MiRNA122a regulation of gene therapy vectors targeting hepatocellular cancer stem cells.

Bijay Dhungel,Christopher J Layton,Jason C Steel,Charmaine A Ramlogan-Steel

doi:10.18632/oncotarget.25280

Abstract

In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3’-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs.

Highlights

Hepatocellular carcinoma (HCC) is the most prevalent form of primary malignancy in the liver and is the third leading cause of cancer related death worldwide [1, 2]
Primary human hepatocytes were found to contain an average of 5655 copies of miRNA122a per 1000 copies of control while the expression of miRNA122a was not detected in HCC cell lines Hep3B, SKHep1, PLC/PRF/5, and SNU423 (Figure 1)
When screening our panel of HCC cell lines, we found that the reduction of miRNA122a, while occurring in most lines, did not occur in all cell lines with HuH7 being the outlier

Summary

Introduction

Hepatocellular carcinoma (HCC) is the most prevalent form of primary malignancy in the liver and is the third leading cause of cancer related death worldwide [1, 2]. Effective therapeutic treatment options for HCC are extremely limited. Less than 20% of HCC patients are diagnosed with early stage tumors [4]. Treatments for the vast majority of patients with unresectable tumors are largely palliative with recurrence often observed after therapeutic interventions [5]. Accumulating evidences suggest that a small subset of HCC possess these stem like properties and are important targets for therapeutic interventions [8]. In this study we use a targeted gene therapy approach to target the CSC population. Designing strategies to deliver therapeutic www.oncotarget.com genes to the hepatocellular CSC population is a new and important approach for HCC therapy

Methods

Results

Conclusion