LncPSCA in the 8q24.3 risk locus drives gastric cancer through destabilizing DDX5 (2021)
Figure 1

Figure 1. Gastric cancer risk SNP rs2978980 alter the enhancer activity of a novel lncRNA lncPSCA in the 8q24 locus.

(A) A schematic view of the lncPSCA and PSCA loci. The risk-associated SNP rs2978980 is shown as the red vertical line. Data from the UCSC Genome Browser, including epigenetic marks for histone H3 lysine 27 acetylation (H3K27ac) are shown.

(B) Abolishment of a RORA binding site in the lncPSCA intronic enhancer by the rs2978980 T>G SNP. The risk allele G of rs2978980 disrupts a RORA binding motif.

(C) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled RORA consensus, rs2978980T or rs2978980G probes and HGC-27 or MGC80-3 nuclear extracts. HGC-27 or MGC80-3 left panel: EMSA with RORA consensus or rs2978980T probes. Lane 1-8: from the first lane at the left side to the right side. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5 and 7, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 1 and 5) or RORA consensus probes (lanes 3 and 7). HGC-27 or MGC80-3 right panel: EMSA with rs2978980T or rs2978980G probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3-5 and 8-10, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 5 and 8), rs2978980G (lanes 3 and 10) or RORA consensus probes (lanes 4 and 9).

(D) Sanger sequencing chromatograms of the SNP region indicated that the MGC80-3 cell line carries the TG genotype and HGC-27 has the GG genotype.

(E) ChIP-qPCR assays using MGC80-3 cells carrying the rs2978980TG genotype. The presence of RORA-binding lncPSCA enhancer region was verified by qPCR with NPAS2 as the positive control. Region a and b: the negative control regions of lncPSCA promoter and intron 1; region c: the lncPSCA enhancer region around rs2978980 SNP; region d: the positive control NPAS2 promoter region. Left subpanel: RORA ChIP-qPCR fragments on lncPSCA (a, b and c) and NPAS2 (d) genes; right subpanel: RORA ChIP-qPCR results of lncPSCA (a, b and c) and NPAS2 (d) genes. Data show one representative example of three biological replicates.

(F) Suppression of RORA using siRNAs (siRORA-1 and siRORA-2) could markedly down-regulate lncPSCA expression in MGC80-3 cells but not in HGC-27 cells. In detail, MGC80-3 and HGC-27 cells were transfected with siRORA-1 and siRORA-2, lncPSCA and RORA expression levels were measured by RT-qPCR and Western Blot.

(G) In gastric cancer or normal tissues of Discovery cohort (n = 96) and Validation cohort (n = 30), there were significantly positive expression correlations between RORA and lncPSCA.

(H) Expression levels of lncPSCA were quantified using RT-qPCR in tumor-normal pairs of both cohorts, respectively. All data of lncPSCA expression were normalized to GAPDH expression levels.

(I) In gastric cancer or normal tissues of both cohorts, there was a significant allele-differential expression between rs2978980G-allele carriers or T-allele carriers.

Data information: The difference between two groups was calculated using Student's t test. One-way ANOVA analysis with Dunnett's test was used for multiple comparisons. The significance of association between gene expression and rs2978980 genotypes was calculated using Spearman's correlation. *P<0.05; **P<0.01; ***P<0.001.