Background
Methods
Patients
Liver histopathology
Clinical parameters
First biopsy | Second biopsy | |
---|---|---|
Demographic data | ||
Number of patients | 36 | 36 |
Gender (Male/Female) | 20/16 | 20/16 |
Histological findings | ||
Steatosis (0/1/2/3) | 0/8/20/8 | 1/15/15/5 |
Lobular inflammation (0/1/2/3) | 4/14/13/5 | 2/20/14/0 |
Ballooning (0/1/2) | 2/21/13 | 4/23/9 |
Stage (0/1/2/3/4) | 5/13/6/11/1 | 2/16/5/12/1 |
Matteoni classification (type 1/2/3/4) | 1/1/3/31 | 0/1/1/33 |
NAFLD activity score (≤2/3, 4/≥5) | 2/12/22 | 4/16/16 |
Clinical parameters | ||
Age (years) | 49 (24-69) | 59 (26-70) |
Body mass index (kg/m2) | 25.6 (20.5-36.5) | 25.5 (19.1-33.6) |
Serum aspartate aminotransferase (IU/l) | 61 (19-152) | 37 (14-132) |
Serum alanine aminotransferase (IU/l) | 104 (28-303) | 49 (8-304) |
Gamma-glutamyl transpeptidase (IU/l) | 65 (17-505) | 43 (9-359) |
Serum albumin (g/dl) | 4.1 (3.4-4.9) | 4.0 (2.8-4.5) |
Platelet count (×103/mm3) | 210 (117-389) | 207 (111-296) |
Fasting plasma glucose (mg/dl) | 94 (65-142) | 106 (73-278) |
Uric acid (mg/dl) | 6.0 (1.8-9.5) | 5.9 (1.5-9.2) |
Total cholesterol (mg/dl) | 209 (130-290) | 202 (131-270) |
Triglycerides (mg/dl) | 137 (62-254) | 131 (54-295) |
High-density lipoprotein cholesterol (mg/dl) | 44 (29-85) | 45 (27-73) |
Low-density lipoprotein cholesterol (mg/dl) | 129 (66-205) | 123 (64-175) |
Non high-density lipoprotein cholesterol (mg/dl) | 162 (95-228) | 150 (95-219) |
Serum ferritin (μg/l) | 265 (<10-1,472) | 202 (13-1,018) |
Hyaluronic acid (μg/l) | 34 (8-561) | 30 (0-222) |
Serum miR-122 (fold change) | 1.03 (0.13-7.63) | 0.66 (0.03-7.65) |
Measurement of serum miR-122
(A) Preparation of the RT reaction master mix | |||
Component | Master mix volume per 15-μL reactiona | ||
100 mM dNTPs (with dTTP) | 0.15 μL | ||
MultiScribeTM Reverse Transcriptase, 50 U/μL | 1.00 μL | ||
10× Reverse Transcription Buffer | 1.50 μL | ||
Rnase Inhibitor, 20 U/μL | 0.19 μL | ||
Nuclease-free water | 4.16 μL | ||
Total volume | 7.00 μL | ||
(B) Performance of reverse transcription | |||
Use the following parameter values to program the thermal cycler: | |||
Step | Time | Temperature | |
Hold | 30 min | 16 °C | |
Hold | 30 min | 42 °C | |
Hold | 5 min | 85 °C | |
Hold | ∞ | 4 | |
(C) Preparation of the qPCR reaction mix | |||
Pipet the following components into each tube: | |||
Component | Single reaction | ||
TaqManⓇ Small RNA Assay (x20) | 1.00 μL | ||
Product from RT reaction | 1.33 μL | ||
TaqManⓇ Universal PCR Master Mix II (x2) | 10.00 μL | ||
Nuclease-free water | 7.67 μL | ||
Total volume | 20.00 μL | ||
(D) Setting up the experiment or plate documentation and running the plate | |||
In real-time PCR system software, create an experiment or plate document on real-time PCR system using the following parameters: | |||
•Run Mode: Standard | |||
•Sample Volume: 20 μL | |||
•Thermal Cycling Conditions: | |||
Enzyme Activation | PCR CYCLE (40 cycles) | ||
Step | HOLD | Denature | Anneal/extend |
Temperature | 95 °C | 95 °C | 60 °C |
Time | 10 min | 15 s | 60 s |
Statistical analysis
Results
Histopathological changes
Steatosis scores | ||||||
Scores at second biopsy | ||||||
0 | 1 | 2 | 3 | Total | ||
Scores at first biopsy | ||||||
1 | 0 | 5 | 3 | 0 | 8 | |
2 | 1 | 7 | 11 | 1 | 20 | |
3 | 0 | 3 | 1 | 4 | 8 | |
Total | 1 | 15 | 15 | 5 | 36 | |
Ballooning scores | ||||||
Scores at second biopsy | ||||||
0 | 1 | 2 | Total | |||
Scores at first biopsy | ||||||
0 | 0 | 2 | 0 | 2 | ||
1 | 1 | 17 | 3 | 21 | ||
2 | 3 | 4 | 6 | 13 | ||
Total | 4 | 23 | 9 | 36 | ||
Lobular inflammation scores | ||||||
Scores at second biopsy | ||||||
0 | 1 | 2 | 3 | Total | ||
Scores at first biopsy | ||||||
0 | 0 | 2 | 2 | 0 | 4 | |
1 | 0 | 8 | 6 | 0 | 14 | |
2 | 2 | 7 | 4 | 0 | 13 | |
3 | 0 | 3 | 2 | 0 | 5 | |
Total | 2 | 20 | 14 | 0 | 36 | |
Stage scores | ||||||
Scores at second biopsy | ||||||
0 | 1 | 2 | 3 | 4 | Total | |
Scores at first biopsy | ||||||
0 | 1 | 4 | 0 | 0 | 0 | 5 |
1 | 1 | 8 | 2 | 2 | 0 | 13 |
2 | 0 | 3 | 2 | 1 | 0 | 6 |
3 | 0 | 1 | 1 | 8 | 1 | 11 |
4 | 0 | 0 | 0 | 1 | 0 | 1 |
Total | 2 | 16 | 5 | 12 | 1 | 36 |
Association of serum miR-122 level with histopathological features
Association of changes in clinical parameters with histopathological scores
ΔSteatosis | ΔBallooning | ΔLobular inflammation | ΔStage | |||||
---|---|---|---|---|---|---|---|---|
r
|
P
|
r
|
P
|
r
|
P
|
r
|
P
| |
ΔAge | 0.4 | 0.011 | 0.3 | 0.049 | 0.3 | 0.055 | 0.4 | 0.026 |
ΔBody mass index | 0.3 | 0.045 | 0.3 | 0.045 | 0.6 | <0.001 | 0.5 | 0.004 |
ΔSerum aspartate aminotransferase | 0.4 | 0.018 | 0.3 | 0.126 | 0.6 | <0.001 | 0.6 | <0.001 |
ΔSerum alanine aminotransferase | 0.4 | 0.018 | 0.3 | 0.108 | 0.5 | 0.001 | 0.3 | 0.054 |
ΔGamma-glutamyl transpeptidase | 0.1 | 0.555 | 0.3 | 0.099 | 0.3 | 0.043 | 0.3 | 0.044 |
ΔSerum albumin | -0.1 | 0.748 | -0.1 | 0.393 | -0.3 | 0.110 | -0.2 | 0.165 |
ΔPlatelet count | 0.1 | 0.740 | 0.0 | 0.918 | -0.2 | 0.145 | -0.1 | 0.416 |
ΔFasting plasma glucose | 0.0 | 0.913 | 0.2 | 0.390 | 0.3 | 0.102 | 0.3 | 0.100 |
ΔUric acid | -0.1 | 0.671 | 0.1 | 0.539 | 0.2 | 0.374 | 0.3 | 0.089 |
ΔTotal cholesterol | 0.1 | 0.636 | 0.3 | 0.096 | 0.0 | 0.992 | 0.1 | 0.546 |
ΔTriglycerides | 0.2 | 0.213 | 0.1 | 0.530 | 0.2 | 0.195 | 0.4 | 0.031 |
ΔHigh-density lipoprotein cholesterol | 0.1 | 0.738 | 0.1 | 0.670 | -0.3 | 0.056 | -0.2 | 0.218 |
ΔLow-density lipoprotein cholesterol | 0.0 | 0.942 | 0.2 | 0.248 | 0.0 | 0.855 | 0.0 | 0.849 |
ΔNon high-density lipoprotein cholesterol | 0.1 | 0.675 | 0.3 | 0.109 | 0.1 | 0.597 | 0.1 | 0.385 |
ΔFerritin | 0.3 | 0.048 | 0.3 | 0.063 | 0.6 | <0.001 | 0.4 | 0.007 |
ΔHyaluronic acid | 0.2 | 0.366 | 0.4 | 0.036 | 0.3 | 0.075 | 0.5 | 0.002 |
Serum miR-122 ratioa | 0.5 | 0.001 | 0.2 | 0.226 | 0.6 | <0.001 | 0.5 | 0.002 |