Fig. 1

Low circ-hnRNPU expression is associated with poor prognosis of gastric cancer. a, Flowchart (left panel) and RT-PCR assay with divergent primers (right panel) indicating the identification of detectable circRNA (asterisk) generated from hnRNPU in gastric cancer AGS cells. b, PCR assay indicating the amplification of circ-hnRNPU with divergent or convergent primers from cDNA or gDNA of gastric cancer cell lines AGS and NCI-N87, while β-actin was used as a negative control. c, Schematic illustration showing the genomic location and composition of circ-hnRNPU generated from its host gene, and validation by Sanger sequencing. d, Northern blot assay using a junction-specific antisense probe indicating the existence of 597-nt circ-hnRNPU in indicated cell lines. e, RNA-FISH using CY3-labeled antisense junction probe showing the localization of circ-hnRNPU (red, arrowheads) in NCI-N87 cells, with or without RNase R digestion (3 U/mg), while sense probe, GAPDH, or U1 were applied as negative or positive controls. Scale bar: 10 μm. f, Real-time qRT-PCR (normalized to β-actin) indicating the distribution of GAPDH, U1, and circ-hnRNPU in the cytoplasmic and nuclear fractions of MKN-45 and AGS cells stably transfected with empty vector (mock) or circ-hnRNPU (n = 5). g, Real-time qRT-PCR assay indicating the levels of circ-hnRNPU (normalized to β-actin, n = 4) in cultured GES-1 and cancer cells. h, Real-time qRT-PCR (left panel, normalized to β-actin) assay indicating the circ-hnRNPU levels in normal gastric mucosa (n = 40) and gastric cancer tissues (n = 81). Kaplan–Meier curves (right panel) indicating the survival of gastric cancer patients (n = 81) with low or high expression of circ-hnRNPU (cutoff value = 0.58). i, MTT colorimetric assay showing the viability of AGS and NCI-N87 cells stably transfected with mock, circ-hnRNPU, linear circ-hnRNPU (lin-hnRNPU), scramble shRNA (sh-Scb), sh-circ-hnRNPU #1, or sh-circ-hnRNPU #2. j, Western blot assay revealing the hnRNPU protein levels in AGS and NCI-N87 cells stably transfected with mock, circ-hnRNPU, lin-hnRNPU, sh-Scb, sh-circ-hnRNPU #1, or sh-circ-hnRNPU #2. Student’s t test or analysis of variance compared the difference in g-i. *P < 0.05. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a, b, d-g, i and j