Circular RNA F-circSR derived from SLC34A2-ROS1 fusion gene promotes cell migration in non-small cell lung cancer

Abstract

Cancer-associated chromosomal translocations are reported to generate oncogenic circular RNA (circRNA), contributing to tumorigenesis. The fusion gene SLC34A2-ROS1 (solute carrier family 34 member 2 and ROS proto-oncogene 1) plays an important role in non-small cell lung cancer (NSCLC) progression. However, whether SLC34A2-ROS1 gene can produce circRNA remains unknown. Here, we identified two novel circRNAs (F-circSR1 and F-circSR2) generated from SLC34A2-ROS1 fusion gene, while F-circSR1 has higher expression than F-circSR2. Functional studies through gain- and loss-of-function strategies showed that both F-circSRs promote cell migration in lung cancer cells, whereas they have little effect on cell proliferation. Using the minigene GFP reporter assay, we verified that the flanking complementary sequences with canonical splicing sites are essential for F-circSR biogenesis. Therefore, our findings demonstrate the oncogenic role of F-circSR in NSCLC and highlight its therapeutic potential.

Keywords: Cell migration; Circular RNA; NSCLC; SLC34A2-ROS1.

Fig. 1

Identification of F-circSR in HCC78 cells. a~b Schematic representation of F-circSR generated from SLC34A2-ROS1 fusion gene. E means exon. c Agarose gel electrophoresis and Sanger sequencing of RT-PCR products from HCC78 cells, the arrows indicate SLC34A2-ROS1 fusion sites. d Identification of F-circSR in HCC78 cells by RT-PCR and Sanger sequencing. The arrows indicate F-circSR junction sites

Fig. 2

F-circSR promotes cell migration in NSCLC cells. a Schematic representation of pCRE5-F-circSR-expressing plasmid. b Agarose gel electrophoresis and Sanger sequencing of RT-PCR products from HEK293T cells transfected with F-circSR-expressing plasmid or empty vector. c Agarose gel electrophoresis of RT-PCR products from A549 and H1299 cells stably expressing F-circSR. d Representative images of Transwell migration assays and quantification in A549 and H1299 cells with or without F-circSR overexpression. e~f Representative images of wound healing assays in H1299 and A549 cells with or without F-circSR overexpression. g~h F-circSR knockdown attenuates the migratory ability in F-circSR1 (g) or F-circSR2 (h) overexpressing H1299 cells. SR1: F-circSR1; SR2: F-circSR2; E: exon

Fig. 3

The flanking complementary sequences are important for F-circSR biogenesis. a Bioinformatics analysis of the cis-elements in the flanking introns of F-circSR. CS: upstream and downstream sequences of F-circSR; SA, splicing acceptor; SD, splicing donor. The circle denotes the junction site. The grey boxes called P indicate complementary sequences. b Schematic representation of GFP-based reporters to examine essential elements for circRNA formation. c~e GFP detection through fluorescence microscope (c), flow cytometry (d) and Western blot (e)