Circ-HuR suppresses HuR expression and gastric cancer progression by inhibiting CNBP transactivation

摘要

Circ-HuR is down-regulated and decreases HuR expression in gastric cancer. a RT-PCR assay with divergent primers indicating the detection of three circRNAs derived from HuR in AGS cells. b PCR assay with divergent and convergent primers showing the amplification of circRNAs from cDNA or genomic DNA (gDNA) of gastric cancer cell lines, while β-actin was used as a negative control. c Schematic illustration indicating the generation of hsa_circ_23897 and hsa_circ_0049027 from its host gene, and validation by Sanger sequencing. d Real-time qRT-PCR assay showing the relative levels (normalized to β-actin) of hsa_circ_23897 or hsa_circ_0049027 in the peritumor and tumor tissues of gastric cancer (n = 81). e Dual-luciferase assay indicating the promoter activity of HuR in AGS and MKN-45 cells stably transfected with empty vector (circ-Mock), hsa_circ-23897 (circ_23897), linear circ-23897 (lin_23897), circ-HuR, or linear circ-HuR (lin-HuR). f and g Real-time qRT-PCR (f, normalized to β-actin, n = 5) and western blot (g) assays revealing the transcript and protein levels of HuR and its downstream target genes in AGS and MKN-45 cells stably transfected with circ-Mock, circ-23897, lin_23897, circ-HuR, or lin-HuR. h and i Western blot (h) and immunofluorescence (i) assays showing the cytoplasmic and nuclear accumulation of HuR in AGS and MKN-45 cells stably transfected with circ-Mock, circ-HuR, or lin-HuR. Nuclei were stained by DAPI (blue). Scale bar, 10 μm. j RNA-FISH assay indicating the nuclear localization of circ-HuR in AGS cells using an antisense probe (green), while sense probe was used as a negative control. U1 and GAPDH were applied as positive controls. Scale bar, 10 μm. Student’s t-test and ANOVA analyzed the difference in (d-f). *P < 0.01 vs. circ-Mock. Data are shown as mean ± SEM (error bars) and representative of three independent experiments in (a-c) and (e-j)