RNA Sequencing Revealed Signals of Evolution From Gallbladder Stone to Gallbladder Carcinoma

Abstract

Gallbladder stone is a major risk factor for gallbladder carcinoma (GBC), while there is still a controversy whether period of follow-up since newly diagnoses of asymptomatic gallstones increases the risk of GBC. In this study, 10 GBC patients and 30 patients with gallstones were admitted to our hospital. Patients with gallstones were divided into 3 groups according to the follow-up time, involving 10 patients with follow-up period of 1-3 years (GS3 group), 10 patients with follow-up period of 5-10 years (GS5 group), and 10 patients with follow-up period of more than 10 years (GS10 group). Tumor and para-tumor tissues of GBC patients, and gallbladder tissues of gallstone patients were collected. RNA sequencing was performed on the 50 samples. Besides, 1,704 differentially expressed genes (DEGs) were identified in tumors compared with para-tumor tissues of 10 GBC patients, which were enriched into some well-known cancer-related pathways, such as PI3K-Akt, mitogen-activated protein kinase (MAPK), Ras, and Wnt signaling pathways, and the most significant pathway was neuroactive ligand-receptor interaction. Patients with gallstones with periods of follow-up equal to 1-3 and > 10 years showed to have higher cancer risk than those with 5-10 years. ALPP and GPR87 are potential biomarkers for predicting cancer risk in patients with gallstones. The in vitro results revealed that GPR-87 can promote the proliferation, migration, and invasion of GBC cells. Herein, we explored the relationship between GBC patients and patients with gallstones with different periods of follow-up in transcriptome level.

Keywords: ALPP; GPR87; RNA sequencing; follow-up time; gallbladder carcinoma; gallbladder stone.

Figure 1

Flowchart of the study process.

Figure 2

Differentially expressed genes (DEGs) in tumor tissues were compared with para-tumor tissues of 10 GBC patients. (A) Volcano plot of the differential expression analysis of genes. (B) Heatmap of unsupervised hierarchical clustering of DEGs in all samples of GBC patients. (C) The top 15 pathways of DEGs of GBC were identified by KEGG enrichment.

Figure 3

(A) Counts of DEGs in GBC tissues were compared with gallbladder tissues of GS3, GS5, and GS10, respectively. (B) A Venn diagram of DEGs for making comparison among three groups of T_vs_GS3, T_vs_GS5, and T_vs_GS10. (C) Principal Component Analysis (PCA) of four groups of samples, including GBC tissues and gallbladder tissues of GS3, GS5, GS10. GS3: patients with gallstones with period of follow-up equal to 1–3 years; GS5: patients with gallstones with period of follow-up equal to 5–10 years; GS10: patients with gallstones with period of follow-up of >10 years.

Figure 4

(A) Taking GS5 as control, counts of DEGs were compared in tumor tissues of GBC, and gallbladder tissues of GS3 and GS10. (B) A Venn diagram of DEGs for making comparison among the three groups. (C) Functional analysis of 40 common DEGs in the three groups by GO enrichment. (D,E) Expression levels of ALPP and GPR87 in the four groups.

Figure 5

Weighted gene correlation network analysis (WGCNA). (A) Clustering of 30 samples and clinical traits of gallstones. The color intensity was proportional to period of follow-up of gallstones, size of stones, and the existence of multiple stones. (B) Clustering tree based on modules' eigengenes. (C) Associations between modules and period of follow-up of gallstones. Each cell contains the corresponding correlation and P-value, and cells were color-coded by correlation. (D,E) Results of GO and KEGG enrichment analyses of genes in pink module.

Figure 6

Inhibition of GPR87 expression attenuates the proliferation of gallbladder cancer cells. (A) Western blotting was used to detect the expression level of GPR87 in gallbladder cancer cells. (B) Quantitative PCR was employed to detect the interference efficiency of GPR87. (C) CCK8 assay was utilized to detect the proliferation ability of gallbladder cancer cells and their control cells after GPR87 knockdown. (D) Clone formation assay was applied to detect the cloning ability of gallbladder cancer cells and their control cells after GPR87 knockdown. (E) Edu experiment detected the proportion of gallbladder cancer cells and their control cells in S-phase after GPR87 knockdown (*P < 0.05).

Figure 7

Inhibition of GPR87 expression attenuates metastatic ability of gallbladder cancer cells. (A) Transwell assay was applied to detect the migration ability of shGPR87 SGC-996 and control cells. (B) Transwell assay was used to detect the migration ability of shGPR87 GBC-SD and control cells. (C) Matrigel invasion assay was applied to detect the invasion ability of shGPR87 SGC-996 and control cells. (D) Matrigel invasion assay was employed to detect the invasion ability of shGPR87 GBC-SD and control cells (*P < 0.05).