Fig. 1

TRPM2-AS is overexpressed in GBC and is associated with high microvascular density and poor prognosis. A Schematic of CD34 immunohistochemistry and lncRNA sequencing of GBC tissues. The microvascular density of 60 GBC tissues was evaluated by CD34 immunohistochemistry and three GBC tissues with the lowest microvascular density and three with the highest microvascular density were selected for lncRNA sequencing. B, C Heatmap (B) and volcano plot (C) of the differential expression of genes in three GBC tissues with high microvascular density or low microvascular density. D RT-qPCR assessment of the top ten lncRNAs with the most significant changes in RNA expression levels. E Relative expression of TRPM2-AS in GBC tissues and in adjacent normal tissues (left)/in low-density GBC tissues and in high-density GBC tissues (right) was quantified using RT-qPCR. F FISH visualization of TRPM2-AS in normal GBC tissues/low-density GBC tissues/ high-density GBC tissues. Blue fluorescence: DAPI-stained nuclei; red fluorescence: Cy3-labeled TRPM2-AS. Scale bar: 60 μm. G Representative images of the immunohistochemical staining of CD34 expression in tissues with high/low TRPM2-AS expression and Pearson’s correlation analysis of the positive correlation between microvascular density and TRPM2-AS expression. Scale bar: 100 μm. H Kaplan–Meier curve analysis of the correlation between the expression level of TRPM2-AS and overall survival (OS)/recurrence-free survival (RFS) in GBC patients. I Multivariate analysis of the prognostic factors for OS and RFS in GBC patients. J Quantification of TRPM2-AS distribution in nuclear and in cytoplasm. K Representative images of FISH analysis showing the subcellular localization of TRPM2-AS in GBC-SD and NOZ cells. Blue fluorescence: DAPI-stained nuclei; red fluorescence: Cy3-labeled U6, 18S and TRPM2-AS probes. Scale bar: 20 μm. Data were assessed with unpaired Student’s t test or one-way ANOVA and presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001