Circ_0018534 Promotes Non-Small Cell Lung Cancer Progression by Upregulating BTBD7 via Sponging miR-153-3p

Background: Non-small cell lung cancer (NSCLC) is the most common malignant tumor in lung. In this study, we aimed to explore the role and underlying mechanism of circular RNA 0018534 (circ_0018534) in NSCLC progression. Methods: Real-time quantitative PCR (RT-qPCR) was used to detect the expression of circ_0018534, microRNA-153-3p (miR-153-3p) and BTB/POZ domain-containing protein 7 (BTBD7) in NSCLC tissues and cells. Protein expression was measured by western blot analysis. The morphological features of obtained NSCLC patient tissues were observed by haematoxylin and eosin staining assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-Ethynyl-29-deoxyuridine, ow cytometry analysis, and transwell migration and invasion assays were used to examine cell viability, proliferation, apoptosis, migration and invasion, respectively. Bioinformatics analysis and dual-luciferase reporter assay were carried out to determine the interaction among circ_0018534, miR-153-3p and BTBD7. Furthermore, lentivector for short hairpin RNA against circ_0018534 (sh-circ_0018534) was used to decrease circ_0018534 expression in an animal tumor model. Results: Circ_0018534 and BTBD7 expression were signicantly increased, whereas miR-153-3p expression was decreased in NSCLC tissues and cells. Circ_0018534 downregulation markedly alleviated cell proliferation, migration, invasion, and elevated cell apoptosis in NSCLC cells in vitro. MiR-153-3p inhibitors partially reversed the effects of circ_0018534 knockdown on cell proliferation, migration, invasion and apoptosis in NSCLC. Moreover, miR-153-3p could directly target BTBD7, and circ_0018534 promoted BTBD7 expression by targeting miR-153-3p. Besides, BTBD7 overexpression attenuated miR-153-3p mimics-mediated effects on NSCLC cell progression. Furthermore, circ_0018534 deletion suppressed NSCLC tumor growth in vivo. Conclusion: Our results demonstrated that circ_0018534 might contribute to the progression of NSCLC by miR-153-3p/BTBD7 axis, providing a potential target for the treatment of NSCLC.


Introduction
Non-small cell lung cancer (NSCLC), a malignant tumor threatening human health and occupying more than 80% of all lung cancer cases, is regarded as the most common type of lung cancers 1 . Despite the remarkable progress of diagnosis and treatment of NSCLC has been made in recent years, the 5-year survival rate of NSCLC patients still remains dismal and just approximately 15%, while there was no signi cant change in the past few years 2 . The reason for the low 5-year survival rate of NSCLC patients might be lack of effective detection ways for NSCLC at an early stage. Therefore, a thorough understanding of the underlying molecular mechanism of the initiation and progression of NSCLC might provide new and effective diagnostic markers for early NSCLC patients.
Previous studies reported that there are at least 75% of the genomes with little protein-coding potential and less than 2% encoding proteins. Circular RNAs (circRNAs) are a type of the noncoding transcripts with closed loop structure, whereas there are other small noncoding transcripts, such as piRNA, miRNA and snoRNA 3 . Many studies have demonstrated that circRNAs could regulate gene expression and act the oncogenic or suppressive roles involving in the initiation and progression of various cancers 4 .
Accumulating evidences have reported that dysregulation of circRNAs is usually observed in a variety of cancers, including esophageal squamous cell carcinoma 5 , anaplastic thyroid carcinoma 6 , gallbladder cancer 7 and also NSCLC 8 . Up to now, several studies have demonstrated that circRNAs contributed to the progression of NSCLC, such as circ_0016760 9 , circ_0000376 10 and circ_0067934 11 . Circ_0018534 is a circRNA and the underlying molecular mechanism of circ_0018534 in NSCLC remains unclear.
MicroRNAs (MiRNAs), a group of small endogenous non-coding RNAs consisting of about 22 nucleotides, are con rmed to regulate the downstream gene expression and serve as tumor regulatory genes in the initiation and development of cancers 12 . Besides, accumulating evidences have indicated that a large number of miRNAs in NSCLC were studied. For instance, Jin and his colleagues detected miR-873 and GLI1 expression in PC9 and PC9/GR cells and discovered the inhibitory effects of miR-873 deletion on ge tinib resistance in NSCLC cells 13 . Jin et al. indicated that miR-1290 upregulation exerted promoting effect on cell growth in vitro and in vivo, and improved the invasive ability of NSCLC cells via degrading the expression of IRF2 14 . Moreover, few researchers observed the dysregulation of miR-153-3p in NSCLC, playing a suppressor role in the tumor progression of NSCLC 15 . However, the involvement of miR-153-3p in the biological processes of NSCLC is little known. Thus, we attempted to explore the functional effects and the relevant mechanism of miR-153-3p in NSCLC.
BTB/POZ domain-containing protein 7 (BTBD7) is a highly conserved protein, belonging to the BTB/POZ domain protein family 16 . Recently, emerging evidence suggested that BTBD7 represented the vital importance in protein degradation, cell metastasis and apoptosis 16 , thus exerted a critical role in the progression of cancers. It has been demonstrated that BTBD7 silencing signi cantly boosted E-cadherin in NSCLC cells and repressed cell migration ability, which was regarded as a tumor promoter in NSCLC 17 .
The effects of BTBD7 on the biological behaviors in NSCLC were further revealed in this research.
In the present study, we detected the expression of circ_0018534, miR-153-3p and BTBD7 in NSCLC tissues and cell lines. And we investigated the interaction among circ_0018534, miR-153-3p and BTBD7. Furthermore, we aimed to explore the functional role of circ_0018534 on NSCLC cells' biological behaviors both in vivo and in vitro.

Materials And Methods
Tissue samples Non-small cell lung cancer tissues (n=50) and the neighboring normal lung tissues (n=50) were collected from 50 NSCLC patients who underwent surgery at Shanghai University of Traditional Chinese Medicine hospital. All NSCLC patients involved in this study gave the signed informed consents. The experiments in the present research were approved by the Human Research Ethics Committee of Shanghai University of Traditional Chinese Medicine hospital. None of the NSCLC patients participating in this research had undergone other therapy.

Cell lines
Two NSCLC cell lines (A549 and H1299) were obtained from American Type Culture Collection (Manassas, VA, USA), and the human bronchial epithelial cell line (16-HBE) used in this study was purchased from Shanghai Fuxiang Biological Technology Co. Ltd. (Shanghai, China). 16-HBE and NSCLC cells were cultured in modi ed Eagle's medium (MEM; Gibco, Rockville, MD, USA) and Dulbecco's modi ed Eagle's medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), and streptomycin/penicillin (100 U/mL; Invitrogen), respectively. All cells were maintained at 37˚C in a humidi ed incubator with an atmosphere of 5% CO 2 .

Haematoxylin and eosin (HE) staining
Tumor tissues from each group of collected NSCLC sufferer specimens were cut into the 5-μm sections, and immobilized with paraformaldehyde (Sigma, Billerica, MA, USA). Then, the section was dehydrated using with ethanol (Millipore, Bradford, MA, USA) and embedded in para n. HE (Beyotime, Shanghai, China) was employed to stain the tissues. The images were captured under a microscope (Nikon, Tokyo, Japan).
Subsequently, the medium was removed, and dimethyl sulfoxide was added to dissolve the blue crystal. Finally, the absorbance was detected at 450 nm by a microplate reader (Bio-Rad Laboratories).

5-Ethynyl-29-deoxyuridine (EdU) assay
The proliferation of A549 and H1299 cells was determined by EdU assay kit (Ribobio, Guangzhou, China). In brief, cells were grown in 12-well plates after transfected with plasmids or oligonucleotides. At 48 h after culture, cells were digested and seeded in 96-well plates. When the con uence of cells was about 30%, the EdU assay was carried out according to the instruction of manufacture. Immunostainings were visualized and captured with a uorescence microscope (Olympus, Tokyo, Japan).

Cell apoptosis assay
Cell apoptosis was detected by ow cytometry analysis. The treated HSCLC cells were collected and centrifuged at 1000 rpm for 8 min. Then phosphate buffered solution (PBS) was used to wash the treated NSCLC cells, and the binding buffer was used to resuspend cells. Subsequently, cells were stained using Annexin V/Propidium Iodide (BestBio, Shanghai China) for 20 min in the dark. Cell apoptosis was measured by ow cytometry (Becton-Dickenson, Franklin Lakes, NJ, USA).

Transwell migration and invasion assay
The transwell chambers used in cell migration and invasion assays were purchased from Corning Incorporated (Big Flats, NY, USA). The chambers pre-coated or non-coated with Matrigel (BD Biosciences, San Jose, CA, USA) were used for cell invasion or migration assay, respectively. In the upper chamber, there were the cells mixed with serum-free medium, while DMEM containing 10% FBS was added into the lower chamber. Then cells not migrated or invaded were scraped, whereas the cells removed to the lower chamber were xed, stained, and analyzed.
Xenograft mouse model BALB/c nude mice were purchased from the Beijing Laboratory Animal Center (Beijing, China). The mice were housed in a standard animal laboratory with a 12 h light-dark cycle, and free access to food and water. A549 cells (2 × 10 6 ) stably transfected with short hairpin RNA (shRNA) against circ_0018534 (sh-circ_0018534) or sh-NC were subcutaneously injected into the right ank of nude mice (5 per group). The transfected nude mice were classi ed into two groups: sh-NC nude mice group and circ_0018534 knockdown nude mice group. After 7 d, the tumor volume was detected for the rst time after the tumors became visible. Subsequently, the volume in every group was measured using a caliper every 4 d. After injection for 28 d, all the mice were euthanized by asphyxia method. The mice were treated with the ow rate of CO 2 to displace 60% air of the chamber volume/min according to the current guideline of American Veterinary Medical Association (AVMA). The tumor weight was determined. The lung tissues were separated from the nude mice and stored at -80˚C. The experiments were approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine hospital.

Statistical analysis
All data were displayed as the means ± the standard deviations (SD). SPSS 18.0 software was used to analyze the data in the present study. Spearman correlation analysis was performed to analyze the correlation between variables. Student's t-test was used to analyze the statistical difference between groups. One-way analysis of variance (ANOVA) was compared the statistical difference among three or more groups. A value of P < 0.05 meant a statistically signi cant difference.

Results
Circ_0018534 expression is upregulated in NSCLC tissues and cells To assess circ_0018534 expression in NSCLC tissues, the morphological features of NSCLC tissues and paracancerous normal lung tissues were observed by HE staining assay (Fig. 1A). We then determined the expression of circ_0018534 in NSCLC tissues. RT-qPCR indicated that circ_0018534 expression was higher in NSCLC tissues than in the corresponding normal tissues (Fig. 1B). Moreover, we found that circ_0018534 was overexpressed in A549 and H1299 cells compared with 16-HBE cells (Fig. 1C). The data suggested that circ_0018534 might play crucial roles in NSCLC.
Circ_0018534 deletion inhibits cell proliferation, migration, invasion, and promotes cell apoptosis in NSCLC To investigate the functional role of circ_0018534 in NSCLC, si-circ_0018534 and si-NC were transfected into A549 and H1299 cells. RT-qPCR was used to evaluate the knockdown e ciency of si-circ_0018534. We found that knockdown of circ_0018534 remarkably decreased circ_0018534 expression in both A549 and H1299 cells ( Fig. 2A). Then, we explored the effects of circ_0018534 on the biological behaviors in NSCLC. Results showed that circ_0018534 deletion inhibited cell viability and proliferation in both A549 and H1299 cells (Fig. 2B-E). Knockdown of circ_0018534 signi cantly elevated the apoptosis rate of NSCLC cells (Fig. 2F). Furthermore, transwell migration and invasion assays were used to measure the migratory and invasive abilities of NSCLC cells. The results showed that circ_0018534 deletion remarkedly reduced the number of migrated and invaded cells (Fig. 2G-H). Meanwhile, we found that circ_0018534 silencing decreased the protein expression of PCNA, Bcl-2, Snail and MMP9, and increased Bax protein expression ( Fig. 2I-J). These results demonstrated that circ_0018534 could promote the progression of NSCLC.
And miR-153-3p was employed as the supposed miRNA associated with circ_0018534 based on its downregulation in NSCLC tissues and the highest expression in NSCLC cells transfected with si-circ_0018534 ( Fig. 3B-C). The binding sites between circ_0018534 and miR-153-3p were shown in Fig. 3D. Then, dual-luciferase reporter assay was performed to determine whether circ_0018534 could function as a molecular sponge to regulate miR-153-3p expression. Results rstly presented the high overexpression e ciency of miR-153-3p (Fig. 3E). Our data then indicated that the luciferase activity was signi cantly decreased in NSCLC cells co-transfected with miR-153-3p mimic and circ_0018534-WT, whereas there was no signi cant change when cells were co-transfected with circ_0018534-MUT and miR-153-3p mimic (Fig. 3F-G). Moreover, the expression of miR-153-3p was assessed in NSCLC tissues and cells. Results showed the expression of miR-153-3p was markedly decreased in NSCLC tissues and A549 and H1299 cells compared with normal lung tissues and 16-HBE cells, respectively (Fig. 3H-I). Furthermore, we found miR-153-3p expression was negatively related to circ_0018534 expression (Fig. 3J). These data demonstrated that circ_0018534 was associated with miR-153-3p.
MiR-153-3p inhibitors reverses the effects of circ_0018534 deletion on the progression of NSCLC To further investigate the relationship between circ_0018534 and miR-153-3p in NSCLC cells, the expression of miR-153-3p was measured in A549 and H1299 cells transfected with anti-miR-NC or anti-miR-153-3p. As described in Fig. 4A, the expression level of miR-153-3p was reduced by anti-miR-153-3p. And results showed that downregulation of circ_0018534 suppressed the cell viability and proliferation, whereas miR-153-3p inhibitors reversed these effects in A549 and H1299 cells (Fig. 4B-E). Our data also noted that miR-153-3p inhibitors signi cantly reversed the promotion effect of circ_0018534 deletion on cell apoptosis (Fig. 4F). In transwell migration assay, we found that the inhibitory effect of circ_0018534 deletion on cell migration ability was markedly attenuated by miR-153-3p inhibitors (Fig. 4G). A similar phenomenon was also discovered in invasion assay, in which miR-153-3p inhibitors reversed the suppressive effect of circ_0018534 downregulation on the invasive ability of A549 and H1299 cells (Fig.  4H). Furthermore, si-circ_0018534 mediated effects on the protein expression of PCNA, Bax, Bcl-2, Snail and MMP9 were also attenuated after miR-153-3p downregulation (Fig. 4I-J). Taken together, circ_0018534 deletion signi cantly suppressed proliferation, migration, invasion, and promoted apoptosis of A549 and H1299 cells by binding to miR-153-3p.
MiR-153-3p targets BTBD7 in A549 and H1299 cells Starbase online database predicted that BTBD7 possessed the complementary binding sites of miR-153-3p (Fig. 5A). Then the reporter plasmids containing the predicted miR-153-3p binding site (BTBD7 3'UTR-WT) and the mutant-type sequence (BTBD7 3'UTR-MUT) were constructed. Our data suggested that the luciferase activity was signi cantly suppressed by miR-153-3p in A549 and H1299 cells transfected with BTBD7 3'UTR-WT; however, for cells transfected with BTBD7 3'UTR-MUT, the luciferase activity was not changed (Fig. 5B-C). Subsequently, results demonstrated that BTBD7 expression at the mRNA and protein levels was markedly increased by anti-miR-153-3p, and was decreased by miR-153-3p in both A549 and H1299 cells (Fig. 5D-E). The mRNA and protein expression of BTBD7 were upregulated in NSCLC tissues and cells relative to normal tissues and cells (Fig. 5F-I). Furthermore, a negative linear relationship between BTBD7 and miR-153-3p expression was observed (Fig. 5J). Overall, miR-153-3p targeted BTBD7 in NSCLC cells.

MiR-153-3p mimics represses NSCLC cell progression by targeting BTBD7
Then, we explored the functional effects between miR-153-3p and BTBD7 on the biological processes in NSCLC. We rstly detected the expression of BTBD7 in A549 and H1299 cells transfected with pcDNA or pcDNA-BTBD7. The data suggested that the mRNA and protein expression of BTBD7 were highly expressed in A549 and H1299 cells transfected with pcDNA-BTBD7 compared with control groups (Fig.  6A-B). Data indicated that miR-153-3p mimics signi cantly suppressed cell viability and proliferation in NSCLC cells, whereas these effects were reversed by BTBD7 overexpression (Fig. 6C-F). Then the apoptosis rate of NSCLC cells was measured by ow cytometry, and we discovered that cell apoptosis was remarkably elevated by miR-153-3p mimics, which was attenuated after BTBD7 overexpression (Fig.  6G). Subsequently, transwell migration and invasion assays were performed. The results indicated that upregulation of BTBD7 signi cantly attenuated miR-153-3p mimics-mediated repression on the migratory and invasive abilities of NSCLC cells (Fig. 6H-I). MiR-153-3p mimics-mediated effects on the protein expression of PCNA, Bax, Bcl-2, Snail and MMP9 were also impaired by BTBD7 overexpression (Fig. 6J). All these data demonstrated that miR-153-3p regulated NSCLC cell progression by targeting BTBD7.

Inhibition of circ_0018534 suppresses NSCLC tumor growth in vivo
Then, we established a xenograft model to investigate the functional role of circ_0018534 in vivo. A549 cells stably transfected with sh-circ_0018534 or sh-NC were injected subcutaneously into nude mice. As shown in Fig. 8A-B, the volume and weight of the tumors were signi cantly suppressed by decreased circ_0018534. Subsequently, we measured the expression of circ_0018534, miR-153-3p and BTBD7 in the tumors of nude mice. The results suggested that the expression of circ_0018534 and the mRNA and protein levels of BTBD7 was markedly suppressed, whereas the expression of miR-153-3p was signi cantly increased in the tumors from nude mice (Fig. 8C-F). Taken together, our results demonstrated that circ_0018534 could signi cantly elevate NSCLC tumor growth in vivo.

Discussion
NSCLC, the most common malignant lung cancer, ranks one of the lethal diseases. The previous studies reported the poor diagnosis methods at an early stage of NSCLC and the poor prognosis caused by the occurrence of tumor metastasis and high mortality 18 . Accumulating evidences indicated that some circRNAs, as well as miRNAs, were involved in the progression of types of cancers 19,20 . Recently, emerging evidences have suggested that many circRNAs could sponge miRNAs to exert functional effects in diseases 21 . Moreover, dysregulation of circRNAs was observed in NSCLC in several researches [22][23][24] . Thus, circRNAs might be novel therapeutic targets for clinical treatment for NSCLC.
In NSCLC progression, multiple circRNAs were involved. For example, circ_0067934 was indicated to promote cell proliferation and metastasis as well as be associated with poor prognosis of NSCLC cases 11 . Circ_0002483 was revealed to enhance the sensitivity of NSCLC to Taxol by binding to miR-182-5p 25 .
Additionally, Ding et al. found that circ_001569 could promote cell proliferation by modulating Wnt/βcatenin pathway in NSCLC 26 . In this study, circ_0018534 was revealed to regulate NSCLC progression for the rst time. We found that the expression of circ_0018534 was higher in NSCLC tissues and cells than the expression in their corresponding normal groups. The downregulation of the expression of circ_0018534 was used to gure out the functional effects of circ_0018534 in NSCLC in vitro, as well as in vivo. Here, we discovered that downregulated circ_0018534 could attenuate cell proliferation, enhance the apoptotic rate, as well as decrease the number of migrated and invaded cells in NSCLC cells in vitro. Besides, we also found the inhibition of tumor growth via downregulating circ_0018534 in the nude mice model in vivo. And circ_0018534 was identi ed to directly target miR-153-3p. Considering all these data, we guessed that circ_0018534 might exert the oncogenic effects in NSCLC by targeting miR-153-3p.
The involvement of miR-153-3p in various types of cancer cells has been well-documented in the previous researches, such as colorectal cancer cells 27 , ovarian cancer cells 28 , and breast cancer cells 29 . Besides, miR-153-3p bound by NEAT1 was the regulatory mechanism for NSCLC progression according to the exploration from Zhao and his colleagues 15 . In this paper, we found that miR-153-3p was lowly expressed in NSCLC tissues and cells, and negatively regulated by circ_0018534. MiR-153-3p inhibitors also reversed circ_0018534 silencing-mediated NSCLC development. Additionally, we observed that BTBD7 was a novel target of miR-153-3p, and increased BTBD7 was discovered in NSCLC tissues and cells relative to the normal groups. In this study, the effects of BTBD7 on the biological behaviors were similar to circ_0018534 in NSCLC cells, and the effects of BTBD7 overexpression, including promotion of cell viability and cell metastasis, repression of cell apoptosis were observed in NSCLC cells in the present research. Subsequently, we performed the reverse experiments and found that overexpression of BTBD7 reversed the effects of miR-153-3p on cell growth, apoptosis and metastasis in NSCLC cells. Furthermore, rescue experiments also demonstrated that circ_0018534 regulated BTBD7 expression by interacting with miR-153-3p.
In summary, we found that the expression of circ_0018534 and BTBD7 were signi cantly boosted, and miR-153-3p expression was decreased in NSCLC tissues and cells. Functionally, circ_0018534 downregulation could suppress cell growth and metastasis, and exert a signi cant promotion effect on cell apoptosis in NSCLC in vitro. Additionally, circ_0018534 deletion also had a suppressive effect on tumor growth in vivo. And circ_0018534 could regulate BTBD7 expression via targeting miR-153-3p. More importantly, we discovered a novel regulatory mechanism in NSCLC that circ_0018534 promoted the progression of NSCLC via miR-153-3p/BTBD7 axis. Our data indicated that circ_0018534 might be a potential target for NSCLC treatment.