Fig. 2

Identification of the circular structure and clinical features of circNRIP1. a. We confirmed the head-to-tail splicing of circNRIP1 in the circHIPK3 RT–PCR product by Sanger sequencing and also determined its genomic size and sequence as reported in the CircBase database. b. We performed a qRT-PCR assay with specially designed divergent and convergent primers and discovered that circNRIP1, rather than linear-NRIP1 or actin, could resist digestion by RNase R. c We detected the expression levels of the back-spliced and canonical forms of NRIP1 in the presence or absence of RNase R supplementation in cDNA and gDNA from GC cells by PCR and an agarose gel electrophoresis assay. d We found that circNRIP1 was more stable than linear NRIP1 in GC cells under treatment with actinomycin D (a transcription inhibitor). (e, f Patients who had high levels of circNRIP1 within their GC tissues had a significantly shorter overall survival (median survival of 21 months vs 51 months; P = 0.0019, log-rank test) and disease-free survival (median survival of 18 months vs 49 months; P = 0.0004, log-rank test). All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001