the most deadly cancer among all gynecologic malignancies [2], and PTX-based treatment is a part of the standard first-line chemotherapy regimen. Different resistance mechanisms have been identified, however, the molecular mechanism of PTX resistance has not been completely elucidated. The development of resistance to chemotherapy in OC patients further complicates OC treatment outcomes [3]. Therefore, understanding the drug resistant mechanism to chemotherapy is critical to eventually improve ovarian cancer treatment efficacy.

Non-coding RNA is a class of RNA transcripts that do not have protein-coding potential, including microRNA(miRNA), long non-coding RNA (lncRNA), and circular RNA(circRNA), which plays an important role in the occurrence and development of cancers [4]. CircRNA has become a research focus in the field of non-coding RNA [5]. Some study has shown that circRNAs can act as a miRNAs “sponge” and play an important role in the progress of cancers [6, 7], expecially in drug resistance. For instance, circCDR1as inhibits the development of platinum resistance in ovarian cancer by competing with miR-1270 [8]. Furthermore, circCELSR1 acts as a molecular sponge and promotes paclitaxel resistance by competition with miR-1252 for binding FOXR2 [9]. For circ_0000231, it was reported [10] that circ_0000231 attenuated Sevoflurane aroused repression impacts on CRC development by sponging miR-622. However, the role of circ_0000231 in OC PTX-resistance remains to be investigated. In this study, we investegated the expression of circ_0000231 in PTX-resistant specimen and cell lines and studied the function and potential mechanism of circ_0000231 in OC.

Specimen Collection

95 ovarian cancer patient samples are obtained from 2012-2019. All patients are diagnosed by surgery and pathology in the Shengjing Hospital of China Medical University: all patients after surgery only received PTX-based chemotherapy, and written informed consent from each patient was obtained prior to study initiation. A PTX-resistant case was distinguished when the primary tumor enlarged or relapsed within 12 months, otherwise, it can be defined as a PTX-sensitive case. Samples were promptly frozen in liquid nitrogen and maintained at -80℃ deep cryogenic refrigerator until use.

Cell culture

Human ovarian cancer cell line (SKOV3) was purchased from Cell Bank (Shanghai Institute for Biological Science) and PTX-resistant OC line(SKOV3-TR30) provided by Zhejiang University affiliated Obstetrics and Gynecology Hospital. SKOV3 and SKOV3-TR30 were maintained in RPMI-1640 medium (Solarbio, Beijing, China) plus 10% fetal bovine serum (FBS, Thermo Fisher, Wilmington, DE, USA) and 1% penicillin/streptomycin (Solarbio, Beijing, China) which were incubated at 37℃ with 5% CO2 and 95% air. SKOV3-TR30 cells were incubated with 50 nM PTX to maintain their resistant phenotype.

RNA extration and Real-time quantitative PCR(qRT-PCR)

RNA was isolated from tissues and cells using Trizol (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The complementary DNA (cDNA) was obtained using the specifc reverse transcription kit (RR047A, Takara, China). The expression level was monitored using SYBR Premix Ex Taq (cat no. RR420A; Takara) and calculated using the 2−ΔΔCt method. β-actin were served as a loading control.

RNase R treatment

For RNase R treatment assay, 10ug of total RNA was incubated with or without RNase R (Epicentre Technologies, USA) at 37 °C for 30 min. Then, circ_0000231 and linear mRNA (ARHGAP12) were calculated by qRT-PCR.

Cell transfection

Small interfering RNAs against circ_0000231, short hairpin RNA (shRNA) against circ_0000231 (sh-circ_0000231), as well as their corresponding negative controls (si-NC and sh-NC) were were synthesized by RiboBio (Guangzhou,China). MiR-140 mimics (miR-140) and the control (miR-NC), miR-140 inhibitor (anti-miR-140) and the control (anti-miR-NC) were synthesized from Hanbio (Shanghai, China). Circ_0000231 (oe-circ) and RAP1B (RAP1B) overexpression vector, and their control groups (oe-NC) (pcDNA) were amplified by GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen) transfection reagent when cells reached to 60%-80%.

CCK-8 Assay

Cell proliferation was measured using CCK-8 (Beyotime, Nantong, China) assays as previously reported. 2 × 10³cells were seeded in 96-well plates and were cultured for 0h, 24 h, 48 h, 72 h and 96 h. For the detection of PTX resistance, ovarian cancer cells in 96-well plates were exposed to different doses of PTX. The absorbance values at 450 nm were then measured using an enzyme immunoassay analyzer. IC50 value represented the half-maximum inhibitory concentration of PTX.

Fluorescence in situ hybridization (FISH)

The subcellular location of circ_0000231 and miR-140 was detected using the FISH assay. Circ_0000231 probe and miR-140 probe were used. Nuclei were stained by 4, 6-diamidino-2-phenylindole (DAPI). The FISH assay was performed using a fluorescent in situ hybridization kit (Servicebio, Wuhan, China) according to the manufacturer’s protocol. Images

were captured with a confocal microscope (Olympus).

Transwell assays

Briefly, the transfected ovarian cells, 2×10⁵ per well, were injected into the upper chamber (Corning, NY, USA) which was used to precoat with Matrigel (Solarbio, Beijing, China). The bottom chamber contained RPMI-1640 with 10% FBS. After culturing for 48 h, the invaded cells were dyed and imaged. In transwell migration assay, cells were directly seeded into the noncoated, and the other steps were similar as above mentioned.

Flow cytometry

The transfected ovarian cells were washed twice in ice cold PBS, and then resuspended in binding buffer. The cells were respectively stained with Annexin V-FITC (BestBio, Shanghai, China) (5ul) and PI (5ul) for 10 min in a dark room and then analyzed with a flow cytometer (FACScan, BD, USA) and apoptotic fractions were analyzed by FlowJo software.

Western b Blot

The proteins of cells and tissues were extracted with RIPA buffer, protease inhibitor and phosphatase inhibitor cocktails (Beyotime, Shanghai, China) and quantifified with a BCA kit (Beyotime, Shanghai, China). Equal loading proteins of cell lysates or tissue lysates were added to each well of SDS PAGE, and blotted on PVDF membranes (Millipore, Billerica, MA).The membranes were incubated with primary antibodies against RAP1B (Proteintech, China), E-cadherin (Proteintech, China), Vimentin (Proteintech, China), MMP9 (Proteintech, China), β-tubulin III (Wanleibio, China), β-actin (Wanleibio, China) at 4 °C overnight. The secondary antibody (Proteintech, China) was then incubated with the membranes for 1h. Enhanced chemiluminescence Western Blotting Substrate (Wanleibio, China) and X-ray films were used to visualize the protein bands.

Immunohistochemical staining (IHC)

Tumor tissues were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. The dealed tissues were sectioned at a thickness of 5 µm and attached to a polylysine slide overnight at 60 °C and dewaxed. The β-tubulin III (Wanleibio,China) and goat anti-rabbit IgG/HRP antibody (Solarbio, China) were used and then incubated with the corresponding secondary antibody. Finally, the numbers of β-tubulin III-positive cells were evaluated under an optical microscope.

Luciferase Reporter Assay

The circ_0000231 and RAP1B wild-type sequence (circ_0000231-WT-3’UTR,RAP1B-WT-3’UTR) containing the bingding site of miR-140 mimic and the mutant-type sequnence (circ_0000231-MUT-3’UTR,RAP1B-MUT-3’UTR) were synthesized and cloned into the psiCHECK vector (Hanbio, Shanghai, China). All these plasmids and miR-NC or miR-140 were transfected into cells using Lipofectamine 2000. After 24h, Firefy luciferase and Renilla luciferase activities were measured via Dual-Lucy Assay Kit (Beyotime, Shanghai, China). Firefy luciferase activity was standardized by Renilla luciferase activity.

RNA-immunoprecipitation (RIP) assay

The interaction between circ_0000231 and miR-140 was assessed by a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA) according to manufacturer’s instruction. Cell lysates were incubated with Argonaute 2 (AGO2; Millipore) or control IgG (Millipore) antibody. Then purified RNA was extracted and analyzed for circ_0000231 and miR-140 expression by qRT-PCR.

Xenograft assay

The ethics of animal research were ratifed by the Animal Ethics Committee of China Medical University. Female BALB/c nude mice (5-week-old) were randomly divided into four groups(n=3). Stably transfected 2×10⁶ SKOV3-TR30 cells (sh-NC, sh-circ_0000231) were subcutaneously injected into the right of mice. After injection for 5 days, mice were treated with PTX (5mg/kg) or normal saline via intraperitoneal injection every 3 days. Tumor Volumn (mm³) = length × width² × 0.5. After 28 days, mice were sacrifced and the xenograft tumors were weighed.

Statistical Analysis

All data were shown as the mean ± standard deviation of at least three independent experiments. Spearman analysis was used to analyze the relationship of associated factors. Statistical analysis was performed using SPSS 26.0. P < 0.05 was considered significant.

Circ_0000231 is highly expressed in PTX-resistant OC tissues and cells

We first measured the expression of circ_0000231 in PTX-resistant OC tissues by qRT-PCR. The expression of circ_0000231 in chemoresistant OC tissues (30cases) was significantly higher than that in the chemosensitive group (65cases) (Fig.1a). We verified in SKOV3 and SKOV3-TR30 that SKOV3 had significantly lower expression levels of circ_0000231 (Fig.1b). Circ_0000231 was mainly located in the cytoplasm of SKOV3 and SKOV3-TR30 cells by FISH analysis (Fig.1c). Moreover, the fragment of linear ARHGAP12 mRNA was digested by RNase R, whereas circ_0000231 remained after the treatment of RNase R (Fig.1de).Using the median expression level of circ_0000231 as cutoff value, we divided the OC patients into low and high expression groups. As the table1 showed, circ_0000231 expression was signifificantly correlated with FIGO stage.

Table 1 Association of circ_0000231 Expression with Clinicopathological Features of OC Patients
	Characteristics	N	Low	High	P-value
	Age (years)				P=0.630
	<50	35	25	10
	≥50	60	40	20
	FIGO				*P=0.000
	I-II	27	26	1
	III-IV	68	39	29
	Differeciation				P=0.576
	High	32	10	6
	Low	63	55	24
	Positive lymph node				P=0.113
	No	43	33	10
	Yes	52	32	20

Circ_0000231 regulates PTX chemosensitivity, Proliferation, Apoptosis and Metastasis in OC in vitro

The effect of circ_0000231 on the sensitivity of OC cells to paclitaxel was investigated. Then, si-circ and si-NC were transfected into SKOV3-TR30 cells (Fig.2a) and oe-circ, oe-NC were transfected into SKOV3 cells (Fig.2b). CCK-8 analyzed that si-circ inhibited SKOV3-TR30 cells proliferation(Fig.2cd). IC50 was analyzed by CCK‐8 assays SKOV3 and SKOV3-TR30 cells were treated with different concentrations of PTX (Fig.2ef). Silencing of circ_0000231 markedly raised apoptosis rates (Fig.2G). The opposite was found with overexpression of circ_0000231 (Fig.2h). Cell migration and invasion were detected using transwell assay. Circ_0000231 knockdown significantly inhibited cell invasion and migration in SKOV3-TR30 (Fig.2i). In contrast, overexpression of circ_0000231 promoted cell migration and invasion (Fig.2j). Furthermore, we found that circ_0000231 knockdown declined protein levels of EMT in SKOV3. Moreover, overexpresion of circ_0000231 significantly increased protein levels of EMT. Collectively, these results suggest that circ_0000231 regulated PTX-resistance, proliferation and metastasis in OC cells.

MiR-140 is a Direct Target of circ_0000231

We used two prediction websites (circBank and circinteractome) to predict potential target miRNAs that might bind to circ_0000231 (Fig.3a). qRT-PCR detected the expression of four possible miRNAs (miR-558, miR-622, miR-140 and miR-135b-5p) in SKOV3-TR30 cells transfected with si-NC and si-circ. The expression of miR-140 was dramatically upregulated in circ_0000231 knockdown cell lines (Fig.3b). Potential binding sites between circ_0000231 and miR-140 were predicted by CircInteractome (Fig.3c). qRT-PCR was used to detect the expression of circ_0000231 in transfected cells with anti-miR-140 and miR-140 (Fig.3d). The prediction that circ_0000231 was targeted by miR-140, dual‐luciferase reporter assay and RIP showed that miR-140 could bind to circ_0000231 (Fig.3e). FISH assay demonstrated that miR-140 and circ_0000231 mainly co-localized in the cytoplasm (Fig.3g). We found that miR-140 was a decreasing trend in PTX-resistant OC tissues and cells (Fig.3 fh). It showed a significant inverse association between miR-140 and circ_0000231 expression in OC samples (Fig.3i).These results supported that miR-140 was a target gene of circ-0000231 in ovarian cancer cells, and circ-0000231 negatively regulated the expression of miR-140.

MiR-140 regulates the PTX chemosensitivity ,Proliferation, Apoptosis and Metastasis in OC

The transfection efficiency of miR-140 were evaluated by qRT-PCR(Fig.4ab). We found that miR-140 regulated cells proliferation (Fig.4cd). The results of IC50 showed that miR-140 supressed the PTX chemosensitivity (Fig.4ef). Flow cytometry revealed that the apoptosis rate of miR-140 mimics group was greatly higher compared with NC group in SKOV3-TR30 cells, and miR-140 suppression could significantly decrease apoptosis rate (Fig.4g). The results of transwell assay indicated that the numbers of migratory cells and invasive cells in the miR-140 inhibit-treated group was markedly increased compared with the NC inhibitor group (Fig.4h). Likewise, the miR-140 mimic and the NC mimic control showed opposite results (Fig.4i). Anti-miR-140 significantly reduced the protein expression levels of vimentin, β-tubulin-III and MMP9, and significantly increased the expression levels of E-cadherin compared with anti-miR-NC. However, miR-140 upregulated the expression levels of vimentin, β-tubulin-III and MMP9, and downregulated the expression levels of E-cadherin (Fig.4j). Collectively, these results suggest that miR-140 knockdown inhibited PTX-resistance, proliferation and metastasis in OC cells. Overexpressed miR-140 exerted an opposite effect.

Inhibition of miR‑140 reverses the effect of circ_0000231 knockdown on OC cell progression and PTX-resistance

MiR-140 inhibition almost reversed circ_0000231 knockdown-induced the upregulation of miR-140 in SKOV3-TR30 cells. Then, results of CCK-8 elucidated that miR-140 inhibition could largely rescued circ_0000231 silence-mediated suppressive function on OC proliferation (Fig.5a). Furthermore, IC50 was analyzed by CCK‐8 assays SKOV3-TR30 cells were treated with different concentrations of PTX. It showed that miR-140 inhibition almost reversed circ_0000231 knockdown-induced the chemosensitivity in SKOV3-TR30 (Fig.5b). The inhibition of miR-140 countervailed the function of circ_0000231 upregulation in apoptosis (Fig.5c). Similarly, miR-140 suppression could reverse the restraining effect of circ_0000231 depletion on cell migration and invasion (Fig.5d). The expression of vimentin, β-tubulin-III and MMP9 in si-NC and anti-miR-140 cotransfection group were upregulated than the other three groups (Fig.5e). The expression of E-cadherin was reduced. The above results suggested that miR-140 silencing could offset the tumor-suppressing effect of circ_0000231 silencing alone.

Circ_0000231 Positively Regulated RAP1B Expression by Interacting with miR-140 in PTX-Resistant OC Cells

Using TargetScan, we predicted that RAP1B might be the target gene of miR-140. RAP1B were dramatically declined in SKOV3-TR30 cells transfected with si-circ or miR-140 mimic , and increased in SKOV3 cells transfected with oe-circ (Supplementary Materials). The result of TargetScan showed that RAP1B possessed complementary sites with miR-140 on its 3’UTR (Fig.6a). Then, we constructed luciferase reporter vectors containing wild-type or mutant sequence of miR-140 MREs and co-transfected with RAP1B. The luciferase assay confirmed that miR-140 targeted RAP1B and negatively regulated RAP1B expression (Fig.6b). RAP1B was higher in SKOV3-TR30 than SKOV3 (Fig.6c). The expression of RAP1B was obviously increased in PTX-resistant ovarian cancer tissues (Fig.6d).Western blot showed the same results (Supplementary Materials). Importantly, we performed Spearman to analysis the relationship between RAP1B and miR-140. The result showed that RAP1B was negatively correlated with miR-140 (Fig.6e). Western blot showed that RAP1B decreased in si-circ and increased in oe-circ (Fig.6f),on the contrary,it increased in anti-miR-140 and decreased in miR-140 (Fig.6g).To further study the effect of RAP1B on PTX-resistant OC, we overexpressed the RAP1B (Supplementary Materials). After RAP1B overexpression, the proliferation rate of SKOV3-TR30 significantly increased (Fig.6h). The IC50 of PTX by CCK-8 showed that overexpression of RAP1B promoted the PTX-resistance (Fig.6i). Overexpression of RAP1B revereses the effect of circ_0000231 knockdown in proliferation, apoptosis, invasion, migration, PTX-resistance and expression of related proteins (Fig.6j-o). To sum up, circ_0000231 positively regulated RAP1B level by sponging miR-140.

Circ_0000231 knockdown enhances PTX sensitivity of ovarian cancer cells in vivo

The xenograft model was constructed to assess the effect of circ_0000231 on OC. Sh-NC or sh-circ-0000231 were subcutaneously injected into the mice. Mice were randomized to four groups : sh-circ_0000231 + PTX, sh-circ_0000231+ normal saline (NS), sh-NC + PTX, and sh-NC + NS (Fig.7a). As shown in figure7a, compared to four groups, the tumor volumes significantly decreased in sh-circ_0000231 + PTX (Fig.7b). Maximum tumor volume was in sh-NC+ NS group. The tumor weight also showed that sh-circ_0000231+PTX group was the samllest in four groups (Fig.c). These results confirmed that the inhibition of circ_0000231 affected PTX-resistant OC cells growth. Furthermore, an IHC assay showed that the tumors treated with sh-circ_0000231 displayed an decreased percentage of β-tubulin III (Fig.7d). Collectively, circ_0000231 knockdown and PTX together resulted in synergistic inhibition in OC cell growth in vivo.

Chemotherapy is widely used for OC nowadays, however, the main obstacle to successful chemotherapy is the development of drug resistance to chemotherapeutics [11]. In this article, we confirmed the mechanism of circRNA-induced OC paclitaxel resistance. This study demonstrated that circ_0000231 was upregulated in PTX-resistant OC cell lines and tissues.

The effects of circ_0000231 were assessed in vitro by overexpressing and silencing experiments. Suppression of circ_0000231 promoted paclitaxel sensitivity, apoptosis and inhibited cell proliferation, migration and invasion, that it may have a vital impact on the metastasis and PTX-resistance of ovarian cancer, whereas overexpression of circ_0000231 had the opposite effect. The same result was also demonstrated in vivo. The above results prove our previous hypothesis that circ_0000231 could be crucial to identify novel diagnostic and therapeutic targets in OC. To explored the molecular mechanism we found that circ_0000231 mainly located in the cytoplasmic fraction of SKOV3, indicating that circ_0000231 might regulate gene expression at post-transcriptional level. In other dieases, circ_0000231 can act as miRNA “sponge” affecting miRNA activity. Liu [12] et al found circ_0000231 knockdown inhibited colorectal cancer cells progression and glycolysis by downregulating MYO6 expression through sponging miR-502-5p. Circ-0000231 promotes atherosclerosis progression through miR-1236-3p/HOXB7 regulatory axis [13]. Thus, we infered circ_0000231 could exert its role in PTX-resistant OC through regulating the miRNA/mRNA axis.

MiR-140 is involved in cell proliferation apoptosis, migration and invasion in various cancers [14], such as breast cancer [15], esophageal squamous cell carcinoma [16], lung cancer [17] as well as in OC [18]. Qiao [19] et al found miR-140 was sponge by LINC00852 and involved in LINC000852 mediated OC progression. MiR-140 also inhibits ovarian cancer growth by repression of PDGFRA [18]. Bioinformatics analysis and luciferase reporter assay showed that circ_0000231 competitively binded to miR-140. Based on our data, miR-140 was downregulated in PTX-resistant OC tissues and cells. In addition miR-140 acts as a tumor suppressor to inhibit the proliferation, migration, invasion and drug resistance of PTX-resistant OC cells. Decreased expression of miR-140 promotes the proliferation, migration, invasion and PTX-resistance in OC cells. We further investigated whether circ_0000231 exerted resistant function in OC through regulating miR-140, circ_0000231 and miR-140 were cotransfected into SKOV3-TR30. MiR-140 inhibitor reversed the tumor suppressive effects induced by silencing of circ_0000231.

We predicted the target gene of miR-140 by TargetScan database. And RAP1B was proved to be a direct target of miR-140 by dual-luciferase reporter assay. Ras-related protein 1 (Rap1) is a universally expressed small GTPase that has two isoforms: Rap1A and Rap1B [20]. In recent years, an increasing number of studies have found that RAP1B acts in regulating the occurrence and development of tumors [21]. RAP1B, as an oncogene, has been reported that it was overexpression in OC cells [22]. Our findings are consistent to the above study. It has been shown that knockdown the RAP1B could inhibit invasion and migration in OC cells. Based on the result, we found that RAP1B was high expression in PTX-resistant OC tissues and cells. Overexpression of RAP1B could promoted the progression of PTX resistance. Furthermore, inhibition of circ_0000231-mediated weak of RAP1B protein expression was signifificantly recuperated following miR-140 inhibitors. Moreover, rescue experiments further confirmed that RAP1B overexpression abrogated the suppressive effect of circ_0000231 knockdown. We were the first to manifest the involvement of circ_0000231/miR-140/RAP1B regulatory axis in OC development and resistance. Our findings highlighted a promising target for OC therapy.

Admittedly, this study has limitations. The mechanism of ceRNA-regulated chemotherapeutic resistance is not fully understood. Meanwhile, the exact mechanism paclitaxel resistance cannot be elucidated by one single circular RNA. Furthermore, only a limited number of ovarian cancer lines were used in this study, which may not be entirely generalizable to other contexts. Nevertheless, this was just an initial exploration of circ_0000231 in OC, and further research of circ_0000231 remained to be explored in future.

To summarize, in this study, circ_0000231 was overexpressed in PTX-resistant OC cells and tissues. Circ_0000231 might act as sponge for anti-oncogenic miR-140 to upregulate RAP1B and promote the PTX resistance progression in OC. Meanwhile, circ_0000231 also activitied the EMT in PTX-resisitant cells. We were the first to manifest the involvement of circ_0000231/miR-140/RAP1B axis in PTX-resistant OC cells. Therefore, our findings were expected to become a significant new target for drug resistance treatment of OC.

Funding Natural Science Foundation of Liaoning Province (Grant NO: 201800832); The Outstanding Scientific Fund of Shengjing Hospital (Grant NO.201705).

Data availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Compliance with ethical standards

Conflict of interests The authors declare that they have no competing interests.

Ethical approval This research was approved by the Ethics Committee of Shengjing Hospital of China Medical University. All the procedures were performed in accordance with the 1964 Helsinki Declaration and its later amendments.

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Circ_0000231 Promotes Paclitaxel Resistance in Ovarian Cancer by Regulating miR-140/RAP1B

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