Transcriptomic Analysis Reveals the Messenger RNAs Responsible for the Progression of Alcoholic Cirrhosis

Abstract

Alcohol-associated liver disease is the leading cause of chronic liver disease. We hypothesized that the expression of specific coding genes is critical for the progression of alcoholic cirrhosis (AC) from compensated to decompensated states. For the discovery phase, we performed RNA sequencing analysis of 16 peripheral blood RNA samples, 4 healthy controls (HCs) and 12 patients with AC. The DEGs from the discovery cohort were validated by quantitative polymerase chain reaction in a separate cohort of 17 HCs and 48 patients with AC (17 Child-Pugh A, 16 Child-Pugh B, and 15 Child-Pugh C). We observed that the numbers of differentially expressed messenger RNAs (mRNAs) were more pronounced with worsening disease severity. Pathway analysis for differentially expressed genes for patients with Child-Pugh A demonstrated genes involved innate immune responses; those in Child-Pugh B belonged to genes related to oxidation and alternative splicing; those in Child-Pugh C related to methylation, acetylation, and alternative splicing. We found significant differences in the expression of heme oxygenase 1 (HMOX1) and ribonucleoprotein, PTB binding 1 (RAVER1) in peripheral blood of those who died during the follow-up when compared to those who survived. Conclusion: Unique mRNAs that may implicate disease progression in patients with AC were identified by using a transcriptomic approach. Future studies to confirm our results are needed, and comprehensive mechanistic studies on the implications of these genes in AC pathogenesis and progression should be further explored.

FIG. 1

Statistical analysis of differentially expressed mRNAs in peripheral blood of patients with AC. (A) Volcano plot displaying differentially expressed peripheral blood mRNAs in (B). Red dots represent significantly up‐regulated (right) and down‐regulated (left) mRNAs. Blue dots represent genes with no significant changes in gene expression (either −2 > FC < 2 or P ≥ 0.05). Bottom panels show expression data of genes with the most significant changes in gene expression between HC (green boxes) and AC (red boxes) groups. (B) Heat map of the top 25 most significantly changed genes compared between HC and AC groups (HC, n = 4; AC, n = 12). Abbreviations: STOX1, storkhead box 1; XRCC6BP1, XRCC6 binding protein 1.

FIG. 2

GSEA of DEGs compared patients with AC to HCs. (A,B) Bubble plot of the selected top (A) 10 up‐ and (B) down‐regulated gene sets in peripheral blood mRNA from patients with AC. The x axis represents the ES, and the size of the bubble represents the number of genes. Coloration from red to yellow represents the P value from low (red) to high (yellow). (C,D) ES plots for the indicated (C) up‐regulated or (D) down‐regulated gene sets. The green line is the running ES of the profile. The score at the peak indicates the ES score for that gene set. Vertical lines refer to individual genes and the position in a gene set. Coloration from red to blue represents the ranked gene list from up‐regulated (red) to down‐regulated (blue) genes in patients with AC compared to HCs. (E,F) Heat map of top 20 genes contributing to the enrichment of their respective pathway. A,C,E consist of analyses for the up‐regulated gene sets, while B,D,F represent analyses for the down‐regulated gene sets. Abbreviations: E2F, E2 transcription factor; ES, enrichment score; mTORC1, mammalian target of rapamycin complex 1; Tgf, transforming growth factor.

FIG. 3

DEG analysis in three study cohorts of human AC compared with HCs. The 12 human AC samples were divided into three groups based on the baseline Child‐Pugh and MELD scores. (A) Volcano plots show DEGs for AC1 versus HCs (upper), AC2 versus HCs (middle), or AC3 versus HCs (bottom). (B) Graphic of the number of up‐regulated or down‐regulated genes. (C) Score plot of the PLS analysis of the samples from HC, AC1, AC2, and AC3. Each dot represents one sample. The highlighted ellipses represent the coverage of 95% of the subjects within each group. (D) Heat map of the top 25 most significantly changed genes based on P value (n = 4/group). (E) Normalized reads of selected genes in peripheral blood samples from different groups. Red boxes, HC; green boxes, AC1; blue boxes, AC2; light blue, AC3; n = 4/group. Abbreviations: LRRC37A3, leucine‐rich repeat‐containing protein 37A3; PPRC1, peroxisome proliferator‐activated receptor gamma coactivator‐related protein 1; TRRAP, transformation/transcription domain‐associated protein; RBM22, RNA binding motif protein 22.

FIG. 4

Unique DEGs in each Child‐Pugh class compared with HCs. (A) Venn diagram indicating the number of unique DEGs in each group and overlapping DEGs among groups. Blue circle, AC1 versus HCs; red circle, AC2 versus HCs; green circle, AC3 versus HCs. (B‐D) Bubble plots of pathway analysis using DAVID (https://david.ncifcrf.gov) for unique DEGs in (B) AC1 versus HCs, (C) AC2 versus HCs, and (D) AC3 versus HCs. The x axis represents the enrichment score. Size of the bubble represents the numbers of genes in each pathway. Coloration from red to green represents the P value from low (red) to high (green). Abbreviations: N/A, not applicable; ubi, ubiquitin.

FIG. 5

Validation of selected genes in peripheral blood in HCs and ACs. (A‐C) qPCR was used to detect selected DEGs in HCs (n = 17), AC1 (n = 17), AC2 (n = 16), and AC3 (n = 15). Each dot representing an individual sample. *P < 0.05, ***P < 0.001, ****P < 0.0001 versus HCs. Abbreviations: ns., not significant; Rel., relative.

FIG. 6

Relative mRNA levels in HC and AC liver tissues. qPCR was used to detect selected genes in HCs (n = 8) and patients with AC (n = 12). *P < 0.05, **P < 0.01, versus HCs. Each dot representing an individual sample.