Differential Expression of Circulating miRNAs Among Healthy, Diseased Controls and HCC Patients—Pilot Study

Abstract

Background and Aim: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Hepatitis B or C is the most common cause of HCC. Majority of the patients of HCC have advanced disease at the time of diagnosis for which locoregional therapy remains the mainstay of treatment. Assessment of post treatment response is based on the imaging modalities like computed tomography or magnetic resonance imaging. There is a need to identify a novel diagnostic and prognostic biomarker for HCC. Circulatory microRNAs (miRNAs) are considered to be potential biomarkers for this purpose due to its ease of detection and high stability in circulation. The aim of the study was to identify the circulatory miRNAs and their differential expression in patients of HCC with Chronic hepatitis B/C (CHB/CHC) patients, diseased (CHB/CHC) and healthy controls. Methods: Serum miRNA profiling was carried out using miScript miRNA PCR Array. This array profiles the expression of a focused panel of 84 miRNAs that differentially expressed in serum. Six endogenous and two exogenous miRNAs were used for normalization. The miRNAs were isolated from serum of HCC patients with CHB/CHC (n = 6), diseased (n = 6) and healthy control (n = 6). These were polyadenylated, reverse transcribed and amplified by real time PCR using universal and miRNA specific primer. PCR array data analysis for differential miRNA expression was carried out using SAbioscience software for scatter plot, heat map, P-value that uses delta Ct values. Results: Out of 84 circulatory miRNAs, we detected 79 miRNAs in healthy and 73 miRNA in serum of HCC patients at cut off of 35 Ct value. Endogenous small nucleolar C/D Box 95 RNA (SNORD95) detection was consistent in serum among six endogenous controls. For normalization, exogenous Cel miR39 and SNORD95 was used. Differential expression between HCC vs healthy and disease control vs healthy had shown down-regulation of 17 and 24 miRNAs; and up-regulation of 10 and 8 miRNAs respectively. A total of 9 serum miRNAs (miR-20a, 885, 148, 30e, 22, 223, 224,130 and 191) were significantly deregulated in HCC patients compared to healthy and disease controls. The fold change of these miRNA expressions varies from 2.06 to 2.44 for upregulation and −2.34 to −2.02 for down regulation. Conclusion: These deregulated miRNAs target genes such as Rho b, mTOR, PTEN, MTPN, MAPK14, cyclin D1 which have significant role in HCC tumor progression and metastasis. The above proposed miRNAs panel including up-and down-regulated miRNAs need validation in large prospective cohort for their utilities as the diagnostic and prognostic markers in HCC patients.