According to global cancer statistics released by the World Health Organization (WHO) in 2020, lung cancer, the leading cancer worldwide, accounts for 11.4% of all cases (Sung et al., 2021). Although carcinogenic factors such as smoking have been controlled, primary lung cancer remains one of the most common malignancies in China. In addition to traditional surgical treatment, in recent years, the research on targeted therapies and immune checkpoint inhibitors has advanced rapidly, and the development and marketing of related drugs have changed the treatment prospects of lung cancer. Unfortunately, most patients are discovered at a late stage or with metastasis, which seriously affects the prognosis (Barash, Peled, Hirsch, & Haick, 2009). It is reported that if lung cancer can be found in time, the 5-year survival rate of patients could reach 36%-70% (Gatteschi, Iannopollo, & Gonfiotti, 2021). Currently, early detection methods of lung cancer mainly include chest X-ray, bronchoscopy, sputum analysis, cytology analysis, and low dose computed tomography (LDCT). Nevertheless, these methods have some limitations and cannot meet our expectations. Nanotechnology has revolutionized the outlook for lung cancer detection and treatment vectors in recent years. However, its immunogenicity, clearance rate, drug-carrying capacity, and targeting create obstacles and challenges for drug transformation into the lung (S. Li et al., 2021; Srinivasan, Rajabi, & Mousa, 2015). In consequence, it’s urgent for better, cheaper, less invasive, and more widespread early lung cancer detection (Haince et al., 2022).
circRNAs are a newly discovered class of RNAs that have re-entered the limelight in recent years due to their critical regulatory roles in the onset and progression of several illnesses. Exploring the expression profiles of circRNAs in tissues and cell lines of different cancer species has led to the discovery of an increasing number of differentially expressed circRNAs, owing to the rapid growth of high-throughput sequencing technology and bioinformatics analysis. Moreover, circRNA has many advantages over other non-coding RNAs (ncRNAs), such as being more stable than linear RNA (Shan et al., 2019), having higher abundance and tissue specificity (Memczak et al., 2013). Thus, circRNAs can be stably present at high levels in body fluids such as serum, urine, and exosomes. Besides, growing evidence also demonstrated the important biological functions of circRNAs (L. L. Chen, 2020; Kristensen et al., 2019). The majority of functional studies have shown that circRNAs contain miRNA response elements (MREs) to act as miRNA sponges. For instance, Li et al. found that circMAT2B activates the circMAT2B/miR-338-3p/PKM2 axis under hypoxic conditions via the sponge miR-338-3p to promote glycolysis and hepatocellular carcinoma malignancy (Q. Li et al., 2019). Besides, some articles confirmed that circRNAs could interact with RNA-binding proteins (RBP) such as circBACH1. It interacts with HuR to promote HuR translocation and facilitates its accumulation in the cytoplasm, thereby downregulating p27 expression (Kullmann, Göpfert, Siewe, & Hengst, 2002). What’s more, circRNAs regulate gene transcription. For example, CircSMARCA5 terminates transcription on exon 15 of SMARCA5 by r-loop formation and upregulates truncated non-functional isoforms (Xu et al., 2020). circRNAs have been thought to be ncRNAs with regulatory roles (C. Y. Chen & Sarnow, 1995). Later, scientists have discovered translatable circRNAs (Pamudurti et al., 2017). circ β-catenin, for instance, was identified to be a protein-coding circRNA, whose translation can induce the progression of HCC by activating the Wnt pathway (Liang et al., 2019).
hsa_circ_0023179 was derived from LRP5 and obtained by circRNA high-throughput sequencing of 3 pairs of NSCLC tissues. It was remarkably upregulated in NSCLC tissues in 12 pairs of NSCLC tissues. First, AGE and Sanger sequencing were used to check that the qRT-PCR product was specific and accurate. The stability of the molecule was identified by the exonuclease digestion experiment assay and actinomycin D assay. Besides, the detection method of hsa_circ_0023179 was evaluated. The linearity and precision of CT values of qRT-PCR products met the requirements. The stability and reproducibility of qRT-PCR were recognized by placement at room temperature and multiple freeze-thaw experiments. The single specific melting curve illustrates the qRT-PCR specificity. After validation, an extensive sample analysis found that hsa_circ_0023179 was overexpressed in the serum of NSCLC patients, and the difference was remarkable compared with healthy donors and BPD patients. Subsequently, the results of ROC curve analysis indicated that hsa_circ_0023179 had higher diagnostic efficacy than CEA, SCC, and Cyfra21-1, while the combination remarkably improved the diagnostic value. In addition, high serum hsa_circ_0023179 expression was correlated with histologic type, TNM stage, lymph node metastasis, and distal metastasis, according to an analysis of clinicopathological data from 126 NSCLC patients. Furthermore, hsa_circ_0023179 expression levels decreased in NSCLC patients after surgery and had a higher survival curve in healthy subjects, suggesting the potential of hsa_circ_0023179 for dynamic monitoring. To verify whether hsa_circ_0023179 was secreted by tumor cells, we examined its expression in NSCLC cell lines (A549, NCI-H1299, NCI-H226, and SPC-A1) and discovered that all were differentially upregulated. Nucleoplasmic separation experiments demonstrated that hsa_circ_0023179 was slightly higher in the cytoplasm than in the nucleus. And in the cytoplasm, some circRNA plays a role in competing for endogenous RNA (ceRNAs) (Salmena, Poliseno, Tay, Kats, & Pandolfi, 2011). We further predicted the hsa_circ_0023179-miRNA-mRNA. Among these predicted miRNAs, hsa-miR-4644 was upregulated in plasma exosomes of bladder cancer patients and promoted bladder cancer progression by targeting UBIAD1 (Pang et al., 2021). hsa-miR-623 was confirmed to be downregulated in lung adenocarcinoma and inhibited lung adenocarcinoma growth and metastasis through ERK/JNK inactivation-mediated MMP-2/9 downregulation (Wei et al., 2016), suggesting that different regulatory networks in NSCLC progression, in which hsa_circ_0023179 may be involved.
Besides, it is worth mentioning that an increasing number of chemotherapeutic agents have been approved by FDA for the clinical treatment of lung cancer. For example, Gefitinib, Crizotinib, and Atezolimumab are important first-line therapeutic agents for lung adenocarcinoma, which inhibit the function of epidermal growth factor receptor (EGFR), mesenchymal lymphoma kinase (ALK) and programmed death-ligand 1 (PDL-1), respectively (Mottaghitalab, Farokhi, Fatahi, Atyabi, & Dinarvand, 2019). However, the resulting drug-resistant mutations are another major problem. Based on this, whether hsa_circ_0023179 plays a role in NSCLC chemotherapeutic drug resistance process may be our next exploration direction. Taken together, this study identified the upregulated hsa_circ_0023179 in NSCLC firstly by circRNA high-throughput sequencing and explored its potential as a tumor marker. However, this study also has some limitations. For instance, all samples were from Nantong University Hospital and Nantong University Cancer Hospital, and the specimens had a certain contingency. In addition, more studies are needed to confirm whether hsa_circ_0023179 can be truly useful in the clinical setting due to the lack of standardized protocols. In summary, the high expression of hsa_circ_0023179 may serve as a potential NSCLC diagnostic marker. Nevertheless, the role of hsa_circ_0023179 in NSCLC biological regulation remains unclear. According to the analysis of hsa_circ_0023179 with clinicopathological features, hsa_circ_0023179 was associated with TNM stage, lymph node metastasis, and distal metastasis, indicating that hsa_circ_0023179 may be involved in the malignant progression of cell proliferation, migration, and invasion in NSCLC, which suggests a new direction for investigation. Our subsequent research will be focus on if hsa_circ_0023179, as ceRNA, is involved in regulating NSCLC cell proliferation, metastasis, and drug resistance, and thus implying the value of hsa_circ_0023179 as a potential therapeutic target for NSCLC.