The expression levels of miR-16, miR-193b, miR-199a, miR-222, and miR-324 in PBMCs were significantly higher in CHC patients compared with healthy controls and significantly different between CHC patients with HCV genotype 1 (GT-1) and non-genotype-1 (non-GT-1). Multivariate logistic regression analysis also showed that patients with high expression levels of the six target miRNAs had an approximately 7.202-fold risk of CHC compared with those with low expression levels of the target miRNAs. We concluded that the expression levels of miR-16, miR-193b, miR-199a, miR-222, and miR-324 target miRNAs in PBMCs of CHC may act as significant risk biomarkers for the development of CHC.
Hepatitis C virus (HCV) infection affects more than 3% of the world’s population [
miRNAs are endogenous, small (approximately 22-nucleotide) noncoding RNAs that downregulate gene expression [
An ideal biomarker of CHC should be released into the systemic circulation or other body fluids, where it can be detected in a blood-based assay or assay of another accessible body fluid [
In this study, we analyzed the expression profiles of miRNAs in PBMCs of CHC patients and healthy controls. Specific expression patterns found in the CHC patients relative to the healthy controls suggested that miRNA activities may potentially contribute to the pathobiology of HCV infection and could be useful diagnostic biomarkers.
Peripheral blood samples were collected from 91 CHC patients who were treated with IFN alpha plus ribavirin at the Department of Internal Medicine, Chung Shan Medical University Hospital, and from 48 healthy controls, between October 2010 and December 2012. Informed written consent was obtained from all of the subjects and/or guardians prior to the use of their blood specimens. The acquisition of the samples and their subsequent examination were approved by the Institutional Review Board of Chung Shan Medical University. None of the participants had a previous history of cancer. The demographic and clinical data of the patients at the time of the sample collection are summarized in Table
The clinical information of CHC patients in this study.
Parameters | |
---|---|
Gender, male/female | 60/31 (2 : 1) |
Age (years) | 55.1 ± 10.6 |
BMI | 24.5 ± 3.4 |
HCV RNA ( |
6.28 ± 0.93 |
AST | 82.7 ± 75.5 |
ALT | 121.3 ± 97.9 |
|
88.6 ± 134.1 |
Cr | 0.79 ± 0.19 |
Cholesterol | 163.1 ± 30.1 |
TG | 136.0 ± 147.8 |
Glucose | 123.7 ± 47.4 |
WBC | 5878.8 ± 1580.8 |
Hb | 14.9 ± 1.5 |
PLT: >15000/<15000 | 53/38 |
Genotype-1/non-1 | 71/20 |
CHC: chronic hepatitis C; BMI: body mass index; AST: aspartate aminotransferase; ALT: alanine aminotransferase; Cr: creatinine; TG: triglyceride; PLT: platelet.
All participants gave their informed consent, and the following criteria were met. (i) All patients had an established diagnosis of CHC. (ii) All patients had an absence of another cause of chronic liver disease and an absence of viral coinfection. (iii) All patients received treatment with either PEG-IFN-
Low-molecular weight RNAs of less than 200 nt were extracted from the PBMCs of 10 CHC patients and a reference pool of 10 paired healthy controls. The total RNA was extracted from the PBMCs of the HCV patients using TRIzol (Invitrogen, Carlsbad, CA), where the TRIzol was supplemented with 0.2 mL of chloroform per 1 mL of reagent, mixed, and centrifuged (15 min; 12,000 g; 4°C). The supernatant was retained. To pellet the RNA, 0.5 mL of isopropanol (per 1 mL of reagent) was added and the mixture was incubated for 10 min at room temperature, followed by centrifugation (15 min; 12,000 g; 4°C). The pellet was washed once in 0.5 mL of 75% ethanol. The final RNA products were quantified by absorbance measurements at 260 nm (A260) and 280 nm (A280). The A260/A280 values were higher than 1.6 for all of the samples. A measure of 5
The detailed methods of isolating short RNAs and the reverse-transcription reaction were the same as those described in Section
All data were analyzed using the Statistical Package for the Social Sciences version 13.0 software (SPSS Inc., Chicago, IL). The Chi-square test (
Previous reports showed that the expression levels of miR-122 were decreased in the liver tissues of CHC patients [
The expression levels of miR-122 in PMBCs of CHC patients and healthy controls were significantly lower than in tumor and nontumor tissues of liver cancer. The miR-122 expression level was detected by real-time PCR.
We analyzed miRNA expression profiles in the PBMCs of 10 CHC patients and 10 paired healthy controls using a miRNAs array. We identified miRNAs that were significantly differentially expressed in the CHC patients and the control group. The results of a global miRNA expression analysis of the PBMCs of the 91 CHC patients compared with the 48 healthy donors are shown in Table
Up- and downexpressed microRNAs in PBMCs of CHC patients compared with the healthy donors.
MicroRNAs | Endogenous control | |||
---|---|---|---|---|
RUN44 | U6B | |||
RQ* |
|
RQ* |
|
|
Downregulated miRNAs | ||||
miR-330-3p | 0.01 |
|
0 |
|
miR-371-3p | 0.02 |
|
0 |
|
miR-501-5p | 0.04 |
|
0 |
|
miR-636 | 0.04 |
|
0 |
|
miR-214 | 0.08 |
|
0 |
|
miR-886-3p | 0.09 |
|
0 |
|
miR-548c-5p | 0.11 |
|
0 |
|
miR-422a | 0.12 |
|
0 |
|
miR-886-5p | 0.24 |
|
0 |
|
miR-545 | 0.27 |
|
0 |
|
miR-326 | 0.29 |
|
0 |
|
miR-9 | 0.29 |
|
0 |
|
miR-579 | 0.38 |
|
0.01 |
|
miR-124 | 0.39 |
|
0.01 |
|
miR-627 | 0.42 |
|
0.01 |
|
miR-449b | 0.5 |
|
— | — |
miR-564 | 0.65 |
|
0 |
|
miR-222* | — | — | 0 |
|
miR-15* | — | — | 0 |
|
miR-16-1* | — | — | 0 |
|
Upregulated miRNAs | ||||
miR-7-1* | 5405.77 | 3.73 | 2.01 | 0.3 |
miR-126* | 2486.35 | 3.4 | 1.3 | 0.11 |
miR-199a-3p | 814.45 | 2.91 | 10.77 | 1.03 |
miR-489 | 592.42 | 2.77 | 7.83 | 0.89 |
miR-194 | 484.64 | 2.69 | 6.41 | 0.81 |
miR-590-5p | 397.96 | 2.6 | 5.26 | 0.72 |
let-7e | 311.16 | 2.49 | 4.11 | 0.61 |
miR-15b | 261.75 | 2.42 | 3.46 | 0.54 |
miR-19a | 245.95 | 2.39 | 3.25 | 0.51 |
miR-19b | 233.54 | 2.37 | 3.09 | 0.49 |
miR-376c | 228.43 | 2.36 | 3.02 | 0.48 |
miR-324-5p | 224.46 | 2.35 | 2.97 | 0.47 |
miR-374a | 218.5 | 2.34 | 2.89 | 0.46 |
miR-27b | 193.3 | 2.29 | 2.56 | 0.41 |
miR-452 | 182.34 | 2.26 | 2.41 | 0.38 |
miR-96 | 176.66 | 2.25 | 2.34 | 0.37 |
miR-210 | 171.29 | 2.23 | 2.26 | 0.35 |
miR-139-5b | 155.02 | 2.19 | 2.05 | 0.31 |
PBMCs: peripheral blood mononuclear cells; CHC: chronic hepatitis C.
*Real-time relative quantification (RQ) values.
Target miRNAs expression in PBMCs of CHC patients with different HCV genotype and healthy controls.
miRNAs | GT-1 | Non-GT-1 | Control |
|
---|---|---|---|---|
miR-16 | 155.065 (10.950)* | 245.167 (25.742) | 13.604 (1.713) | <0.001 |
>1.713 | 54 | 15 | 24 | |
≦1.713 | 12 | 5 | 24 | |
|
0.001 | |||
miR-122 | 0.0016 (0.0002) | 0.0000003 (0) | 0.0031 (0.0004) | <0.001 |
>0.0004 | 17 | 0 | 22 | |
≦0.0004 | 54 | 20 | 26 | |
|
<0.001 | |||
miR-193b | 0.2175 (0.0070) | 0.0068 (0.00076) | 0.0033 (0.00013) | <0.001 |
>0.00013 | 64 | 13 | 24 | |
≦0.00013 | 6 | 7 | 24 | |
|
<0.001 | |||
miR-199a | 1366.614 (513.816) | 95.7169 (33.5522) | 479.1835 (9.4969) | <0.001 |
>9.4969 | 65 | 13 | 24 | |
≦9.4969 | 4 | 7 | 24 | |
|
<0.001 | |||
miR-214 | 159.1452 (0.4719) | 677.42 (0.5559) | 20.2853 (0.2282) | 0.943 |
>0.2282 | 41 | 12 | 24 | |
≦0.2282 | 26 | 8 | 24 | |
|
0.469 | |||
miR-222 | 3.9360 (0.4564) | 2.0039 (0.1394) | 0.4898 (0.0103) | <0.001 |
>0.0103 | 61 | 18 | 23 | |
≦0.0103 | 5 | 2 | 24 | |
|
<0.001 | |||
miR-324 | 49.4397 (2.2015) | 21.9596 (4.2781) | 9.4064 (0.0524) | |
>0.0524 | 53 | 16 | 24 | |
≦0.0524 | 12 | 4 | 24 | |
|
0.001 |
PBMCs: peripheral blood mononuclear cells; CHC: chronic hepatitis C.
*Data were presented by mean (medium).
Target miRNAs expression in PBMCs of CHC patients with different HCV genotype and healthy control groups.
To further confirm whether the changes in the expression levels of these miRNAs could be used as a useful biomarker in HCV infection, we analyzed the association of the miRNA levels and clinical factors (such as ALT, AST, and liver cirrhosis) of the CHC patients. As shown in Table
The association of the targets miRNAs expression levels and clinical factors (such as ALT, AST, and liver fibrosis) of the CHC patients.
Parameters | miR-193b | miR-199a-3p | miR-122 | miR-16 | miR-214 | miR-222 | miR-324-3p |
---|---|---|---|---|---|---|---|
ALT | |||||||
<40 U/L | 0.169 ± 0.838 | 777.105 ± 2178.070 | 0.003 ± 0.010 | 21.762 ± 69.405 | 18.220 ± 65.577 | 0.425 ± 0.954 | 7.771 ± 39.245 |
≧40 U/L | 0.214 ± 1.464 | 1113.280 ± 497.265 | 0.001 ± 0.002 | 212.344 ± 504.989 | 54.410 ± 200.481 | 3.425 ± 11.770 | 26.781 ± 67.231 |
|
0.001 | 0.009 | <0.0001 | <0.0001 | 0.235 | <0.0001 | 0.001 |
AST | |||||||
<40 U/L | 0.027 ± 0.005 | 426.223 ± 1115.870 | 0.003 ± 0.012 | 13.217 ± 40.283 | 17.412 ± 64.851 | 0.496 ± 1.135 | 11.157 ± 48.112 |
≧40 U/L | 0.174 ± 1.297 | 882.580 ± 436.044 | 0.001 ± 0.003 | 168.314 ± 445.087 | 45.757 ± 177.139 | 2.631 ± 10.244 | 20.214 ± 58.935 |
|
<0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.760 | <0.0001 | 0.001 |
Liver cirrhosis | |||||||
No | 0.228 ± 1.410 | 964.452 ± 454.544 | 0.017 ± 0.122 | 197.502 ± 490.549 | 150.749 ± 891.345 | 2.529 ± 5.821 | 45.846 ± 209.718 |
Yes | 0.012 ± 0.029 | 1340.147 ± 440.160 | 0.001 ± 0.003 | 109.524 ± 243.190 | 40.417 ± 79.656 | 6.452 ± 19.406 | 34.221 ± 86.321 |
|
0.221 | 0.800 | 0.866 | 0.996 | 0.054 | 0.584 | 0.508 |
AST: aspartate aminotransferase; ALT: alanine aminotransferase.
Multivariate logistic regression analysis of the risk of CHC.
Parameters | Favorable/unfavorable | OR | 95% CI |
|
---|---|---|---|---|
Gender | Female/male | 0.227 | 0.080–0.648 | 0.006 |
Age | Per year | 1.182 | 1.1062013;1.262 | <0.001 |
BMI | ≧27/<27 | 2.090 | 0.394–11.074 | 0.386 |
Six target miRNAs | High/low | 7.202 | 2.014–25.727 | 0.002 |
Six target miRNAs were including miR-16, miR-122, miR-193b, miR-199a-3p, miR-222, and miR-324-3p.
In CHC patients’ PBMC, the expression levels of miR-16, miR-122, miR-193b, miR-199a-3p, miR-222, and miR-324-3p were increased and miR-122 expression was decreased compared with healthy controls.
Several studies have demonstrated a relationship between HCV and host miRNA, especially miR-122, the most abundant miRNA in the liver [
Changes of microRNAs in the liver have been reported in disease processes such as hepatocarcinogenesis and liver fibrosis [
There is however only limited information about their detection in blood and their correlation with histological disease severity in patients with CHC. Previous studies have elected to use miRNA from serum instead of miRNA from exosome as the candidate for diagnosing diseases [
A previous report showed that after full-length HCV was transfected into HepG2 cells, levels of miR-193b increased, whereas those of the predicted downstream target Mcl-1 gene decreased compared with a parental control [
In conclusion, we found specific expression patterns of miRNAs in CHC patients compared with healthy controls, suggesting the potential contribution of miRNA activities to the pathobiology of HCV infection. Changes in the levels of miRNAs in PBMCs in CHC may act as significant diagnosis biomarkers for CHC.
The authors do not have any commercial or other associations that might pose a conflict of interests.
Ya-Wen Cheng and Chun-Che Lin designed the study and wrote the paper; Chiu-Chun Chang and Wan-Ling Hsieh designed the experiments and prepared the figures; Chun-Che Lin collected the CHC and control samples; Chiu-Chun Chang and Wan-Ling Hsieh analyzed data. All authors gave final approval for the paper to be submitted for publication. Chiu-Chun Chang and Chun-Che Lin equally contributed to this study.
This study was supported by grant of the National Science Council (NSC 100-2314-B-040-012) and Chung Shan Medical University Hospital (CSH-2012-C-016).