Circular RNA hsa_circ_0008003 facilitates tumorigenesis and development of non‐small cell lung carcinoma via modulating miR‐488/ZNF281 axis

Abstract As one of the most aggressive malignancies, non‐small cell lung carcinoma (NSCLC) has high risks of death. It has been demonstrated that circRNAs accelerate NSCLC progression, but the underlying molecular mechanisms of circRNAs in NSCLC were still obscure. In the first place, the circRNA microarray of NSCLC was investigated in this study, and hsa_circ_0008003 (circ‐0008003) was chosen as the research object. Then, it was unveiled that the expression of circ‐0008003 examined via qRT‐PCR was elevated in tumour tissues relative to the non‐tumour tissues, which was associated with TNM stage and lymphatic metastasis in NSCLC. Additionally, the prognosis of NSCLC patients with high circ‐0008003 level was poor. Besides, circ‐0008003 silencing dampened the invasion and proliferation of NSCLC cells. Next, according to the mechanistic studies, circ‐0008003 functioned as a ceRNA of ZNF281 in NSCLC by acting as the endogenous sponge for miR‐488, which was proved to be a tumour suppressor in NSCLC. Additionally, ZNF281 overexpression and miR‐488 suppression recovered the influences of repressed circ‐0008003 on NSCLC cellular processes. It was validated in this research that circ‐0008003 triggered tumour formation in NSCLC, which was adjusted via miR‐488/ZNF281 axis, casting a novel light on the resultful target for treating NSCLC and predicting the prognosis.

of non-coding RNA (ncRNA) implicated in different biological processes. 2,3 LncRNAs and miRNAs are verified in previous research to function in the evolution of lung cancer, especially in adjustment of apoptosis, proliferation and invasion. [4][5][6][7] Herein, ncRNAs need to be researched to continuously confirm the mechanism associated with the cancer from the aspect of epigenetics.
As a newfound type of RNAs, circular RNAs (circRNAs) display a wide expression in eukaryotes. As bioinformatics and high-flux sequencing have boomed in recent years, our sight on circRNAs has been notably broadened. Most circRNAs reported recently are non-coding, but some of them encode proteins or polypeptides according to reports. 8,9 Through non-canonical splicing, circRNAs generate with relative resistance to degradation by exonucleases. 10,11 Inextricable associations are found between circRNAs and the development of multiple disorders, especially that of cancer. 12,13 As circRNAs have gradually been extensively analysed, their functions as tumour oncogenes or suppressors are identified through adjusting the apoptosis, migration, proliferation, invasion and metastasis of tumour cells. [14][15][16][17] For instance, circRNA hsa_circ_0001649 inhibits HCC progression via multiple miRNA sponge 18 ; circRNA 0001785 functions as a competing endogenous RNA (ceRNA) to adjust the mechanism of osteosarcoma by sponging miR-1200 so as to elevate HOXB2 19 ; hsa_circ_0091570 acts as a ceRNA to impede the evolution of HCC by sponging hsa-miR-1307. 20 However, the effect of cir-cRNAs in NSCLC needs continuous explorations.
In the current research, the expression of circ-0008003 in NSCLC was discussed, and the function and potential regulatory mechanism of circ-0008003 were then investigated. The findings unfolded that circ-0008003 served as a ceRNA to mediate ZNF281 by sponging miR-488 in NSCLC.

| Cell lines and transfection
The Cell Bank of Chinese Academy of Sciences provided five human NSCLC cell lines (CAL-12T, HCC827, A549, H460 and H1299) and one human bronchial epithelioid cell line (16HBE).
They were cultured in RPMI 1640 medium (HyClone) with 1% penicillin/streptomycin (Gibco) and 10% foetal bovine serum (FBS; Pan Life Sciences) and maintained under a humid circumstance with 5% CO 2 at 37°C. Subsequently, 1 × 10 6 recombinant lentivirus-transducing units and 6 μg/mL Polybrene (Sigma) were utilized to infect cells in the case of the fusion of 60%-80%. Treatment with 2 μg/mL puromycin was adopted for the selection of cells with 2 weeks of stable transfection, and these cells were chosen through flow cytometry for later experiments. GenePharma provided lentiviruses and plasmids applied in this research.

| Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
In line with the regimen of the manufacturer, the total RNA extraction from tissues and cells was performed using TRIzol reagent (Invitrogen), followed by qRT-PCR mentioned above. 21

| Cell proliferation assay
Based on the regimen of the manufacturer, cell proliferation was examined via Cell Counting Kit-8 (CCK8) (Beyotime) assay.

| Cell invasion assay
By reference to the regimen of the manufacturer, the invasion capacity of cells was assessed by Transwell assay. Transwell chambers (Millipore Corporation) pre-coated with matrigel were then used.
During invasion assessment, the upper chamber was inoculated with 2 × 10 5 cells in medium with no serum, whereas the lower chamber was added with 600 μL of medium with 10% FBS as a chemoattractant. Following 24 hours of incubation, cells subjected to migration were fixed and dyed with a 5% crystal violet solution (Beyotime), and the images of cells undergoing migration were captured by an optical microscope (Olympus Corporation). Moreover, Image-pro Plus 6.0 (Media Cybernetics) was utilized to count the migrated cells.

| Luciferase reporter assay
Wild-type plasmids circ-0008003-WT and ZNF281-WT containing binding sites for miR-488 and mutant plasmids circ-0008003-MUT and ZNF281-MUT were consolidated into the pGL3 promoter vector (GenePharma). After the seeding of NSCLC cells into a 24well plate, the cells underwent cotransfection with 80 ng plasmids + 5 ng pRL-SV40 and 50 nmol/L miR-488 mimics or negative control (NC) with the help of Lipofectamine 2000 reagent. After that, the luciferase activity was assessed using the dual-luciferase reporter assay kit (Promega). All experiments were carried out thrice, respectively.

| RNA immunoprecipitation (RIP) assay
By reference to the guidance of the manufacturer, RIP assay was carried out with the use of the Magna RIP kit (Millipore).
Specifically, lysis buffer (Gibco) containing RNase inhibitor was used to lyse H460 and H1299 cells. After that, protein A/G agarose beads with AGO2 antibody were added to incubate cell lysates, with normal rabbit IgG as NC. Then, the RNAs that were co-precipitated underwent washing, purification and examination using qRT-PCR.

| RNA-Fluorescence In Situ Hybridization (RNA-FISH)
RNA-FISH was conducted using circ-0008003 probes conjugated with fluorescence by reference to the former research. 22 Then, DNA probe sets (Biosearch Technologies) were employed for hybridization based on the regimen of Biosearch Technologies. Ultimately, a FV1000 confocal laser microscope (Olympus) was utilized for probing cells.

| In vivo assays
Shanghai SLAC Laboratory Animal Co. Ltd. provided female nude mice aged 4 weeks, which were randomly allocated into two groups: 1 ×

| Immunohistochemistry staining
Ki-67 antibody and PCNA antibody (Abcam) were used for IHC staining by reference to the regimen mentioned above. 23

| Abnormal circ-0008003 expression was observed in NSCLC tissues and cells
GSE11 2214 microarray analysis demonstrated that circ-0008003 displayed a high expression in NSCLC tissues. On the basis of the criteria of P < .05 and fold change >4, the different expression of circRNAs including circ-0008003 was confirmed in NSCLC tissues ( Figure 1A). To verify the role of circ-0008003 in NSCLC, qRT-PCR was adopted to test its expression in NSCLC tissues and matched non-tumour tissues. According to the findings, NSCLC tissues had remarkably raised circ-0008003 expression relative to non-tumour tissues ( Figure 1B). And circ-0008003 level in TNM stage Ⅰ-Ⅱ was evidently lower than that in TNM stage Ⅲ-Ⅳ ( Figure 1C). Besides, tissues undergoing lymphatic metastasis had higher circ-0008003 expression than those undergoing no lymphatic metastasis ( Figure 1D). Additionally, circ-0008003 relative expression in NSCLC cells was evidently raised relative to human bronchial epithelioid cell line 16HBE ( Figure 1E). Because of the highest or lowest circ-0008003 expressions in H460 and H1299 cells, they were picked for later assays. 24,25 Circ-0008003 has resistance to RNase R digestion, so it was considered to possess circRNA features ( Figure 1F). These results suggest the elevated aberrant expression of circ-0008003 and its role in NSCLC as an oncogene.

F I G U R E 1
Circ-0008003 is raised in NSCLC tissues and cells. GSE11 2214 (platform GPL19978) microarray analysis is conducted for three NSCLC samples and three matched adjacent normal samples. On the basis of the criteria of P < .05 and fold change >4, the differential expression of circRNAs including circ-0008003 is confirmed in NSCLC tissues. The expression levels of circ-0008003 in human NSCLC tissues and cell lines are detected by qRT-PCR. A, Heat map shows differentially expressed circRNAs with statistically significance between NSCLC samples and three matched adjacent normal samples. Circ-0008003 expression is elevated in NSCLC samples. B, Up-regulation of circ-0008003 in NSCLC tissues (n = 30) compared with matched para-carcinoma tissues (n = 30). C, Expression of circ-0008003 in high TNM stage (n = 15) is higher than in low TNM stage (n = 15). D, The level of circ-0008003 is raised in tissues undergoing lymphatic metastasis (n = 17) compared with those undergoing no lymphatic metastasis (n = 13). E, Circ-0008003 expression in NSCLC cell lines (CAL-12T, H460, HCC827, H1299 and A549) and the human bronchial epithelioid cell line 16HBE. F, Resistance of circ-0008003 in NSCLC cells to RNase R digestion. Data are the mean ± SEM. *P < .05, **P < .01, ***P < .001

| The promotive effect of circ-0008003 on NSCLC cellular processes
Because of the confirmed increase in circ-0008003 expression in NSCLC, its influences in NSCLC cell biological processes were tested by silencing or raising circ-0008003 expressions in H460 and H1299 cells. QRT-PCR findings illustrated that circ-0008003 was remarkably pulled down or raised in NSCLC cells after the addition of circ-0008003 shRNA or overexpressing vector (Figure 2A).
According to CCK8 and 5-Ethynyl-2'-deoxyuridine (EDU) assays, the proliferation ability of NSCLC cells was decreased after silencing circ-0008003 and increased after up-regulating circ-0008003 ( Figure 2B-C). Besides, the invasion abilities of the two cell lines became weak after silencing circ-0008003 and strong after upregulating circ-0008003 unfolded by Transwell assay ( Figure 2D).
These data reveal that circ-0008003 promotes NSCLC cell invasion and proliferation in vitro.

| Circ-0008003 was considered to be an endogenous sponge for miR-488 in NSCLC
CircRNAs expressed in the cytoplasm have been demonstrated to substantially affect pathological and physiological processes by sponging miRNAs to some extent. 28,29 To ascertain the cellular localization of circ-0008003, cells were isolated into the nucleus and cytoplasm fractions, in which the controls, U6 and GAPDH, could be seen. QRT-PCR validated that circ-0008003 was monitored mainly in the cytoplasm fractions in NSCLC cells ( Figure S1A).
Identically, a substantial staining of circ-0008003 in the cytoplasm of H1299 cells was also determined in RNA-FISH ( Figure S1B).  Figure S2A illustrated that the expression of miR-488 was greatly inhibited or enhanced in NSCLC cells treated with miR-488 inhibitor or mimics relative to that in cells treated with NC. Besides, miR-488 impeded cell invasion and proliferation ( Figure S2B-D). Collectively, the results indicate that miR-488 influences NSCLC evolution as a tumour suppressor.

| Circ-0008003 accelerated NSCLC development by repressing miR-488
To ascertain that the role of circ-0008003 required miR-488 reduction in a direct manner, miR-488 inhibitor was transfected into circ-0008003-repressed H460 and H1299 cells, and the observably reduced miR-488 was discovered by qRT-PCR ( Figure 5A). A foregone conclusion was obtained from CCK8 and EDU assays that the proliferation capacities of circ-0008003-repressed H460 and H1299 cells F I G U R E 4 MiR-488 is adjusted by circ-0008003 in NSCLC in a direct and negative manner. A, The forecasting binding sites between miR-488 and circ-0008003 are obtained from Circinteractome. B, The relative luciferase activities of NSCLC cells treated with WT or MUT circ-0008003 reporter plasmids and miR-488 mimics or NC mimics. C, RNA reduction experiment continuously verifies the interaction between miR-488 and circ-0008003 in NSCLC cells. D, QRT-PCR analysis of miR-488 expression levels in H460 and H1299 cells following the inhibition of circ-0008003. E, Measurement of miR-488 expressions in NSCLC cell lines (CAL-12T, H460, HCC827, H1299 and A549) and the human bronchial epithelioid cell line 16HBE via qRT-PCR. F, Measurement of miR-488 expressions in cancerous and para-carcinoma tissues via qRT-PCR. G, Association between circ-0008003 expression and miR-488 in NSCLC tissues is estimated by Pearson's correlation analysis. Data are the mean ± SEM. *P < .05, **P < .01, ***P < .001 were promoted by miR-488 suppression ( Figure 5B,C). Lastly, silencing miR-488 was proved by Transwell assay to evidently boost the invasion capacities of circ-0008003-repressed H460 and H1299 cells ( Figure 5D). The above discovery implies that miR-488 participates in the carcinogenic process regulated by circ-0008003 in NSCLC cells.

| ZNF281 was a direct target of miR-488
Next, the specific potential mechanism of circ-0008003/miR-488 axis in stimulating NSCLC evolution was discussed. With bioinformatics prediction tools (TargetScan, miRDB and Starbase), ZNF281, a carcinogene in cancers, was found to be a target of miR-488 ( Figure 6A). Besides, the direct binding of miR-488 to ZNF281 3'UTR at the forecasting sites was ascertained in NSCLC cells ( Figure 6B). From immunoprecipitate conjugated with Ago2 in H460 and H1299 cells, miR-488 and ZNF281 mRNA were obtained, validating the existence of the interaction between miR-488 and ZNF281 mRNA in RNA-induced silencing complex (RISC) adjusted by miR-488 ( Figure 6C). Next, the expression of ZNF281 was disclosed to notably rise in NSCLC cells and tissues relative to controls ( Figure 6D-E). What's interesting was that miR-488 level was shown as an inverse correlator while circ-0008003 expression as a positive correlator of ZNF281 expression in NSCLC tissues ( Figure 6F,G). As a result, ZNF281 may affect circ-0008003/miR-488 axis in NSCLC evolution.

| ZNF281 positively adjusted circ-0008003 in NSCLC evolution
To figure out the adjustment of ZNF281 on the role of circ-0008003 in NSCLC evolution, the function of ZNF281 overexpression in the biological processes of circ-0008003-repressed H460 cells was F I G U R E 5 Effects of circ-0008003 inhibitor in NSCLC cells are reversed by silencing miR-488 to some extent. A, Findings of miR-488 relative expression in circ-0008003-repressed H460 and H1299 cells treated with miR-488 inhibitor or NC inhibitor through qRT-PCR. B-C, Cell proliferation and (D) invasion capacities of circ-0008003-inhibited H460 and H1299 cells with or with no suppression of miR-488 are analysed (scale bar = 100 μm). Data are the mean ± SEM. *P < .05, **P < .01, ***P < .001 analysed. In the first place, it was revealed that mRNA and protein expression levels of ZNF281 were remarkably impeded after the repression of circ-0008003 and became normal after cotransfection with ZNF281 overexpression vector ( Figure 7A,B). Afterwards, circ-0008003-repressed H460 cells predominantly regained their repressed proliferation abilities following cotransfection with ZNF281 overexpression vector ( Figure 7C,D). Additionally, ZNF281 overexpression alleviated the hampering of circ-0008003-repressed invasion processes in H460 cells ( Figure 7E). Basically, circ-0008003 facilitates NSCLC evolution via circ-0008003/miR-488/ZNF281 axis.

| D ISCUSS I ON
CircRNAs have been demonstrated to substantially influence different biological processes in cells. 30,31 According to reports, aberrant adjustment of circRNAs exerts effects on cancer evolution and is associated with patient's prognosis. 32,33 CircRNAs have been recently proved to pivotally affect NSCLC. 34  Verified by multiple reports, circRNAs adjust different cancers through targeting mRNAs or miRNAs. 37,38 CircRNA hsa_circ_0091570 F I G U R E 6 ZNF281 is the direct target of miR-488. A, The binding sites between miR-488 and ZNF281 3'UTR are forecasted by bioinformatics prediction. B, Luciferase reporter assay shows the specific forecasting binding sites between miR-488 and ZNF281 in NSCLC cells. C, Interaction between miR-488 and ZNF281 mRNA in H460 and H1299 cells is confirmed by RIP assay. D, Measurement of ZNF281 expressions in NSCLC cell lines (CAL-12T, H460, HCC827, H1299 and A549) and the human bronchial epithelioid cell line 16HBE via qRT-PCR. E, ZNF281 expressions in 30 NSCLC tissues and matched para-carcinoma tissues are measured using qRT-PCR. F-G, Pearson's correlation analysis is employed to figure out the correlation between ZNF281 expression and the level of miR-488 or circ-0008003 in 30 NSCLC tissues. Data are the mean ± SEM. *P < .05, **P < .01, ***P < .001 blocks HCC progression by sponging hsa-miR-1307 like a ceRNA. 20 CircRNA hsa_circRNA_100290 adjusts cell growth in oral squamous cell carcinoma by GLUT1 and glycolysis like a ceRNA for miR-378a. 39 CircRNA CircCACTIN accelerates the evolution of gastric cancer by sponging miR-331-3p and mediating the expression of TGFBR1. 40 Also, circRNAs serve as an endogenous sponge for miRNAs in NSCLC. For instance, circRNA 100146 exerts an oncogenic effect by binding to miR-615-5p and miR-361-3p in NSCLC in a direct manner. 41 Currently, it was uncovered that circ-0008003 with expression primarily in the cytoplasm accelerates NSCLC evolution by sponging miR-488 that represses NSCLC, 42,43 which was identified as a suppressor gene in NSCLC in this research.
ZNF281 is located at chromosome 1q32.1 and is a critical regulator of embryonic stem cell differentiation and tissue development. 44 Multiple studies have demonstrated the diverse roles of ZNF281 in stem cell pluripotency and development processes. 45,46 Therefore, ZNF281 is regarded as a relevant gene for cellular stemness. Notably, recent investigations have revealed that ZNF281 is a novel oncoprotein that is frequently overexpressed in human malignancies, including colorectal and pancreatic cancers. 47,48 More importantly, high ZNF281 expression in cancer tissues enhances metastatic potential and reduces patient survival. 46,49,50 However, its actual role and underlying mechanism in NSCLC have not yet been elucidated.
This research showed that ZNF281 was a target of miR-488 and circ-0008003 affected NSCLC via adjusting miR-488/ZNF281 signalling like an oncogene.
To sum up, circ-0008003 plays a carcinogenic role in NSCLC development via sponging miR-488, thus releasing and increasing ZNF281. This research firstly evidences circ-0008003's identities as an underlying treatment marker and a prognostic target in NSCLC, F I G U R E 7 ZNF281 overexpression restores the repression of circ-0008003 inhibition in NSCLC. A-B, The mRNA and protein levels of ZNF281 in H460 cells with NC shRNA, circ-0008003 shRNA, or together with ZNF281 elevation, are assessed using qRT-PCR and Western blotting, respectively. C-D, CCK8 and EDU assays are carried out to ascertain the proliferation capacity of H460 cells under different conditions (scale bar = 100 μm). E, Invasion ability of H460 cells receiving various treatments is tested through Transwell assay (scale bar = 100 μm). Data are the mean ± SEM. **P < .01, ***P < .001 vs control group, △ P < .05, △△ P < .01 vs circ-0008003 shRNA group but its clinical significance still needs in-depth investigations in the future.

ACK N OWLED G EM ENTS
This work was supported by the grants from the Foundation of the Top-Notch Scientific Research of the High-Level Health Talents Project (Grant No. LGY2018042).

CO N FLI C T O F I NTE R E S T
The authors state that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.