Circular RNA F-circEA-2a derived from EML4-ALK fusion gene promotes cell migration and invasion in non-small cell lung cancer

We recently identified the existence of EML4-ALK-derived circRNA F-circEA-4a. To evaluate whether EML4-ALK fusion gene could produce other circRNAs, we deeply Sanger-sequenced the reverse transcription PCR (RT-PCR) products using divergent F1/R1 primers (Fig. 1a) from H2228 (harboring EML4-ALK-v3b) and A549 cells (without the fusion gene, negative control). Besides F-circEA-4a, we found the presence of another circRNA named as F-circEA-2a with “AA” motif at the junction site in the RNA sample from H2228 cells, which was treated with RNase R to remove linear RNAs (Fig. 1b). F-circEA-2a, about 0.55 kb in length, is the back-splicing product between 5′-head of EML4 exon4 and 3′-tail of ALK exon22 (Fig. 1a). Dot blot hybridization using ³²P-labeled probes across the respective junction sites indicated that the enrichment of F-circEA-2a in H2228 cells was less than that of F-circEA-4a (Fig. 1c, left), which was further confirmed by quantitative PCR (qPCR) (Fig. 1c, right). Subcellular fractionation and qPCR assays showed that F-circEA-2a was mainly located in the cytoplasm (Fig. 1d). These data demonstrated the presence of another circRNA F-circEA-2a produced from EML4-ALK fusion gene.

To investigate cellular function of F-circEA-2a, we constructed F-circEA-2a-expressing plasmid, in which F-circEA-2a sequences (red arrows) and the flanking sequences (green arrows, favorable for circRNA formation) were cloned into pCDH-CMV-MCS-EF1-puro vector (Fig. 2a). Sanger sequencing of RT-PCR products from the cells transfected with F-circEA-2a-expressing plasmid indicated that F-circEA-2a was successfully expressed and correctly back-spliced (Fig. 2b). The ectopically expressed F-circEA-2a was predominantly located in the cytoplasm of both H1299 and A549 cells (Fig. 2c and d), same as the endogenous one in H2228 cells.

Both MTT and colony formation assays suggested that ectopically expressed F-circEA-2a had no significant effect on cell proliferation in A549 and H1299 cells (Fig. 2e, f and g). However, F-circEA-2a enhanced cell migration and invasion in both cells by Transwell assays and wound-healing experiments (Fig. 2h and i). Because both F-circEA-4a and F-circEA-2a can promote cell migration and their difference is that F-circEA-4a has extra AA dinucleotides at the junction site, so they may exert the cellular function through the same mechanism. Emerging evidence shows that circRNAs play an important role under physiological or pathological conditions. For example, circMTO1, down-regulated circular RNA in hepatocellular carcinoma (HCC), suppresses HCC progression by acting as the sponge of oncogenic miR-9 to promote p21 expression [9]. These data reveal a novel circRNA F-circEA-2a enhances cell migration and invasion without any influence on cell proliferation, enlarging the understanding of circRNA as oncogenic molecule participating tumor development.

Compared to linear RNAs, the covalently-bonded circRNAs are relatively resistance to RNase R digestion. Some circRNAs (such as circ-N4BP2L2, circ-GSE1) originated from cancer tissues may enter the circulating system and stably be present in intercellular fluid [10]. Our recent work indicates that F-circEA-4a is a potential liquid biopsy biomarker for EML4-ALK-positive NSCLC patients [8]. To investigate the clinical significance of F-circEA-2a, we measured F-circEA-2a levels in tumor tissues and plasma of three NSCLC patients with EML4-ALK-v3b translocation and two NSCLC patients without such fusion gene. The convergent primers (F2/R2) were also used to detect EML4-ALK fusion mRNA. To improve specificity, we designed nested divergent primers (Fig. 3a, first round PCR primers: F1/R1; nested PCR primers for F-circEA-4a: F3/R3, nested PCR primers for F-circEA-2a: F4/R4). The R3 and R4 primers were designed to cross the junction sites of F-circEA-4a and F-circEA-2a, respectively, facilitating specific detection of these circRNAs (Fig. 3a). RT-PCR and Sanger sequencing data demonstrated that F-circEA-4a, F-circEA-2a and EML4-ALK mRNA were all detected in the tumor tissues of NSCLC patients with EML4-ALK-v3b translocation, whereas they were absent in the patients without such fusion gene (Fig. 3b), indicating the specific existence of F-circEA-4a and F-circEA-2a in the EML4-ALK-positive NSCLC tumors. However, in contrast to F-circEA-4a, we failed to detect F-circEA-2a in the plasma of EML4-ALK-positive NSCLC patients (Fig. 3c), which may be caused by low enrichment or a discrepant junction motif of F-circEA-2a. These results provided a rebuttal that F-circEA-4a is a promising biomarker for EML4-ALK-v3b translocation, highlighting its uncontroversial role in the liquid biopsy of EML4-ALK-positive NSCLC patients.