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

Abstract

Oncogenic fusion gene Echinoderm Microtubule-associated protein-Like 4-Anaplastic Lymphoma Kinase (EML4-ALK) contributes to tumorigenesis of a subset of non-small cell lung cancer (NSCLC). Recently, we demonstrated that F-circEA-4a, a tumor-promoting circular RNA (circRNA) generated from the back-splicing of EML4-ALK variant 3b (v3b), is a novel liquid biopsy biomarker for NSCLC. However, circRNAs produced from EML4-ALK gene and their roles in NSCLC are not well-characterized. Here, we identify another EML4-ALK-v3b-derived circRNA, F-circEA-2a, harboring "AA" (rather than "AAAA" in F-circEA-4a) motif at the junction site. F-circEA-2a mainly locates in the cytoplasm and promotes cell migration and invasion, but has little effect on cell proliferation. Moreover, F-circEA-2a exists in tumor, but not in the plasma of NSCLC patients with EML4-ALK fusion gene, further supporting the significant diagnostic value of F-circEA-4a for EML4-ALK-positive NSCLC. This work finds a novel oncogenic circRNA generated from EML4-ALK fusion gene, highlighting the pivotal role of circRNA in EML4-ALK-positive NSCLC development.

Keywords: Cell migration/invasion; Circular RNA; EML4-ALK; Non-small cell lung cancer.

Fig. 1

Identification of F-circEA-2a in NSCLC. a Schematic representation of F-circEA-2a/4a generated from EML4-ALK gene. The divergent primers (F1/R1) were used to detect F-circEA-2a/4a. b RT-PCR and Sanger sequencing of F-circEA-2a in H2228 cells. The arrow indicates the junction site of F-circEA-2a. c Measurement of F-circEA-2a/4a in H2228 cells by dot blot hybridization (left) and qPCR (right, normalized to GAPDH mRNA). d qPCR analysis of F-circEA-2a after nucleus/cytoplasm fractionation of H2228 cells. GAPDH mRNA and U6 RNA were used to indicate the cytoplasmic and nuclear RNA, respectively. Western blotting confirmed good nucleus/cytoplasm fractionation. Data are shown as the mean ± SD. See Additional file 1: Table S1 for the information of the primers and oligonucleotides in this figure. The experimental protocols are described in the Additional file 2

Fig. 2

F-circEA-2a promotes cell migration and invasion in NSCLC cells. a Schematic representation of F-circEA-2a-expressing plasmid with the flanking sequence of laccase 2 to facilitate RNA circularization. b Agarose gel electrophoresis and Sanger sequencing of RT-PCR products from H1299 cells transfected with F-circEA-2a-expressing plasmid and empty vector. c.d Nucleus/cytoplasm fractionation and qPCR analysis of F-circEA-2a in A549 (c) and H1299 (d) cells. Western blotting against laminA/C and tubulin showed efficient nucleus/cytoplasm fractionation. Data are shown as the mean ± SD. e.f.g MTT (e.f) and colony formation assays (g) in A549 and H1299 cells transfected with F-circEA-2a-expressing plasmid or empty vector (Ctrl). h.i Representative images of Transwell (h) and wound-healing assays (i) in A549 and H1299 cells transfected with F-circEA-2a-expressing plasmid or empty vector (Ctrl). See Additional file 1:Table S1 for the information of the primers and oligonucleotides in a and b. The experimental protocols are described in the Additional file 2

Fig. 3

Identification of F-circEA-2a in NSCLC patients’ samples. a Primers used to detect EML4-ALK mRNA and F-circEA-2a/4a. The convergent primers (F2/R2) were used to detect EML4-ALK mRNA, the divergent primers F3/R3 and F4/R4 were used to detect F-circEA-4a/2a, respectively. b.c Agarose gel electrophoresis and Sanger sequencing of RT-PCR products from tumor tissues (b) or plasma (c) of NSCLC patients with (patients 1–3) or without (patients 4–5) EML4-ALK variant 3b translocation. See Additional file 1:Table S1 for the information of the primers and oligonucleotides in this figure. The experimental protocols are described in the Additional file 2