Investigation of the Extracellular Vesicles as a Liquid Biopsy Based Biomarkers for Viral Induced Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is globally the second leading cause of cancer deaths after lung cancer. Various risk factors associated with HCC includes chronic infections with hepatitis B and C viruses, nonalcoholic steatohepatitis, alcohol, obesity, diabetes, exposure to aflatoxin and smoking while cirrhosis plays a primary role in HCC development. The high prevalence of viral hepatitis and consequently HCC in Pakistan makes the need to diagnose the disease at its early stage and treat the patient a prime importance. Presently, diagnostic methods and therapies against HCC have been comparatively improved but the survival of HCC patients still remains a major issue. Some of the improved diagnostic methods come with a limitation of false positive or false-negative thus cannot be relied on. This brings in a dire need of development of new diagnostic markers that could detect HCC at early stages Recently, cell to cell communication via Extracellular Vesicles (EVs) has drawn attention of many as it has been found to be involved in HCC progression. Increased number of EVs are released in HCC with altered cargo in comparison to normal cells. As a result, role of EVs in HCC indicates that the increased number of these vesicles can serve as a biomarker for HCC progression and metastasis. The aim of the study was to provide basis for the introduction of a non-invasive and effective procedure for HCC diagnosis using EVs. In the first part of the current study mRNA derived from EVs extracted from HCC patients as compared to healthy control was observed. HCC patients’ samples were collected from Holy family Hospital (HFH), Rawalpindi, Pakistan and Military Hospital (MH), Rawalpindi, Pakistan. Angiopoietin-like protein-3 (ANGPTL3), SH3 domain-binding glutamic acid-rich-like protein 3 (SH3BGRL3) and Interferon-induced transmembrane protein 3 (IFITM3) were selected as potential biomarkers. The selected genes are well known for their involvement in HCC progression, metastasis and inflammation and their presence in EVs was confirmed from the EVs database, Vesiclepedia. Transcriptional analysis of the selected genes depicted that in the EVs from HCC patients, the expression level of ANGPTL3, SH3BGRL3 and IFITM3 was significantly increased by 2.62, 4.3 and 9.03 folds, respectively. Significant alteration in mRNA levels of the selected biomarkers reveals their potential as a possible biomarkers for diagnosing HCC at its early stages. In the second part of the study, EVs from human plasma of HCC patients were incubated with Huh7 cell lines to confirm their intercellular communication property. EVs signaling downstream targets of each selected biomarker showed a significant expected difference in expression. ANGPTL3 and its target AKT showed a significant increase of 2.62 & 4.1 folds, respectively. SH3BGRL3 was significantly increased by 4.3 folds while tumor necrosis factor-α (TNF-α) showed a fold change of 1.46 with a comparatively low significance. This was because of the inhibitory effect of SH3BGRL3 gene on its target TNF-α gene. Expression of IFITM3 and matrix metalloproteinase-9 (MMP-9) significantly increased with a fold change of 9.03 & 5.05, respectively. Findings of the current study shows that the selected biomarkers showed a promising result and can serve as a biomarker for viral induced HCC diagnosis. This is because the mRNA levels of each selected biomarker showed a significance change in HCC patients as compared to healthy controls. Furthermore, it can be suggested through in-vitro study that EVs through their downstream signaling contribute in HCC progression, inflammation and metastasis.