A growing number of lncRNAs have been identified recently. Their expressions are usually tissue-specific [25]. They regulate gene expression through one or more of several mechanisms that include the induction of chromatin remodeling, transcriptional interference, the regulation of alternative splicing, and the alteration of protein localization or activity (such as scaffolding) in the assembly of multiple protein complexes [26].
Tumorigenesis and progression are results of gene expression disorders, usually involving the activation of oncogenes or the inactivation of tumor suppressor genes. LncRNAs, which can function as both oncogenes and tumor suppressor genes, may become future biomarkers and consequential therapeutic targets in the clinical management of lung cancer patients.
Carcinogenic lncRNAs that promote the growth of cancer cells, increase their invasiveness, and inhibit apoptosis, such as HOTAIR1, MALAT1 and PVT1, function as oncogenes in non-small cell lung cancer. These lncRNAs are upregulated in NSCLC and promote growth, proliferation, and invasion [26–28]. CBR3-AS1 is highly expressed and functions as an oncogene in many cancer types [9–11, 29–32]. For example, CBR3-AS1 expression is significantly increased in hepatocellular carcinoma and is correlated with tumor size, vascular invasion, and advanced TNM stage, and may be an independent indicator of poor prognosis [11]. CBR3-AS1 expression is significantly increased in colon cancer tissues and cell lines, and is correlated with the depth of invasion, lymph node metastasis, and TNM stage [31]. CBR3-AS1 expression is significantly upregulated in retinoblastoma tissue and cell lines, and correlated with disease progression [29]. CBR3-AS1 is also highly expressed in osteosarcoma tissue and cell lines, and associated with Enneking stage, distant metastasis, histologic grade, and prognosis [10]. CBR3-AS1 expression is also significantly increased in glioma tissue and cell lines compared to adjacent non-cancerous tissues and normal cells, and correlated with shorter OS and PFS [30]. In contrast, the CBR3-AS1 levels in breast cancer tissues and serum are significantly lower than those of healthy tissues and controls [32]. These findings indicate that CBR3-AS1 has regulatory functions that may differ among cancer types. Our bioinformatics analysis results also suggested that CBR3-AS1 expression varied among specific cancers. In addition, our research suggested that CBR3-AS1 was highly expressed in NSCLC, and is higher in patients with malignant phenotypes. Our experimental results showed that CBR3-AS1 could inhibit NSCLC apoptosis, and promote cell proliferation, and promote NSCLC cellular transition into the mitosis stage (G2-M). Moreover, our murine xenograft model demonstrated that CBR3-AS1 overexpression promoted in vivo NSCLC cell growth and proliferation. These results are consistent with other reports that implicate CBR3-AS1 as a cancer gene in NSCLC.
CeRNA regulatory mechanisms mediated by lncRNAs have recently become a major research focus. The ceRNA hypothesis was introduced by Professor Pandolfi in 2011 [33]. Endogenous RNAs, such as mRNA, lncRNA, and pseudogenes, which contain the same miRNA binding sites, may inhibit miRNA attachment to target genes through competitive binding, obviating the silencing effect of miRNA on its target gene. This hypothesis has been confirmed in many important biological processes, such as tumorigenesis, which suggests that lncRNAs in NSCLC can bind to miRNAs in a targeted manner, thus preventing miRNAs from regulating target genes and their corresponding pathways.
MiRNAs silence mRNAs by pair-binding with the 3' UTR region of the target sequence, while lncRNAs affect microRNA-induced gene silencing through binding with miRNA response elements [33]. Qu R et al. found that ZEB1-AS1 was upregulated in NSCLC cells, and that the upregulated ZEB1-AS1 negatively regulated miR-409-3p through the mechanism of ceRNA, thereby affecting the expression and function of its target gene ZEB1 [34]. LncRNA CASC11 is highly expressed in lung cancer tissues and can promote lung cancer development by combining with miRNA-302 to upregulate CDK1 protein expression [35]. Our study found that in NSCLC, cytoplasmic CBR3-AS1 functioned as a ceRNA to provide an oncogenic microenvironment. Moreover, our bioinformatics analysis suggested that CBR3-AS1 and miR-409-3p attached at specific binding sites, and their expressions in NSCLC cells were negatively correlated, suggesting that both might exert regulatory functions through ceRNA-based mechanisms. Our dual luciferase reporter gene experiment confirmed that CBR3-AS1 and miR-409-3p could combine, and our qRT-PCR results confirmed their mutual inhibitory effect.
By evaluating miR-409-3p expression in NSCLC tissue from 48 patients, we found that miR-409-3p was significantly lower in NSCLC cells, and that low miR-409-3p expression was associated with higher tumor stages and worse prognosis. These findings suggested that miR-409-3p might function as a cancer suppressor in NSCLC, which is consistent with the results reported by Qu R et al. [36]. Previous studies have reported that miR-409-3p can inhibit breast cancer by downregulating Akt1 [37]. Wu et al. also confirmed that miR-409-3p can function as a tumor suppressor gene in osteosarcoma by downregulating ZEB1 expression [38]. miR-409-3p may also delay the proliferation of tongue squamous cell carcinoma by inhibiting RDX, thus reducing migration and invasion [39]. Our results indicated that miR-409-3p was poorly expressed in NSCLC, and that miR-409-3p could inhibit CBR3-AS1 expression in NSCLC, thus antagonizing the CBR3-AS1-mediated promotion of proliferation and invasion. In another word, CBR3-AS1 can function as a ceRNA in NSCLC by binding miR409-3p.
SOD1 overexpression can promote cell cycling and inhibit apoptosis, thus accelerating tumor proliferation and metastasis. In addition, our previous results confirmed that SOD1 was one of the target genes of miR-409-3p and its function in NSCLC is regulated by miR-409-3p [18]. miR-409-3p mimic reduced the mRNA and protein expressions of SOD1 in NSCLC cells, SOD1 expression in CBR3-AS1 + miR-409-3p mimic was reserved, indicating that CBR3AS-1 expression antagonized the inhibitory effect of miR-409-3p on SOD1 expression. In contrast, reduced CBR3-AS1 expression could reverse the promotion of SOD1 by an miR-409-3p inhibitor. These results indicate that CBR3-AS1 can combine competitively with miR-409-3p as a ceRNA to regulate the expression of its target gene SOD1 in NSCLC.
Radiotherapy plays a vital role in the treatment of NSCLC. Consequently, the enhancement of NSCLC radiosensitivity is imperative. Previous studies have confirmed that lncRNA could affect the radiosensitivity of various cancer cell types through the mechanism of ceRNA [40]. Wang et al. found that inhibiting PVT1 expression might improve the radiosensitivity of NSCLC through the miR-424-5p/PVT1/CARM1 signaling pathway [40–42]. LincRNA ROR can reduce DNA repair capacity through the LincRNA-ROR/miR-145/Rad18 pathway, thereby enhancing the radiosensitivity of hepatoma cells [41]. Elevated GAS5 increases RECC expression by downregulating miR-21, and accelerates the apoptosis of esophageal squamous cell carcinoma cells after radiotherapy, thus improving radiosensitivity [42]. Therefore, we hypothesized that CBR3-AS1 could affect the radiosensitivity of NSCLC cells through the CBR3-AS1/miR-4093p/SOD1 pathway. Our results confirmed that in NSCLC cells after irradiation, CBR3-AS1 knockdown promotes apoptosis and thereby decreases radioresistance, Furthermore, CBR3-AS1 can regulate miR-409-3p levels, and thereby influence the SOD1 expression after radiotherapy. In addition, our clone forming assay results also showed that CBR3-AS1 expression could eliminate the miR-409-3p-mediated inhibition of SOD1 upregulation and radioresistance. The accumulation of ROS caused by radiotherapy is the primary determinant of radiosensitivity [43]. SOD1 expression can significantly reduce ROS accumulation after radiotherapy, thereby attenuating the ROS-induced cascade that ends in apoptosis [42]. Based on our results combined with those of other studies, it is reasonable to conclude that CBR3-AS1 knockdown can inhibit SOD1 expression in NSCLC cells after radiotherapy through the CBR3-AS1/miR-409-3p/SOD1 pathway, thus leading to ROS accumulation and thereby enhancing radiosensitivity.
DNA repair capacity is also an important determinant of radiosensitivity. The lncRNA-mediated alteration of DNA repair capacity has been the focus of radiosensitivity research in recent years. PVT1knockdown can induce apoptosis of nasopharyngeal carcinoma cells after radiotherapy by affecting the DNA repair pathway [44]. Wang et al. found that lncRNA-LINP1 knockdown may affect Ku80 and DNA-PKs protein expressions and delay the repair of DNA double-strand breaks after radiotherapy, thus increasing apoptosis after ionizing radiation. Our study also found that CBR3-AS1 knockdown increased γH2AX foci significantly and also promoted apoptosis after irradiation, suggesting that apoptosis was accelerated by impaired DNA repair capacity.