Serum was collected from 192 patients attending the Outpatient Department of Department of Gastroenterology, Gauhati Medical College and Hospital, Guwahati India with informed consent. Patients with Anti-HCV antibodies and HCV RNA positive were included in the study. All patients were confirmed to have HCV genotype 3 infections. Patients with HIV or HBV co-infection, diabetes, alcoholic liver disease, autoimmune or haematological diseases and abnormal liver ultrasounds were excluded from the study.
72 patients were categorised as CHC (Chronic Hepatitis C), while 64 had liver cirrhosis and 56 were diagnosed with HCC. The presence of liver cirrhosis was ascertained by the occurrence of ascites, esophageal varices, splenomegaly, jaundice, imaging and liver biopsies (when available). HCC was diagnosed with 4-phase multi-detector computed tomography (CT) scan, dynamic contrast-enhanced magnetic resonance imaging (MRI) as mentioned elsewhere 18.
Serum from N=40 sex and age matched healthy individuals were taken as controls. All of the controls were selected on the basis of normal liver function tests, normal ultrasound of liver, and absence of viral markers, autoimmune hepatitis and diabetes. Informed consent was obtained from all individual participants included in the study. The study was approved by Institutional ethics committee of Gauhati Medical College and Hospital.
HCV RNA extraction, quantification and genotyping
QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) was used for extracting HCV RNA from serum of HCV infected patients according to manufacturer’s protocol and stored at −80 °C. HCV viral load was quantified using Artus HCV RG RT-PCR Kit (Qiagen, Valencia, CA, USA) on Rotor-Gene Q 5plex Platform (Qiagen, Valencia, CA, USA). For HCV genotyping 5’UTR-core region was PCR amplified with specific primers and 173bp product was sequenced to determine the HCV genotype.
Total RNA was isolated from whole blood samples using the miRNeasy Minikit (Qiagen, Valencia, CA, USA) followed by miRNA enrichment. Briefly, 100 µl of patient serum was taken with 500 µl of QIAzol lysis reagent and incubated at room temperature for 5 minutes. 140 µl of chloroform was added and vortex to mix for 15 s and incubated at room temperature for 2 minutes. Two separate phases were obtained by centrifuging at 12000 x g at 4⁰C for 15 minutes. For purification of only miRNA Upper aqueous phase was transferred into new sterile tubes and one volume of 70% ethanol was added and mixed by pipetting. Up to 700 µl of the mixture was added onto RNeasy mini spin column placed in a clean 2ml collection tube and centrifuged at 8000xg at room temperature for 15s. 450 µl of 100% ethanol was added to the flow through and mixed thoroughly by vortex. RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA) was used for the purification of miRNA fraction from total RNA. 700 µl of the mix was added onto the RNeasy MinElute spin column placed in a 2ml collection tube, and centrifuged at 8000xg at room temperature for 15s. The flow through was discarded and the process repeated for the whole mix volume. 700 µl buffer RWT was added onto the column and centrifuged at 8000xg for 15s. 500 µl of buffer RPE was added onto the column, centrifuged at 8000xg at room temperature for 15 Secs and the flow through discarded. 500 µl of 80% ethanol was added to the column, centrifuged at 8000xg at room temperature for 2 min and flow through was discarded. A new 2ml spin column was used and again centrifuged at 8000xg at room temperature for 5 min. 14 µl of RNase-free water was added onto the column and centrifuged at 8000xg to elute miRNA enriched fraction.
10ng of miRNA was used in total volume of 20 µl to carry out reverse transcription by incubating for 60min at 37⁰C and 5min at 95⁰C using miScript PCR Starter Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s protocol.
Quantitative real-time PCR
Mature miR-122 expression was carried out by quantitative real time PCR using miScript PCR Starter Kit and miScript SYBR Green PCR Kit (Qiagen, Valencia, CA, USA) with Hs_miR-122a_1 miScript Primer Assay (Qiagen, Valencia, CA, USA) which targets hsa-miR-122-5p. Relative quantification was done using 2-∆∆CT method using U6-snRNA as the internal reference.
Statistical calculations were carried out using Statistical Package for the Social Sciences version 16.0 software (SPSS Inc., Chicago, IL) or GraphPad Prism-5.0 (GraphPad Software, CA, USA). A Chi square test or Fischer exact test was performed to compare categorical data. Comparison of independent samples from two groups was performed using the Mann-Whitney U-test. Spearman’s nonparametric rank test was used to determine nonparametric statistical significance. The correlation coefficients (𝑟) were calculated using Spearman correlation. Receiver operating characteristic (ROC) analysis was performed to assess the diagnostic and prognostic accuracy and area under the curve (AUC) was calculated. AUROC less than 0.60 were considered unreliable for ROC curve. p < 0.05 was considered to be statistically significant.