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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1361-1372 |
| Number of pages | 12 |
| Journal | Hepatology Communications |
| Volume | 6 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 2022 |
ASJC Scopus subject areas
- Hepatology
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Transcriptomic Analysis Reveals the Messenger RNAs Responsible for the Progression of Alcoholic Cirrhosis. / Yang, Zhihong; Han, Sen; Zhang, Ting et al.
In: Hepatology Communications, Vol. 6, No. 6, 06.2022, p. 1361-1372.Research output: Contribution to journal › Article › peer-review
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TY - JOUR
T1 - Transcriptomic Analysis Reveals the Messenger RNAs Responsible for the Progression of Alcoholic Cirrhosis
AU - Yang, Zhihong
AU - Han, Sen
AU - Zhang, Ting
AU - Kusumanchi, Praveen
AU - Huda, Nazmul
AU - Tyler, Kelsey
AU - Chandler, Kristina
AU - Skill, Nicholas J.
AU - Tu, Wanzhu
AU - Shan, Mu
AU - Jiang, Yanchao
AU - Maiers, Jessica L.
AU - Perez, Kristina
AU - Ma, Jing
AU - Liangpunsakul, Suthat
N1 - Funding Information: Supported by the National Institutes of Health (K01AA26385 to Z.Y., K01DK112915 to J.L.M.), Indiana University Research Support Fund (Grant IU RSFG to Z.Y.), Ralph W. and Grace M. Showalter Research (to Z.Y.), Indiana University School of Medicine Dean’s Scholar in Medical Research (to S.L.), Indiana Institute for Medical Research (to Z.Y., P.K.), National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK107682 to S.L.), National Institute on Alcohol Abuse and Alcoholism (R01 AA025208, R21AA024935, U01 AA026917, UH2/UH3 AA026903, U01AA026817 to S.L.), and Veterans Administration Merit Award (1I01CX000361 to S.L.). Funding Information: HCs were recruited from outpatient clinics at the Roudebush Veterans Administration Medical Center (VAMC), Indianapolis, IN. Patients with AC were those who attended liver clinics at Indiana University Hospital or Roudebush VAMC. These patients had a history of alcohol consumption averaging at least 80 g/day (for men) or 50 g/day (for women) for at least 10 years. This criterion is based on epidemiological evidence of the relationship between alcohol consumption and cirrhosis. The diagnosis of AC has been described.(12,14,20,21) In brief, it was made using radiographic imaging, history of portal hypertension complications, or biopsy-proven cirrhosis, with the exclusion of other known causes of liver diseases, such as viral hepatitis B or C, autoimmune liver disease, hemochromatosis, or Wilson disease. The study was approved by the Institutional Review Board (IRB) at Indiana University–Purdue University Indianapolis (IUPUI) and Roudebush VAMC Research and Development Program. Written informed consent was obtained from each participant. HCs were recruited from outpatient clinics at the Roudebush Veterans Administration Medical Center (VAMC), Indianapolis, IN. Patients with AC were those who attended liver clinics at Indiana University Hospital or Roudebush VAMC. These patients had a history of alcohol consumption averaging at least 80 g/day (for men) or 50 g/day (for women) for at least 10 years. This criterion is based on epidemiological evidence of the relationship between alcohol consumption and cirrhosis. The diagnosis of AC has been described.(12,14,20,21) In brief, it was made using radiographic imaging, history of portal hypertension complications, or biopsy-proven cirrhosis, with the exclusion of other known causes of liver diseases, such as viral hepatitis B or C, autoimmune liver disease, hemochromatosis, or Wilson disease. The study was approved by the Institutional Review Board (IRB) at Indiana University–Purdue University Indianapolis (IUPUI) and Roudebush VAMC Research and Development Program. Written informed consent was obtained from each participant. Baseline demographics, clinical characteristics, and laboratory tests of the study cohort were obtained at the time of enrollment, as reported.(14) Baseline Child-Pugh and Model for End-Stage Liver Disease (MELD) scores were calculated for patients with AC to further categorize them into compensated (Child-Pugh class A, AC1) and decompensated (Child-Pugh class B or C, relating to AC2 or AC3, respectively) liver diseases. Peripheral blood was collected from venipuncture at enrollment into the PAXgene Blood RNA tube (Qiagen; catalog #762165). The sample was gently inverted and stored at −80°C within 2 hours of collection. Peripheral blood RNAs were extracted using a QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. Blood RNAs from HCs (n = 4) and patients with AC (n = 12, with 4 patients for each Child-Pugh classification) were subjected to global transcriptomic profiling using the Arraystar human microarray, version 3.0 (Arraystar, Rockville, MD). The original raw data were uploaded to GitHub (https://github.com/yangjoe-iu/ALD_Blood-RNASeq). Quantitative polymerase chain reaction (qPCR) was used to validate the expression of mRNAs in a separate cohort of 17 HCs and 48 patients with AC. The baseline demographic and clinical characteristics are provided in Supporting Table S1. The primer’s sequences are provided in Supporting Table S2. We also collected de-identified human liver specimens from a different cohort of patients with AC for mRNA analysis. The collection was performed under the IRB-approved protocol at the IUPUI. The raw intensities of samples were grouped and uploaded to the online software (https://www.metaboanalyst.ca/MetaboAnalyst/home.xhtml) for the statistical analysis using algorithms they provided.(22) After the integrity check, the data were normalized by a pooled sample from HCs and logarithmically transformed. The partial least squares-discriminant analysis (PLS-DA) was performed using the plsr function provided by the R pls package. Classification and cross-validation were conducted using the caret package. Differentially expressed mRNAs were identified through an adjusted P value of 0.05 and at least 2-fold differential gene expression to generate the volcano plot. The heat map and correlation analysis were generated using the default setting. Statistical analyses of differentially expressed mRNAs among HCs and patients with AC with different severities stratified by Child-Pugh classification were performed using one-way analysis of variance analysis (ANOVA). GSEA was conducted using the GSEA software downloaded from https://www.gsea-msigdb.org/gsea/index.jsp. Raw intensities data were processed following the guidelines for RNA sequencing (RNA-Seq) data sets with GSEA.(23) Bubble plots were generated using the ggplot2 package in R. Enrichment plots and heat maps were provided from the GSEA software. Functional protein association networks were analyzed using STRING (https://string-db.org). Venn diagrams were generated using the Venn Diagram package in R. The list of unique differential gene expression from each group was submitted to DAVID (https://david.ncifcrf.gov) for the gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Bubble plots were generated by the R ggplot2 package. We used descriptive statistics, such as mean, SEM, and frequencies (percentages). Statistical analysis was performed using a Student t test for unpaired data or ANOVA to determine the mean difference. We used survival analyses to determine the prognostic significance of the selected mRNA transcripts and the survival outcome in subjects with AC. The survival analysis was conducted to determine the significance of the variable of interest and time to mortality as calculated by the date of enrollment until the time of death. Patients who survived were censored. Cox regression models were used to determine the hazard ratio (HR) and its 95% confidence interval (CI). The cut-off P value < 0.05 was considered statistically significant. Publisher Copyright: © 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.
PY - 2022/6
Y1 - 2022/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85124541540&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85124541540&partnerID=8YFLogxK
U2 - 10.1002/hep4.1903
DO - 10.1002/hep4.1903
M3 - Article
C2 - 35134262
AN - SCOPUS:85124541540
SN - 2471-254X
VL - 6
SP - 1361
EP - 1372
JO - Hepatology Communications
JF - Hepatology Communications
IS - 6
ER -