The current prospective study involved 150 patients out of 2140 patients who attended the tropical medicine, hepatology, oncology, and general medicine outpatient clinics in the Alexandria University Hospital (Alexandria, Egypt) in the period from December 2017 till January 2020. The control group involved 50 apparently healthy volunteers.
The proposal was explained to all participants, and they signed a written consent. The proposal was accepted by the committee of ethics (Faculty of medicine, University of Alexandria).
Exclusion criteria included patients with coinfections with hepatitis B virus (HBV) or HIV, organ transplantation, autoimmune disease or use of immunosuppressant or antiviral drugs, diabetes mellitus, Schistosoma, and other malignant comorbidities.
All participants were subjected to full history taking, thorough clinical examination, laboratory investigations including complete blood picture, liver transaminases (alanine aminotransferase (ALT), and aspartate aminotransferase (AST)), serum albumin, total bilirubin, and alpha-fetoprotein, and abdominal ultrasonography.
Two hundred participants were divided into three groups: group I, 100 patients with HCV-related liver cirrhosis; group II, 50 patients with early-stage hepatocellular carcinoma (HCC); and group III, 50 healthy controls.
Ultrasound examinations were performed by experienced ultra-sonographers in the Radiology Department of the main Alexandria University Hospital. Items to be observed in ultrasound examination were determination of the liver size, nodularity of the liver surface, the coarseness of the parenchyma, size of lymph nodes around the hepatic artery, patency and flow of veins and arteries, and probable hepatocellular carcinoma.
HCV antibody testing was done by using a commercial recombinant immunoblot assay and confirmed by real-time quantitative HCV RNA PCR (more than 15 IU/ml).
All the HCC patients were diagnosed by abdominal ultrasound and alpha-fetoprotein . Triphasic CT scan examination and Barcelona clinic liver cancers (BCLC)  staging was done for the selection of group II patients. Based on BCLC; patients at a very early stage (stage 0) and early stage (stage A) were selected as candidates for study with single tumors less than 2 cm or multinodular tumor less than 3 cm in size, with the absence of clinically relevant portal hypertension.
Real-time quantitative PCR for miR-331-3p and miR-23b expression levels
Total RNA including microRNA was immediately isolated from plasma samples using miRNeasy Mini Kit (Qiagen, Maryland, USA) according to manufacturers’ instructions. The concentration and purity of the extracted total RNA were assisted using NanoDrop2000 Spectrophotometer (Thermo Scientific, USA). Single-stranded cDNA was synthesized from purified RNA samples using the Taqman miRNA reverse transcription Kit (Applied Biosystems, USA) for RNA reverse transcription according to the manufacturer’s protocol. The real-time amplification was performed using TaqMan MicroRNA assays for miR-331-3p, miR-23-3p, and TaqMan Fast Advanced Mater Mix (Applied Biosystems, USA) on the RotorGene Q Real-Time PCR System (Qiagen, Germany). RNU6 was used as endogenous references; its expression was stable in all the samples and independent of the analyzed variables. The relative expression levels were determined using the 2−ΔΔCT method.
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). The Kolmogorov- Smirnov was used to verify the normality of distribution of variables. Comparisons between groups for categorical variables were assessed using the Chi-square test. ANOVA was used for comparing the four studied groups and followed by post hoc test (Tukey) for pairwise comparison. While Kruskal-Wallis test was used to compare different groups for abnormally distributed quantitative variables and followed by post hoc test (Dunn’s for multiple comparisons test) for pairwise comparison. The significance of the obtained results was judged at the 5% level.