Circular RNA hsa circ 0072309 inhibits non-small cell lung cancer progression by sponging miR-580-3p

Wenguang Pang1,2, Fengliu Huang3, Xin Zhang4, Min Ye2, Yanming Huang3, Xiufang Huang5, Jingzhuo Pang2, Chengjie Cai2 and Zheng Wang6 1Department of Thoracic Surgery, The First Affiliated Hospital of Jinan University, Guangzhou 510630, China ; 2Department of Thoracic Surgery, Jiangmen Central Hospital, Jiangmen 529030, China ; 3Department of Respiration Medicine, Jiangmen Central Hospital, Jiangmen 529030, China ; 4Clinical Experimental Center (Clinical Biobanks), Jiangmen Central Hospital, Jiangmen 529030, China ; 5Department of Pathology, Jiangmen Central Hospital, Jiangmen 529030, China ; 6Department of Thoracic Surgery, The 2nd Clinical Medical College of Jinan University, Shenzhen 518020, China


Introduction
Lung cancer is one of the most commonly found carcinoma type and is an aggressive tumor with high incidence and mortality rate worldwide [1].Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer diagnoses and is the major cause of lung cancer-associated mortality due to late-stage detection [2].Despite advances in clinical and experimental oncology have been made in recent years, the 5-year overall survival rate of NSCLC is still limited [3].Thus, to explore the molecular mechanisms of NSCLC and to identify effective biomarkers is essential for NSCLC early diagnosis and treatments.
Circular RNAs (circRNAs) are a new class of noncoding RNA molecules with the length of at least a few hundred nucleotides lacking 5 caps or 3 poly-A tails [4].CircRNAs are reported to closely associate with the progression of a large number of cancers [5].Aberrant regulation of circRNAs has been noted in gliomas [6], renal cell carcinomas [7] and other human malignancies [8].With the development of high-throughput sequencing and bioinformatics analysis, increasing numbers of novel circRNAs have been identified to be potential cancer prognostic biomarkers [9].CircRNA has circ 0072309 was identified to be located on chromosome 5 between the base sites 38523520 and 38530768 [10].Some reports showed that has circ 0072309 was lowly expressed in breast cancer [11,12], human intracranial aneurysms [10] and renal carcinoma [13].However, the function of has circ 0072309 in the progression of NSCLC remains elusive.
In the present study, we screened the circ 0072309 from GSE112214 dataset.Quantitative real-time PCR (qRT-PCR) results showed that circ 0072309 was down-regulated in NSCLC tissues and cell lines.The overexpression of circ 0072309 significantly blocked the cell proliferation, migration and invasion in A549 and H1299 cells.Luciferase reporter and RNA pull down assays indicated that circ 0072309 directly bound to miR-580-3p.The blockage of cell proliferation, migration and invasion induced by circ 0072309 was mitigated by miR-580-3p mimic.Taken together, our study indicated that circ 0072309 blocks NSCLC tumor progression by sponging miR-580-3p.These findings provided a novel biomarker and potential treatment strategy for NSCLC diagnosis and clinical treatment.

Clinical samples
The clinical specimens including 30 pairs of NSCLC tissues and adjacent normal tissues were isolated from patients who underwent surgery between 2016 and 2018 at the Department of Thoracic Surgery, The 2nd Clinical Medical College of Jinan University.Patients did not receive any preoperative therapy, including radiotherapy and chemotherapy before surgery.The pathological diagnosis of tissue samples were performed by two pathologists according to WHO grade criteria.All tissues were isolated and immediately transferred into liquid nitrogen for follow-up experiments.All patients have signed the informed consents prior to their participation in the study.The study was reviewed and approved by the Ethics Committee of The 2nd Clinical Medical College of Jinan University.

Cell viability assay
Cell viability was determined by using cell counting kit 8 (CCK-8, Donjindo, Kumamoto, Japan).Cells were seeded into a 96-well plate at the density of 2 × 10 3 cells/well.After 0, 24, 48, 72 and 96 h, cells were incubated with 10 μl of CCK-8 reagent and incubated at 37 • C for 2 h.Finally, the optical density was measured using EnSpire™ 2300 Multilabel Reader (PerkinElmer Inc., Waltham, MA, U.S.A.) at the wavelength of 450 nm.The experiment was repeated three times independently.

Transwell assay
Matrigel-coated (BD Biosciences, Franklin Lakes, NJ, U.S.A.) Transwell inserts (Costar, Manassas, VA, U.S.A.) were used to measure the invasive capacity of cells.The inserts were coated with 50 μl of 1 mg/ml Matrigel according to the manufacturer's protocol.A total of 2 × 10 5 cells in 200 μl of Dulbecco's modified Eagle's medium (DMEM) without serum were plated to the upper chamber.DMEM with 10% FBS was added to the lower compartment as a chemoattractant.After 24-h incubation, cells invading the lower surface of the membrane were fixed with cold methanol-glacial acetic acid and stained with 1% Crystal Violet for 15 min at room temperature.For each membrane, five fields (up, down, center, left and right, ×200) were selected for cell counting and photographed under the microscope (Canon, Tokyo, Japan).Transwell assays were performed with two technical replicates and three independent biological replicates.

Biotinylated-RNA pull down
Biotinylated miR-580-3p probe was synthesized by GenePharma (Shanghai, China).Cells were seeded into six-well plates and transfected with bio-miR-580-3p or bio-miR NC at a final concentration of 20 nM for 24 h.The probe was incubated with M280 streptavidin dynabeads (Life Technologies Inc., Rockville, Maryland, U.S.A.) at 25 • C for 2 h to generate probe-coated beads.The biotin-conjugated RNA complex was pulled down after incubation of the cell lysates with streptavidin-coated magnetic beads and extracted with TRIzol reagent.The level of circ 0072309 in bound fractions was evaluated by qRT-PCR analysis.

Luciferase report assay
The luciferase reporter plasmid (Luc) was purchased from OBiO Technology (Shanghai, China).Sequences containing potential wild-type or mutant binding sites for miRNAs in circ 0072309 were cloned downstream of the luciferase gene promoter (Luc-circ 0072309 or Luc-circ 0072309-mutant).Cells were seeded into 24-well plates at the concentration of 5 × 10 4 cells/well and co-transfected with Luc-circ 0072309 or Luc-circ 0072309-mutant, with miR-580-3p mimic or miRNA NC.After 48 h, cells were extracted and the Dual Luciferase Reporter Assay was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, U.S.A.) by normalizing the firefly luminescence to Renilla luminescence.The experiment was repeated three times independently.

Statistical analysis
Data were presented as the mean + − standard deviation.Statistical analysis was carried out with IBM SPSS version 18.0 (SPSS, Inc., Chicago, IL, U.S.A.).Difference between two groups was analyzed using Student's t test and the analysis of multiple groups was carried out by using one-way analysis of variance followed by Dunnett's post-hoc analysis.*P<0.05 was considered to indicate a statistically significant difference.All experiments in the present study were repeated three times at least.

Circ 0072309 is down-regulated in NSCLC tissues and cell lines
We analyzed the microarray data (GSE112214 containing three pairs of NSCLC tissues and matched non-tumor tissues) from the GEO database (http://www.ncbi.nlm.nih.gov/gds/).The criteria for selection of differentially expressed circRNAs were |log2FC (fold change)| ≥ 1 and P<0.05.The top 15 up-and 15 down-expressed circRNAs were displayed in Figure 1A by using heatmaps.We found that the expression of circ 0072309 in NSCLC tissues was the lowest.In addition, in GSE112214 dataset, the expression of circ 0072309 in three NSCLC tissues was lower than that in matched non-tumor tissues (Figure 1B).We further collected 30 patients and confirmed the expression of circ 0072309 in NSCLC tissues and non-tumor tissues in Figure 1C.Similar results observed that circ 0072309 was down-regulated in patient-derived NSCLC tumor specimens versus adjacent healthy lung tissues.We extended our analysis from NSCLC tumor tissues to NSCLC cell lines.Compared with HBE, the expression of circ 0072309 in NSCLC cell lines (CALU3, H125, A549 and H1299) was significantly blocked (Figure 1D).Among the four NSCLC cell lines, circ 0072309 expression in A549 and H1299 cells was lower than that in CALU3 and H125 cells.We selected A549 and H1299 cells for subsequent cir 0072309 overexpression experiments.Taken together, these data indicated that circ 0072309 is poorly expressed in NSCLC tissues and cell lines.

circ 0072309 inhibits cell proliferation, migration and invasion in A549 and H1299 cells
To explore the role of circ 0072309 in NSCLC carcinogenesis, we transfected A549 and H1299 cells with pcDNA3.1-circ0072309 and empty pcDNA3.1 (Vector), and then performed CCK-8, wound-healing and Transwell assays.qRT-PCR was employed to confirm the transfection efficiency.In Figure 2A, compared with Vector group, the transfection of pcDNA3.1-circ0072309 significantly enhanced the expression of circ 0072309, suggesting that pcDNA3.1-circ0072309 was successfully overexpressed in A549 and H1299 cells.In Figure 2B, CCK-8 assay results showed that circ 0072309 overexpression significantly inhibited A549 and H1299 cell viability compared with that in Vector-transfected cells.These data showed that circ 0072309 inhibited cell proliferation in NSCLC cells.
We further examined the effect of circ 0072309 on migration and invasion capabilities through wound healing and Transwell assays.In Figure 2C, compared with the Vector group, cells transfected with pcDNA3.1-circ0072309 showed a wider wound area at 24 h after cell propagation.Similarly, the overexpression of circ 0072309 significantly decreased the number of invasive cells compared with Vector group in Figure 2D.These results showed that circ 0072309 prevented cell migration and invasion in NSCLC cells.Taken together, the overexpression of circ 0072309 significantly blocks cell proliferation, migration and invasion in A549 and H1299 cells.

circ 0072309 serves as a sponge for miR-580-3p
To determine the mechanism of circ 0072309 functioning in NSCLC, we first analyzed the subcellular location of circ 0072309 by qRT-PCR and found that circ 0072309 was mainly located in the cytoplasm shown in Figure 3A.Many circRNAs may act as competitive endogenous RNAs and modulators of miRNA activity by competing for miRNA-binding sites [14].Based on the prediction provided by publicly available bioinformatics algorithms Circinteractome tool (https://circinteractome.nia.nih.gov),we fortunately found a putative miR-580-3p binding site on the circ 0072309 sequence.To explore the relationship between circ 0072309 and miR-580-3p, we first constructed a wild-type (circ 0072309 wt) or a mutant (circ 0072309 mut) luciferase reporter plasmid (Figure 3B).Cells co-transfected with circ 0072309 wt and miR-580-3p mimic significantly decreased the luciferase signal in comparison with cells transfected with circ 0072309 wt and miR NC (Figure 3C).However, no significant difference was detected in the luciferase signal intensity of circ 0072309 mut between the miR-580-3p mimic and miR NC (P>0.05).These findings indicated that circ 0072309 bound to miR-580-3p.We further performed an RNA pull down using a biotin-labeled miR-580-3p probe.In Figure 3D, circ 0072309 was enriched within the miR-580-3p probe pull-down RNA complex in comparison with miR NC.These data suggested that circ 0072309 directly interacted with miR-580-3p.In A549 and H1299 cells, the overexpression of circ 0072309 significantly blocked the expression of miR-580-3p (Figure 3E), indicating that the negative relationship between circ 0072309 and miR-580-3p.Taken together, these data indicated that circ 0072309 serves as a sponge for miR-580-3p in A549 and H1299 cells.

circ 0072309 blocks cell proliferation, migration and invasion through miR-580-3p in NSCLC cells
After confirming the interaction between circ 0072309 and miR-580-3p, we further examined whether the anti-tumor function of circ 0072309 is attributed to its repression of miR-580-3p.We downloaded the data from The Cancer Genome Atlas (TCGA, https://cancergenome.nih.gov/).A total of 565 samples were included in the present study, containing 519 NSCLC samples and 46 matched non-tumor tissues.The expression of miR-580-3p in NSCLC tissues were significantly up-regulated than those in non-tumor tissues (Figure 3F).The similar trend was found in the patient-derived NSCLC tumor specimens versus adjacent healthy lung tissue (Figure 3G).These data suggested that miR-580-3p might exert as an oncogene in NSCLC progression.We transfected miR-580-3p mimic in circ 0072309-overexpressing cells and performed functional assays in Figure 4A.CCK-8 assay results showed that the overexpression of circ 0072309 significantly blocked cell viability, however, the addition of miR-580-3p mimic rescued the blockage of cell viability (Figure 4B).Similar results were observed in wound healing and Transwell assays.The administration of miR-580-3p mimic mitigated the blockage of the migration and invasion induced by circ 0072309 (Figure 4C,D).These data showed that the transfection of miR-580-3p mimic into circ 0072309 overexpressing cells rescued proliferation, migration and invasion defects, suggesting that circ 0072309 inhibited NSCLC progression through blocking the function of miR-580-3p.

Discussion
Lung cancer is an aggressive tumor with high incidence and mortality rate [15].NSCLC is responsible for poor survival of a majority of lung cancer patients [16].Due to non-specific symptoms and no effectual diagnostic tests, approximately 70% of NSCLC are diagnosed at an advanced stage [17].Non-invasive prognostic biomarkers are needed for the diagnosis of the advanced NSCLC patients with different histological types [18].With the advance in the study of the molecular basis about lung cancer progression, targeted therapies have been used for NSCLC treatment [19].CircRNAs are a group of non-coding RNAs with a stable closed-loop structure which prevents them from decomposing by the enzymes [20].Increasing evidence indicated that circRNAs are closely associated with the progression of NSCLC [21].For example, inhibition of circRNA VANGL1 suppresses the progression of bladder cancer [22].Circ 000984 promotes cell proliferation and metastasis in NSCLC by modulating Wnt/β-catenin pathway [23].CircRNA circFOXO3 promotes the prostate cancer progression [24].The down-regulation of circRNA ciRS-7 [25] or circ 0067934 [26] blocks cell proliferation, migration and invasion of NSCLC cells.In our study, we found circ 0072309 was down-regulated in NSCLC tissues and cells, and the overexpression of circ 0072309 significantly blocked cell proliferation, migration and invasion, suggesting that circ 0072309 serves as a tumor suppressor in NSCLC.These data provided new evidence about the role of circRNA during the progression of cancers and provided a new biomarker for NSCLC diagnosis and therapy.

Figure 1 .
Figure 1.circ 0072309 is poorly expressed in NSCLC tissues and cell lines (A) The heatmaps displayed the top 15 up-and down-expressed circRNAs between NSCLC and adjacent non-tumor tissues in GSE112214 based on the criteria of |log2FC| > 1 and P<0.05.Red in the two plots denoted up-regulation and blue stood for down-regulation.(B) circ 0072309 was lowly expressed in three NSCLC tissues in comparison with adjacent non-tumor tissues in GSE112214 dataset.(C) circ 0072309 was down-regulated in patient-derived NSCLC tumor specimens versus adjacent healthy lung tissues (n=30).(D) The expression of circ 0072309 was blocked in NSCLC cell lines.Total RNA was extracted from tissues or cells and qRT-PCR was performed to detect the expression of targets.**P<0.01.

Figure 2 .
Figure 2. The overexpression of circ 0072309 significantly blocks cell proliferation, migration and invasion (A) The transfection of pcDNA3.1-circ0072309 up-regulated the expression of circ 0072309 determined by qRT-PCR in A549 and H1299 cells.(B-D) The overexpression of circ 0072309 blocked the cell viability, migration and invasion determined by CCK-8, wound healing and Transwell assays in A549 and H1299 cells.Cells were transfected with pcDNA3.1-circ0072309 (circ 0072309) or pcDNA3.1 (vector).Total RNA was extracted for qRT-PCR and cells were collected for CCK-8, wound healing and Transwell assays.**P<0.01.