Fig. 2.

CircURI1 represses GC metastasis in vitro and in vivo. (A) RT-qPCR analysis of circURI1 and URI1 mRNA expression in AGS cells treated with two independent siRNAs against circURI1. siNC, siRNA with scrambled sequences; siCirc-1 and siCirc-2, two siRNAs against the junction sites of circURI1. (B and C) Wound-healing and Transwell assays of AGS cells treated with siRNAs targeting circURI1. (Scale bars, 100 μm.) (D) Strategy of KO reverse complementary repeats in human URI1 intron 4 using CRISPR-Cas9 in AGS cells. Gel image of PCR products from cell genotyping is performed, and the corresponding amplifications are confirmed by Sanger sequencing. WT, wild-type; KO, deletion of the complementary repeat sequences in human URI1 intron 4. (E) RT-qPCR analysis of circURI1 and URI1 mRNA expression in WT and circURI1 KO AGS cells. (F and G) Wound-healing and transwell assays of WT and circURI1 KO cells. (Scale bars, 100 μm.) (H) Construction of circURI1 overexpression with its endogenous flanking sequences including the complementary Alu element pairs. EV, empty vector; CMV, cytomegalovirus promoter; pA, polyadenylation signal. (I) RT-qPCR analysis of circURI1 and URI1 mRNA expression upon circURI1 overexpression. EV, empty vector. (J and K) Wound-healing and Transwell assays of AGS cells after circURI1 overexpression. EV, empty vector. (Scale bars, 100 μm.) (L) Severe combined immunodeficient mice were administered an intravenous injection of the AGS stable cell line with circURI1 knockdown and the control (n = 5 per group), and sectioning of the lung followed by H&E staining was performed to visualize lung metastasis. Black arrows indicate the metastatic tumor. sh-KD circURI1, stable cell line with lentivirus shRNA to knockdown circURI1; sh-NC, negative control cells for knockdown circURI1. Error bars indicate SEM from three independent experiments. N.S., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by two-tailed Student’s t test.