{'fraction_non_alphanumeric': 0.044365572315882874, 'fraction_numerical': 0.027373558118899733, 'mean_word_length': 4.826053243732231, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Circular RNA circ‑ACACA regulates proliferation, migration and glycolysis in non‑small‑cell lung carcinoma via miR‑1183 and PI3K/PKB pathway.

Non-small cell lung carcinoma (NSCLC) accounts for 85% of all lung cancers and the five-year survival rate is ~1% in the late stage. Circular RNAs (circRNAs) were reported to be involved in the progression of diverse human cancers. However, the role of circ-ACACA in NSCLC progression remains elusive. Quantitative polymerase chain reaction was conducted to detect the expression levels of circ-ACACA and microRNA (miR)-1183 in NSCLC tissues and cells. A Cell Counting Kit-8 assay and transwell assay were employed to check proliferation and migration, respectively. Metabolic alternations in NSCLC cells were monitored by the Seahorse XFe96 analyzer. The protein levels of cellular myelocytomatosis, matrix metallopeptidase 9, glucose transporter 1, phosphatase and tensin homolog, phosphoinositide 3-kinases (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (PKB) and p-PKB in samples were measured by western blotting. The interaction between circ-ACACA and miR-1183 was predicted by circular RNA Interactome, which was verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Xenograft tumor model was established to investigate the biological roles of circ-ACACA in vivo. The level of circ-ACACA was markedly upregulated in NSCLC tissues and cells, which was contrary to the expres-sion of miR-1183. Knockdown of circ-ACACA inhibited proliferation and migration of NSCLC cells and also reduced the glycolysis rate. In addition, miR-1183 was a target of circ-ACACA and its downregulation reversed circ-ACACA silencing-mediated inhibitory impact on NSCLC progression. Further studies indicated that circ-ACACA regulated the PI3K/PKB pathway through interacting with miR-1183 and downregulation of circ-ACACA suppressed tumor growth. Knockdown of circ-ACACA impeded NSCLC progression by sponging miR-1183 and inactivating the PI3K/PKB signaling pathway.


Introduction
Non-small cell lung carcinoma (NScLc) is a growing threat to humans and the dominant cause of cancer-related deaths worldwide (1). The five-year overall survival is not optimistic due to metastasis and chemoresistance (2,3). Hence, it is pressing to find novel molecular targets for NSCLC treatment.
Circular RNAs (circRNAs), a type of single-stranded RNAs which form a covalently closed continuous loop, are produced by backsplicing (4) and they are resistant to exonuclease-mediated degradation (5). Growing evidence has proved that circRNAs are associated with colon cancer (6), gastric cancer (7), bladder cancer (8) and NScLc (9,10). Microarray data showed that hsa_circ_0106705 (circ-ACACA) was upregulated in human lung cancer according to Yang et al (11). However, the regulatory mechanism of circ-ACACA in NSCLC is hardly reported and needs to be investigated further.
MicroRNAs (miRNAs/miRs) are short (~22 nucleotides) and highly conserved noncoding RNAs, which mediate gene expression by binding to the 3'-untranslated region of mRNA at the post-transcriptional level (12). Numerous studies have emphasized the core position of miRNAs in governing cancer development (13,14). miR-1183 was reported to be dysregulated in numerous human diseases (15)(16)(17) and Zhou et al (18) reported that miR-1183 functioned in the tumorigenesis of NSCLC. Nevertheless, the precise mechanism of miR-1183 in NScLc progression remains to be studied.
The phosphoinositide 3-kinases/protein kinase B (PI3K/PKB) pathway is essential for cell survival and apoptosis (19) and their aberrant activation is usually correlated with malignancy (20). A previous study indicated that the PI3K/PKB pathway was associated with the initiation of endometrial cancer (21) and cisplatin-resistance in NScLc (22). Therefore, in-depth studies of the PI3K/PKB signaling pathway could contribute to the development of new effective therapeutic methods for NScLc.
In this study, the expression level of circ-ACACA in NSCLC tissues and cells was first measured. Afterwards, the function and potential regulatory mechanism of circ-ACACA in NSCLC were further investigated by subsequent experiments.

Materials and methods
Specimens and cell culture. A total of 60 NScLc tissues and paired nearby healthy tissues were sourced from patients with NSCLC (36 males and 24 females; 20-65 years old) who had undergone surgical resection between April 2015 to August 2017 at Liaocheng People's Hospital, and the 60 NSCLC tissues included 37 early stages (I and II) tissues and 23 advanced stages (III and IV) tissues or contained 21 lymphoid node metastasis tissues and 39 controls. Tumor, node and metastasis (TNM) staging was classified according to the 7th edition of the American Joint committee on cancer TNM classification based on information obtained regarding the tumor during tumor surgery and histological or imaging studies. Every patient signed the informed consent and the official approval from the Ethics Committee of Liaocheng People's Hospital was obtained in this study. Human normal bronchus epithelium cell line (BEAS-2B) and NSCLC cell lines (A549, H1975, H1395, H1793, H1299 and H1792) were purchased from the American Type Culture Collection. McCoy's 5A medium (Sigma-Aldrich; Merck KGaA), containing 5% cO 2 and 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA) was used to culture cells.
Purified RNAs were treated with RNase R (Beijing Solarbio Science & Technology Co., Ltd.) for subsequent experiments.
Cell Counting  assay. cell proliferation was tested with CCK-8 reagent (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. After transfection, A549 and H1299 cells were seeded into 96-well plates and then incubated with 10 µl CCK-8 solution for 2 h. Optical density values were examined at 450 nm wavelength using a microplate reader (Bio-Rad Laboratories, Inc.).
Transwell assay. A Transwell chamber was employed to check the capacity of cell migration. Transfected cells (5x10 4 cells) were seeded into the upper chamber and medium containing FBS was placed in the lower chamber. After being treated with 0.1% crystal violet for 20 min at room temperature (Beijing Solarbio Science & Technology Co., Ltd.) following incubation for 24 h, migrated cells were analyzed under an inverted light microscope (MTX Lab Systems).
Dual-luciferase reporter assay. The potential complementary sequences of circ-ACACA and miR -1183 were forecasted by circular RNA Interactome (24). The wild type (WT) sequence of circ-AcAcA harboring the binding sites of miR-1183 was inserted into the pGL3 vector (Promega Corporation) to establish the luciferase reporter vector WT-circ-ACACA. Similarly, the mutant (MUT)-circ-ACACA reporter vector was established by mutating the potential target sites of miR-1183. Then, the luciferase reporter vectors (100 ng) were cotransfected with 50 ng miR-1183 or miR-Nc into A549 and H1299 cells for 24 h using Lipofectamine 2000 (Beijing Solarbio Science & Technology, Co., Ltd.). Firefly luciferase activities were normalized by comparison with Renilla luciferase. The Dual-Glo Luciferase Assay System kit (Promega Corporation) was utilized to measure luciferase activity.
RNA immunoprecipitation (RIP) assay. RIP was carried out using Magna RIP RNA-Binding Protein Immunoprecipitation kit (EMD Millipore) following the manufacturer's protocols. Briefly, harvested cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) and incubated with magnetic beads conjugated with anti-Argonaute 2 (Anti-Ago2) antibody (1:5,000; cat. no. MABE253; EMD Millipore) for 8 h at 37˚C, and immunoglobulin G (IgG; 1:5,000; cat. no. 12-370; EMD Millipore) was used as a negative control. The protein was removed by Proteinase K. The immune precipitated RNA was purified and analyzed by RT-qPCR.
RNA pull-down assay. A biotin-labeled probe against miR-1183 (named as Bio-miR-1183) and its negative control (named as Bio-NC) were obtained from Sangon Biotech Co., Ltd. Transfected cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) and incubated with streptavidin-coupled beads (Sangon Biotech Co., Ltd.). After being treated by proteinase K, circ-ACACA was isolated and checked by RT-qPCR.
Xenograft mice model. BALB/c nude mice (male; 5 weeks old; ~18-23 g; n=10) were acquired from Shanghai LingChang Biotech Co., Ltd. (Shanghai SLAC Laboratory Animal Co., Ltd.) and kept in specific pathogen free conditions (temperature 23±2˚C, relative humidity 55±5%, 12 h/12 h light/dark cycle with ad libitum access to water and food). The mice were randomly grouped into two groups (n=5 each). Lentivirus harboring short hairpin RNA targeting circ-AcAcA (named as sh-circ-ACACA) and negative control (sh-NC) were constructed by GeneCopoeia, Inc. A549 cells at a density of 1x10 6 cells/well in 6-well culture plates were infected with 4 µg of filtered lentivirus plus 8 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) for 48 h and subsequently selected by 2.5 µg/ml puromycin (Invitrogen; Thermo Fisher Scientific, Inc.) for at least 3 days to establish stable cell lines. A total of ~2x10 6 A549 cells infected with sh-circ-AcAcA or sh-Nc plasmid dNA in 200 µl of FBS-free culture medium were injected subcutaneously into the flank of the nude mice. The tumor volume was calculated every 7 days according to the formula: 0.5 x length x width 2 . The tumor weight was measured after the mice were euthanized.
The mRNA or protein levels of corresponding genes in tumors were checked by RT-qPCR or western blotting, respectively. The animal experiment was approved by the Animal Care and Use Committee of Liaocheng People's Hospital and executed referring to the instructions of the National Animal Protection and Ethics Institute.
Statistical analysis. Experimental data were calculated by GraphPad Prism 8.0 (GraphPad Software, Inc.) and presented as the mean ± standard deviation. Two independent groups were compared by using Student's t-test. For more than two groups, the one-way analysis of variance followed by Tukey post hoc test was utilized to assess the difference. Receiver operating characteristic (ROC) curve analysis was performed referring to a previous study (25). The Kaplan-Meier method was utilized to assess overall survival and the log-rank test was used to analyze the differences between survival curves. Pearson's correlation coefficient was applied to analyze the correlation between circ-AcAcA and miR-1183 in NScLc tissues. Every experiment was repeated at least three times independently. P<0.05 was considered to indicate a statistically significant difference.

circ-ACACA is significantly upregulated in NSCLC and
correlates with the poor prognosis. To explore the role of circ-ACACA in NSCLC, the expression patterns were first checked. The RT-qPCR data showed that circ-ACACA was significantly upregulated in NSCLC tissues compared with paired normal tissues (Fig. 1A). Next, the correlation between circ-AcAcA expression and NScLc progression was evaluated and the results showed that the level of circ-ACACA was increased in advanced stages (III and IV) compared with early stages (I and II; Fig. 1B). In addition, metastatic samples displayed an upregulated level of circ-ACACA (Fig. 1C) and the higher level of circ-ACACA led to the lower survival rate (Fig. 1D). Also, the diagnostic accuracy of circ-ACACA was assessed using the ROC curve analysis and the data showed that the area under the ROC curve was 0.7822 (Fig. 1E), which indicated that circ-ACACA might be a hallmark of NSCLC. Moreover, circ-ACACA was also significantly upregulated in NSCLC cells (Fig. 1F). Further analysis indicated that the mRNA of circ-ACACA (c-ACACA) was conspicuously resistant to RNase R compared with the mRNA of ACACA (m-ACACA; Fig. 1G and H). Collectively, these results illuminated that circ-ACACA might act an oncogene in NSCLC and have clinical diagnostic value.  Knockdown of circ-ACACA inhibits proliferation and migration and retards glycolysis rate. The levels of c-ACACA and m-ACACA were checked and the data showed that they were abundant in the cytoplasm of NSCLC cells ( Fig. 2A and B). To investigate the function of circ-ACACA in NSCLC, A549 and H1299 cells were first infected with si-circ-ACACA or si-NC and then the knockdown efficiency was confirmed ( Fig. 2C and D). The CCK-8 assay showed that downregulation of circ-AcAcA inhibited proliferation of NScLc cells ( Fig. 2E and F) and the transwell assay indicated that knockdown of circ-ACACA weakened the ability of migration of NSCLC cells (Fig. 2G and H). Glycolysis analysis indicated that circ-AcAcA silencing decreased EcAR in NScLc cells ( Fig. 2I and J). Afterwards, the protein levels of c-myc (related to proliferation), MMP9 (related to migration) and GLUT-1 (related to glycolysis) were measured and the results indicated that downregulation of circ-ACACA significantly reduced the expression of these proteins in NScLc cells (Fig. 2K and L).  Altogether, these results demonstrated that circ-AcAcA silencing suppressed proliferation and migration and alleviated the Warburg effect in NScLc cells.

Downregulation of miR-1183 promotes proliferation and migration and elevates glycolysis rate.
To probe the function of miR-1183 in NScLc, its expression was detected and the data showed that miR-1183 was significantly downregulated in NScLc tissues and cells compared with normal tissues and cells (Fig. 3A and B). Thereafter, NSCLC cells were transfected with anti-miR-1183 or anti-Nc and the results indicated that miR-1183 was significantly decreased in the anti-miR-1183 group (Fig. 3c and d). Next, a ccK-8 assay and transwell assay were performed and the data showed that downregulation of miR-1183 promoted proliferation ( Fig. 3E and F) and enhanced the migration ability of NScLc cells (Fig. 3G and H). In addition, miR-1183 inhibitor increased EcAR in NScLc cells (Fig. 3I and J). Simultaneously, the protein levels of c-myc, MMP9 and GLUT1 were significantly elevated in the anti-miR-1183 group (Fig. 3K and L). To sum up, these results demonstrated that miR-1183 might act as a tumor suppressor in NScLc progression in vitro.
circ-ACACA targets and negatively regulates miR-1183 in NSCLC. The interaction between circRNAs and miRNAs in cancer is documented in numerous reports (8,9). To probe the relationship between the two, Pearson's correlation coefficient was analyzed and the result indicated that the expression of miR-1183 was negatively associated with circ-AcAcA in NScLc tissues (Fig. 4A). Afterwards, the miR-1183 mimic was introduced to NScLc cells and the overexpression efficiency was verified (Fig. 4B). By using circular RNA Interactome, circ-AcAcA was found to harbor the binding sites of miR-1183 (Fig. 4c). The dual-luciferase reporter assay showed that miR-1183 significantly diminished the luciferase activity of WT-circ-ACACA in NSCLC cells, rather than MUT-circ-ACACA ( Fig. 4D and E). The RIP assay indicated that the relative enrichment of circ-ACACA and miR-1183 was increased in the Anti-Ago2 group compared with the control group ( Fig. 4F and G). Simultaneously, RNA pull-down assay showed that circ-ACACA was notably enriched in NSCLC cells transfected with Bio-miR-1183 ( Fig. 4H and I). Further study indicated that knockdown of circ-ACACA significantly increased the expression of miR-1183 in NScLc cells (Fig. 4J and K). All in all, these results demonstrated that miR-1183 was a target of circ-ACACA and negatively modulated by circ-ACACA in NScLc cells.
Downregulation of miR-1183 reverses circ-ACACA silencing-mediated effects on proliferation, migration and glycolysis rate. To further dissect the impact of the interaction between circ-AcAcA and miR-1183 on NScLc progression, the level of miR-1183 in NSCLC cells infected with si-circ-AcAcA or si-circ-AcAcA + anti-miR-1183 was first measured, as well as the corresponding controls. The result showed that miR-1183 was significantly upregulated in the si-circ-AcAcA group, while its expression was significantly decreased following infection with anti-miR-1183 ( Fig. 5A and B). The CCK-8 assay indicated that downregulation of miR-1183 inverted the circ-ACACA silencing-mediated inhibitory effect on proliferation of NSCLC cells (Fig. 5C and D). The Transwell assay revealed that circ-ACACA silencing-mediated the repressive impact on migration of NSCLC cells and was reversed by the miR-1183 inhibitor ( Fig. 5E and F). Similarly, the decreased ECAR in the si-circ-ACACA group was reversed after infection with anti-miR-1183 ( Fig. 5G and H). Meanwhile, the declined protein levels of c-myc, MMP9, GLUT-1 in si-circ-ACACA group were reversed by the miR-1183 inhibitor ( Fig. 5I and J). From these results, it could be concluded that circ-AcAcA mediated the progression of NSCLC by interacting with miR-1183 in vitro.
circ-ACACA regulates the PI3K/PKB signaling pathway by interacting with miR-1183 in NSCLC cells. To investigate whether circ-ACACA could affect the PI3K/PKB pathway, the protein levels of PTEN, total PI3K (t-PI3K), p-PI3K, total PKB (t-PKB) and p-PKB in NSCLC cells infected with si-circ-AcAcA, si-circ-AcAcA + anti-miR-1183 or matched controls were detected. The results indicated that PTEN was significantly upregulated and the levels of p-PI3K/t-PI3K and p-PKB/t-PKB were notably downregulated in the si-circ-ACACA group, whereas the situation was reversed after infection with anti-miR-1183 ( Fig. 6A and B). In summary, these results demonstrated that the circ-AcAcA/miR-1183 axis regulated the PI3K/PKB pathway in NSCLC.

circ-ACACA silencing inhibits tumor growth in vivo.
To verify the function of circ-ACACA in NSCLC cells in vivo, the xenograft mouse model was established using A549 cells transfected with sh-circ-AcAcA or sh-Nc. The data showed that knockdown of circ-ACACA led to an obvious shrink in tumor volume (Fig. 7A) and decline in tumor weight (Fig. 7B). Also, the level of circ-ACACA was significantly decreased in the sh-circ-AcAcA group (Fig. 7c), the opposite effect to the expression of miR-1183 (Fig. 7d). In addition, knockdown of circ-ACACA reduced the protein levels of c-myc, MMP9 and GLUT-1 in tumors (Fig. 7E). Similarly, the protein level of PTEN was conspicuously elevated and the protein levels of p-PI3K/t-PI3K and p-PKB/t-PKB were clearly declined in sh-circ-AcAcA group (Fig. 7F). Taken together, these results suggested that downregulation of circ-AcAcA suppressed NScLc progression in vivo.

Discussion
NScLc accounts for ~85% of all lung cancers and the five-year survival rate can be very low due to metastasis and drug-resistance (2,3). Therefore, it is essential to find new molecular targets and investigate potential mechanisms. Recently, circRNAs have been verified to regulate the progression of numerous cancers. Liu et al (7) found that circular RNA YAP1 inhibited gastric cancer progression via modulating miR-367-5p. Lu et al (8) reported that circSLc8A1 suppressed bladder cancer progression via regulating PTEN. Chen et al (9) found that circRNA 100146 acted as an oncogene in NScLc. To explore the function of circ-AcAcA in NScLc, its expression level was checked and it was found that circ-ACACA was upregulated in NScLc tissues and cells and contributed to poor prognosis. Also, ROC curve analysis indicated that circ-ACACA could be a biomarker for NSCLC. Further analysis showed that downregulation of circ-AcAcA hindered proliferation and migration of NSCLC cells. Alteration of energy metabolism, especially abnormal activation of the glycolysis pathway was observed in diverse human cancers (26,27). circRNAs are reported to be involved in the regulation of the Warburg effect in human cancers (28,29). Hence, the level of EcAR were checked in NScLc cells and it was found that circ-ACACA silencing reduced the glycolysis rate. In addition, the protein levels of c-myc, MMP9 and GLUT-1 also declined in NScLc cells infected with si-circ-AcAcA. Furthermore, in vivo experiments showed that knockdown of circ-AcAcA repressed tumor growth. All in all, these results suggested that circ-AcAcA might function as an oncogene and could be a potential therapeutic target in NScLc.
Growing evidence has clarified the fact that circRNAs could serve as the sponges of miRNAs to function in numerous cancers (30,31) and the present research showed that circ-ACACA was mainly expressed in the cytoplasm in NSCLC cells. In this study, miR-1183 was forecasted to be a target of circ-AcAcA and this interaction was confirmed. A previous report showed that miR-1183 was involved in the regulation of NSCLC progression (18). In this study, the level of miR-1183 was decreased in NSCLC tissues and cells. Moreover, the miR-1183 inhibitor repressed proliferation and migration of NSCLC cells and reduced the glycolysis rate. In-depth studies illustrated that the repressive impact of circ-AcAcA silencing-mediated NScLc progression was reversed by downregulating miR-1183. A previous report indicated that the PI3K/PKB signaling pathway participated in the regulation of NSCLC (32). To investigate whether circ-ACACA affected this pathway, the expression of related proteins in NScLc cells infected with si-circ-AcAcA, si-circ-AcAcA + anti-miR-AcAcA or corresponding controls was checked. The results indicated circ-ACACA silencing elevated the expression level of PTEN, a major antagonist of PI3K activity and also decreased the levels of p-PI3K/t-PI3K and p-PKB/t-PKB, while downregulation of miR-1183 reversed the effect. These data indicated that the silencing of circ-ACACA inactivating the PI3K/PKB pathway, whereas this effect was abolished by the miR-1183 inhibitor. Taken together, these results suggested that circ-ACACA mediated the proliferation, migration and glycolysis via sponging miR-1183 and the circ-ACACA/miR-1183 axis regulated the PI3K/PKB pathway.
In conclusion, the current research demonstrated that circ-AcAcA was upregulated in NScLc tissues and cells. Also, downregulation of circ-AcAcA restrained NScLc progression via interacting with miR-1183 and inactivating the PI3K/PKB pathway. This novel mechanism may provide a theoretical basis for research into circRNA-directed treatment in NScLc.

Acknowledgement
Not applicable.

Funding
No funding was received.

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.