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Identification of circ_0058357 as a regulator in non‐small cell lung cancer cells resistant to cisplatin by miR‐361‐3p/ABCC1 axis

Abstract Background Drug resistance is a major clinical drawback behind the failure of chemotherapy in non‐small cell lung cancer (NSCLC). In this study, we undertook to identify the precise role of circular RNA (circRNA) circ_0058357 in the functional properties of DDP‐resistant NSCLC cells. Methods Circ_0058357, miR‐361‐3p and ATP‐binding cassette (ABC) subfamily C member 1 (ABCC1) were quantified by qRT‐PCR and western blot. Cell survival and viability were gauged by MTT assay. Cell proliferation, apoptosis, invasion and migration were measured by EdU, flow cytometry, transwell and wound‐healing assays, respectively. The direct relationship between miR‐361‐3p and circ_0058357 or ABCC1 was validated by dual‐luciferase reporter assay. Results Our data showed that circ_0058357 was highly expressed in DDP‐resistant NSCLC tissues and cells. Inhibition of circ_0058357 repressed cell growth, invasion, migration, and promoted DDP sensitivity and cell apoptosis of H1299/DDP and A549/DDP cells in vitro. Moreover, inhibition of circ_0058357 diminished the growth of A549/DDP cells and sensitized them to the cytotoxic effect of DDP in vivo. Mechanistically, circ_0058357 contained a miR‐361‐3p binding site and miR‐361‐3p was identified as a molecular mediator of circ_0058357 regulation. MiR‐361‐3p suppressed ABCC1 expression by binding to ABCC1 3′UTR, and miR‐361‐3p‐mediated inhibition of ABCC1 affected the growth, invasion, migration, apoptosis and DDP sensitivity of H1299/DDP and A549/DDP cells. Furthermore, circ_0058357 regulated ABCC1 expression by competitively binding to shared miR‐361‐3p. Conclusions Our findings identified that inhibition of circ_0058357 suppresses the growth and metastasis of H1299/DDP and A549/DDP cells and sensitizes them to DDP therapy partially by targeting the miR‐361‐3p/ABCC1 axis.

INTRODUCTION Non-small cell lung cancer (NSCLC), accounting for the majority of lung cancer, is one of the biggest causes of cancer mortality worldwide. 1 Cisplatin (DDP) is a clinically used chemotherapeutic drug and DDP-based therapy has been the standard-of-care for NSCLC patients, with a good performance status. 2,3 Unfortunately, DDP resistance frequently arises, giving rise to a major clinical drawback. Despite intensive efforts, mechanisms of resistance to DDP are still not fully understood. Thus, to develop novel therapeutic approaches to improve DDP efficacy in NSCLC, identifying the mechanisms of DDP resistance is required.
Circular RNAs (circRNAs), a new kind of noncoding RNAs, are covalently closed RNA transcripts with neither 5 0 caps nor 3 0 tails. 4 Numerous studies have unveiled the post-transcriptional regulation of circRNAs in gene expression by competitively binding to shared microRNAs (miRNAs), highlighting the importance of such interactions in cancer pathogenesis and drug resistance. [5][6][7] Moreover, abnormal expression of circRNAs in DDP-resistant NSCLC cells has been reported. 8 For instance, Lu et al. 9 uncovered that circ_0096157 could regulate the malignant behaviors of DDP-resistant A549 cells (A549/DDP). Pang et al. 10 ascertained that downregulation of circ_0031250 restrained the growth and invasion of A549/DDP and H460/DDP cells and sensitized them to DDP therapy depending on the modulation of the miR-4458/REV3L axis. Zhong et al. 11 demonstrated that circ_100565 was able to affect the functional properties, including DDP sensitivity, in DDP-resistant NSCLC cell lines by binding to miR-337-3p to induce ADAM metallopeptidase domain 28. Circ_0058357, formed by the head-to-tail splicing of exons of autophagy related 9A (ATG9A) transcript, has been identified as a potent tumor driver in NSCLC by upregulating AVL9 cell migration associated (AVL9) by competing for shared miR-24-3p. 12 In the preliminary survey, we found that circ_0058357 is differentially expressed in DDP-resistant NSCLC (A549/DDP) cells versus the sensitive parental cells ( Figure S1). Herein, we undertook to explore the precise action of circ_0058357 in the functional properties of DDP-resistant NSCLC cells.
Deregulated expression of miRNAs in DDP-resistant NSCLC cells has also been uncovered. 13 Recent studies have identified the antitumor role of miR-361-3p in NSCLC, [14][15][16] suggesting its tremendous therapeutic potential in NSCLC. Moreover, downregulation of miR-361-3p is reported to be associated with gefitinib resistance in NSCLC. 17 ATPbinding cassette (ABC) subfamily C member 1 (ABCC1, multidrug resistance-associated protein 1/MRP1) is a pivotal player in cancer-related multidrug resistance. 18 Furthermore, resistance of NSCLC to DDP involves ABCC1, wherein ABCC1 operates as a contributor to resistance development. [19][20][21] Here, we provide evidence that circ_0058357, an overexpressed circRNA in DDP-resistant NSCLC, affects the functional properties and DDP sensitivity of A549/DDP and DDP-resistant H1299 (H1299/DDP) NSCLC cells. We also identify circ_0058357 as a post-transcriptional regulator of ABCC1 expression through miR-361-3p, highlighting a novel circRNA/miRNA/mRNA network in DDP-resistant NSCLC cells.

Human samples and cell lines
We obtained all human NSCLC samples from the First Affiliated Hospital of Zhengzhou University with written informed consent provided by all subjects. Use of human samples was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. In total, we analyzed 21 primary tumors (defined as sensitive) collected from NSCLC primary patients before therapeutic treatment and 23 NSCLC tissues (defined as resistant) obtained from recurrent patients after treatment of DDP-based chemotherapy to evaluate the expression levels of circ_0058357, miR-361-3p and ABCC1.
RNA preparation, ribonuclease R (RNase R) treatment and quantitative real-time PCR (qRT-PCR) We prepared total RNA using the NucleoSpin RNA II from tissue samples and cultured cells as recommended by the manufacturers (Macherey-Nagel). We isolated the cytoplasmic and nuclear RNA using the cytoplasmic and nuclear RNA purification kit from H1299/DDP and A549/DDP cells based on the manufacturer's protocols (Norgen Biotek). RNase R treatment was performed by adding 5 U of RNase R (Geneseed) into 2 μg total RNA and 10 min incubation at 37 C.
For the quantification of circ_0058357 and mRNAs, reverse transcription (RT) PCR generated cDNA was done using the random hexamers with QuantiTect RT Kit (Qiagen); amplification of gene-specific products was achieved by qRT-PCR using SYBR Green (Qiagen) and designed primers (Table S1). For miRNA analysis, RT PCR was done using the stem-loop RT primers and miScript II RT Kit (Qiagen) and cDNA products were amplified and quantified with miRNA-specific primers (Table S1) and miScript SYBR Green Kit as per the manufacturer's recommendations. Using the comparative Ct method (2 ÀΔΔCt ), we determined the relative expression of target genes relative to the housekeeping gene β-actin or U6.
We plated H1299/DDP and A549/DDP cells in 12-well dishes at 2 Â 10 5 cells per well. The next day, the cells were transfected with 30 nM miRNA mimic, 30 nM miRNA inhibitor, or 100 nM siRNA and RNAiMAX or 1 μg of plasmid DNA and lipofectamine 3000 as per the manufacturer's recommendations (Invitrogen) for transfection. We harvested the transfected cells for cell viability, proliferation, migration, invasion assays after 12 h and for apoptosis and expression analyses after 72 h.

MTT assay for cell survival and viability
The transfected H1299/DDP and A549/DDP cells were seeded in 96-well dishes at 3000 cells per well and cultured at 37 C for 48 h. The cells were then challenged with (survival analysis) or without (viability analysis) various concentrations of DDP (Sigma-Aldrich) for 24 h. The medium was replaced by fresh growth media containing 0.5 mg/ml of MTT solution (Roche). Following a 4-h incubation at 37 C, DMSO was used to dissolve formazan crystal. The number of viable cells was proportional to the optical density measured by a PowerWave340 reader (BioTek Instruments). We estimated the IC 50 value of DDP from a plot of the percentage (50%) of viable cells versus DDP concentration.

Cell proliferation and apoptosis assays
We evaluated cell proliferation with the 5-ethynyl-2 0deoxyuridine (EdU) assay and cell apoptosis using flow cytometry. For proliferation analysis, the transfected H1299/ DDP and A549/DDP cells were seeded in 96-well dishes at 3000 cells per well and cultured at 37 C for 48 h. After that, cells were orderly incubated with 50 μM of EdU solution (Invitrogen) for 2 h, Apollo488 staining solution (Ribobio) and 4 0 ,6-diamidino-2-phenylindole (DAPI, Invitrogen) nuclear staining solution for 20 min. Fluorescence microscopy was performed in 10 random fields with a fluorescence microscope (Leica). The ratio of EdU-positive nuclei (green) to total nuclei (blue) was calculated as the proliferation rate of transfected cells. For apoptosis analysis, the transfected H1299/DDP and A549/DDP cells were stained using a Roche Annexin V-FITC/propidium iodide (PI) staining kit as recommended by the manufacturers (Roche). Stained cells were analyzed with a MUSE Cell Analyzer (Merck Millipore). We considered the cells that were positive for Annexin V as the apoptotic cells.

Measuring cell invasion by transwell assay
We plated the transfected H1299/DDP and A549/DDP cells at 2 Â 10 5 cells per well into 24-well transwell inserts precoated with Matrigel (8 μm pore size, Corning). We placed the inserts into the culture dishes containing the fresh growth media. Following a 24-h culture at 37 C (cell proliferation was not significantly inhibited by circ_0058357 silencing in this time point ( Figure S2), the invaded cells (5 randomly selected fields per sample) on the undersurface of the membrane were counted using 100Â bright-field microscopy with a microscope (Nikon).

Measuring cell migration by wound-healing assay
The transfected H1299/DDP and A549/DDP cells were seeded into six-well dishes and cultured at 37 C for 6 h to create a monolayer. Subsequently, a homogeneous wound track was made using the tip of a fine forceps. We acquired images at 0 and 24 h of at least five randomly selected microscopic fields and quantified cell migration by analyzing the area of wound repair with NIS Elements software (Nikon).

Western blot
We obtained total protein from tissue samples and cultured cells using the RIPA buffer (Merck Millipore) supplemented with protease inhibitors (Thermo Fisher Scientific) as per the accompanying recommendations. For immunoblotting, proteins were resolved on the Mini-Protean TGX gels (Bio-Rad) by electrophoresis, and the resulting gels were electrotransferred to nitrocellulose membranes (Bio-Rad). The primary antibodies (Abcam) employed were: rabbit monoclonal CyclinD1 (ab16663), rabbit monoclonal ABCC1 (ab260038), rabbit polyclonal matrix metalloproteinase 9 (MMP9, ab38898), rabbit polyclonal multidrug resistance 1 (MDR1, ab129453) and rabbit monoclonal GAPDH (ab181602). The anti-rabbit IgG labeled by HRP was used as the secondary antibody (ab205718, Abcam). Immunoreactivity was detected by ECL (Merck Millipore), and band densitometry was analyzed with the ChemiDoc MP Imager and Image Lab software as described by the manufacturers (Bio-Rad).

Bioinformatics
To search the miRNA-binding sites to circ_0058357, we used the StarBase and Circinterctome computational algorithms. We also used the StarBase target prediction software to predict the molecular targets of miR-361-3p.

Dual-luciferase reporter assay
The segment of circ_0058357 or ABCC1 3 0 UTR harboring the predicted miR-361-3p complementary sites, provided by Abiocenter, was cloned into Xho I and Pme I sites downstream of the SV40 promoter-driven renilla luciferase cassette in psiCHECK-2 vector (Promega). Mutations in the seed sequence were generated by TaKaRa MutanBEST Kit as described by the manufacturers (TaKaRa). MiR-361-3p mimic (30 nM) or negative control and the indicated reporter construct (200 ng) were cotransfected into H1299/DDP and A549/DDP cells using lipofectamine 3000. We tested the luciferase activity after 48 h using the dual-luciferase reporter system with a luminometer based on the manufacturer's guidance (Promega).

Generation of stable circ_0058357 depletion cell line
We obtained lentiviral particles coding circ_0058357 shRNA (sh-circ_0058357) or mock shRNA (sh-NC) from Geneseed. A stable circ_0058357 depletion cell line was achieved by transducing the virus particles into A549/DDP cells in media containing 8 μg/ml polybrene. Two days after infection, virus-positive cells were selected with puromycin (2 μg/ml) for 2 weeks.

Xenograft model studies
All animal experiments followed international guidelines and the study was approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. We used 6-8-week-old BALB/c female nude mice (Ai Ling Fei Biotechnology Co., Ltd.) for examining the effect of circ_0058357 on the tumorigenicity of A549/ DDP cells and DDP cytotoxic activity. For xenograft formation, we gave BALB/c nude mice a 200 μl volume of PBS containing 4 Â 10 6 transduced A549/DDP cells by subcutaneous injection into their right flanks. After eight days, we performed DDP (30 mg/kg, every 3 days) or PBS administration by intraperitoneal injection and measured tumor volumes (length Â width 2 /2) using a digital caliper every three days. Each group included six mice. On day 23, mice were sacrificed by CO 2 overdose according to institutional ethics guidelines. Postmortem examination included weight measurement, and the tumors were analyzed for circ_0058357, miR-361-3p, ABCC1 and MMP9 expression levels by qRT-PCR and western blot as above and immunohistochemistry assay with antibodies against ABCC1 (ab260038, Abcam) and MMP9 (MA5-15886, Invitrogen), as described elsewhere. 22

Statistical analysis
Data are presented as means of at least three independent experiments performed in quintuplicate, with the standard error of the mean. In general, we used a two-tailed Student's t-test (two groups) or analysis of variance with Dunnett's post hoc test (multiple groups) to compare significant differences. We employed the Pearson's correlation coefficients to analyze the expression correlation between miR-361-3p and circ_0058357 or ABCC1 in NSCLC tissues obtained from recurrent patients after treatment of DDP-based chemotherapy. Values of p < 0.05 were considered statistically significant.

Overexpression of circ_0058357 in DDP-resistant NSCLC
To observe the relevance of circ_0058357 in adaptive DDP resistance, we initially examined its expression pattern by qRT-PCR analysis and found a clear upregulation of circ_0058357 in a panel of DDP-resistant NSCLC tissues compared with the primary sensitive NSCLC tumors (Figure 1(a)). We also assessed circ_0058357 expression in H1299/DDP and A549/DDP NSCLC cells. H1299/DDP and A549/DDP cells showed increased expression of circ_0058357 compared to the parental cells (Figure 1(b)). Additionally, we performed RNase R treatment to validate the RNase R resistance of circ_0058357. Circ_0058357 was more tolerant of RNase R digestion than the linear GAPDH transcript (Figure 1(c),(d)), suggesting that circ_0058357 has a higher tolerance to exonucleases. Moreover, subcellular localization analysis revealed that circ_0058357 predominantly localized to the cytoplasm of H1299/DDP and A549/ DDP cells (Figure 1(e),(f)).

Inhibition of circ_0058357 impedes cell growth, invasion, migration, and promotes DDP sensitivity and cell apoptosis of H1299/DDP and A549/DDP cells in vitro
To elucidate the precise role of circ_0058357, we decreased circ_0058357 expression with siRNA-targeting circ_0058357 (si-circ_0058357) in H1299/DDP and A549/DDP cells, which expressed high levels of circ_0058357. The circ_0058357 inhibition efficacy of si-circ_0058357 was verified by qRT-PCR analysis (Figure 2(a)). H1299/DDP and A549/DDP cells exhibited higher IC 50 values of DDP compared with the sensitive parents ( Figure 2(b)). Intriguingly, circ_0058357 loss of function markedly lessened the IC 50 for DDP of H1299/DDP and A549/DDP cells (Figure 2(c)), indicating that inhibition of circ_0058357 sensitized H1299/ DDP and A549/DDP cells to DDP therapy. Moreover, downregulation of circ_0058357 potently hindered the viability (Figure 2(d)) and proliferation (Figure 2(e)) of H1299/DDP and A549/DDP cells. Conversely, downregulation of circ_0058357 strongly enhanced cell apoptosis of H1299/DDP and A549/DDP cells compared with the si-NC controls (Figure 2(f )). Furthermore, circ_0058357-silenced H1299/DDP and A549/DDP cells showed suppressed invasion (Figure 2(g)) and migration (Figure 2(h)) rates compared to the controls. Additionally, inhibition of circ_0058357 resulted in decreased levels of cycle-related cyclin D1, migration-related MMP9 and MDR1 in H1299/DDP and A549/DDP cells compared with the si-NC controls (Figure 2(i),(j)). Together, these findings establish that circ_0058357 affects cell functional properties and DDP sensitivity of H1299/DDP and A549/ DDP cells.
F I G U R E 2 Legend on next page.

Circ_0058357 operates as a regulator of ABCC1 expression through miR-361-3p
The above observations demonstrated that circ_0058357 and ABCC1 harbor a shared binding sequence for miR-361-3p. Hence, we next decided to investigate whether circ_0058357 could affect ABCC1 expression. Indeed, ABCC1 protein level was remarkably reduced in circ_0058357-silenced H1299/ DDP and A549/DDP cells; however, this effect was strongly abated by miR-361-3p downregulation (Figure 7(a),(b)). All these findings suggest that circ_0058357 affects ABCC1 expression by competitively binding to shared miR-361-3p.

Inhibition of circ_0058357 diminishes the growth of A549/DDP cells and sensitizes them to the cytotoxic effect of DDP in vivo
To determine whether circ_0058357 possesses regulatory effects in vivo, we transduced A549/DDP cells with sh-circ_0058357 or sh-NC and injected them subcutaneously into the right flanks of BALB/c nude mice before DDP or PBS administration. Strikingly, sh-circ_0058357 transduction or DDP administration repressed tumor growth (Figure 8(a),(b)). Interestingly, simultaneous sh-circ_0058357 transduction and DDP administration led to a clearer repression in tumor growth (Figure 8(a),(b)). Furthermore, sh-circ_0058357transduced tumors showed reduced levels of circ_0058357 and ABCC1 protein and increased expression of miR-361-3p with or without DDP administration (Figure 8(c)-(e)). Our immunohistochemistry analysis also showed a strong reduction in the level of metastasis-related MMP9 protein 23,24 in sh-circ_0058357-transduced tumors with or without DDP administration (Figure 8(e)), suggesting that circ_0058357 depletion may weaken the metastatic potential of the tumors.
All these data suggest that downregulation of circ_0058357 expression diminishes tumor growth and enhances DDP sensitivity in vivo.

DISCUSSION
Drug resistance has become a major reason behind the failure of chemotherapy in NSCLC. 25 However, little is known about DDP resistance mechanisms. Intense efforts have led to the identification of deregulated circRNA activity in the development of DDP resistance in NSCLC. 8,10,26 In this study, we have demonstrated the influence of circ_0058357 on the functional properties and DDP sensitivity of H1299/ DDP and A549/DDP cells. More importantly, we have established a new circRNA/miRNA/mRNA network implicated in the regulation of DDP-resistant NSCLC cells.
Our findings showed the overexpression of circ_0058357 in DDP-resistant NSCLC. Consistent with the recent work showing the tumor-inhibitory effect of circ_0058357 F I G U R E 7 Circ_0058357 affects ABCC1 expression through miR-361-3p. H1299/DDP (a) and A549/DDP (b) cells were transfected with si-circ_0058357 + anti-miR-361-3p, si-circ_0058357 + anti-miR-NC, si-circ_0058357 or si-NC and checked for ABCC1 protein level by western blot. **p < 0.01, ***p < 0.001, ****p < 0.0001 F I G U R E 8 Inhibition of circ_0058357 represses tumor growth and sensitizes A549/DDP cells to DDP in vivo. (a)-(e) A549/DDP cells infected with sh-circ_0058357 or sh-NC were injected (4 Â 10 6 cells each mouse) subcutaneously into BALB/c nude mice. After eight days, DDP (30 mg/kg, every 3 days) or PBS administration and tumor volume measurement began. Each group included six mice. Twenty-three days later, tumors were collected. Scale bar: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 deficiency on NSCLC, 12 we first demonstrated that inhibition of circ_0058357 impedes the growth and metastasis of H1299/DDP and A549/DDP cells and sensitizes them to the cytotoxic effects of DDP. As previously reported for other circRNAs, 27,28 circ_0058357 is RNase R resistant because of its covalently bonded structure. 4 Additionally, the cytoplasmic localization of circ_0058357 in H1299/DDP and A549/ DDP cells provides the possibility for the interactions between circ_0058357 and miRNAs because miRNAs silence gene expression in the cytoplasm in the RNAinduced silencing complex (RISC). 29 Numerous circRNAs form a crucial type of posttranscriptional regulators by inhibiting miRNA activity. 30 Here, we first ascertained that circ_0058357 harbors a miR-361-3p binding site, and the effects of circ_0058357 inhibition are partially due to the elevation of miR-361-3p. The dual role of miR-361-3p in human carcinogenesis as either tumor inhibitors or tumor drivers has been highlighted (for instance): miR-361-3p contributes to the development of pancreatic cancer 31 and breast cancer 32 ; miR-361-3p works as a strong anticancer factor in lymphoma 33 and ovarian cancer. 34 The contradictory findings may be attributed to the different types of tumors in these reports. In line with previous studies showing the anticancer activity of miR-361-3p in NSCLC, [14][15][16] we uncovered that miR-361-3p hinders the growth and metastasis of H1299/ DDP and A549/DDP cells and sensitizes them to DDP therapy. A recent study has also discovered that miR-361-3p can promote enzalutamide (Enz) sensitivity of Enz-resistant prostate cancer by targeting androgen receptor splicing variant 7 (ARv7). 35 ABCC1 participates in the development of drug resistance in cancers, such as hepatocellular, colon and ovarian cancers. [36][37][38] Moreover, ABCC1 exerts an important promoting effect on the development of DDP resistance in NSCLC. [19][20][21] In this study, we first showed that miR-361-3p inhibits ABCC1 expression through the perfect binding sequence in ABCC1 3 0 UTR, and inhibition of ABCC1 by miR-361-3p is partially responsible for the regulation of miR-361-3p in the growth, metastasis and DDP sensitivity of H1299/DDP and A549/DDP cells. Pei et al. 21 reported that miR-185-5p affected DDP sensitivity of NSCLC by targeting ABCC1. Furthermore, we pointed to the role of circ_0058357 as a posttranscriptional regulator of ABCC1 expression by competing for binding to miR-361-3p. ABC efflux transporters, including ABCC1, contribute to drug resistance in cancer cells by limiting the exposure to anticancer drugs. 39 Thus, developing the inhibitors of ABC efflux transporters is an effective approach to overcome drug resistance. With our findings, we envision that inhibition of circ_0058357 may be a promising point for the development of circRNA-based therapies against DDP resistance in NSCLC.
Taken together, we have demonstrated that inhibition of circ_0058357 suppresses the growth and metastasis of H1299/DDP and A549/DDP cells and sensitizes them to DDP therapy. We have identified a novel circ_0058357/ miR-361-3p/ABCC1 network which regulates the DDP resistance of NSCLC.