Noninvasive Visualization of MicroRNA-16 in the Chemoresistance of Gastric Cancer Using a Dual Reporter Gene Imaging System

Fu Wang,Xinxing Song,Jie Tian,Kaichun Wu,Xiaoyuan Chen,Jimin Liang,Weidong Yang,Jing Wang,Xiujuan Li,Feng Cao,Jing Xin,Shenxu Wang,Javier S Castresana

doi:10.1371/journal.pone.0061792

Abstract

MicroRNAs (miRNAs) have been implicated to play a central role in the development of drug resistance in a variety of malignancies. However, many studies were conducted at the in vitro level and could not provide the in vivo information on the functions of miRNAs in the anticancer drug resistance. Here, we introduced a dual reporter gene imaging system for noninvasively monitoring the kinetic expression of miRNA-16 during chemoresistance in gastric cancer both in vitro and in vivo. Human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) genes were linked to form hNIS/Fluc double fusion reporter gene and then generate human gastric cancer cell line NF-3xmir16 and its multidrug resistance cell line NF-3xmir16/VCR. Radioiodide uptake and Fluc luminescence signals in vitro correlated well with viable cell numbers. The luciferase activities and radioiodide uptake in NF-3xmir16 cells were remarkably repressed by exogenous or endogenous miRNA-16. The NF-3xmir16/VCR cells showed a significant increase of 131I uptake and luminescence intensity compared to NF-3xmir16 cells. The radioactivity from in vivo 99mTc-pertechnetate imaging and the intensity from bioluminescence imaging were also increased in NF-3xmir16/VCR compared with that in NF-3xmir16 tumor xenografts. Furthermore, using this reporter gene system, we found that etoposide (VP-16) and 5-fluorouracil (5-FU) activated miRNA-16 expression in vitro and in vivo, and the upregulation of miRNA-16 is p38MAPK dependent but NF-κB independent. This dual imaging reporter gene may be served as a novel tool for in vivo imaging of microRNAs in the chemoresistance of cancers, as well as for early detection and diagnosis in clinic.

Highlights

Gastric cancer remains the first leading cause of cancer death in China and the fourth most common malignancy worldwide despite a dramatic decrease in its mortality and morbidity over the past three decades [1]
A scrambled nucleotide sequence of similar length to 3xmir16 was inserted at the 39UTR of Human sodium iodide symporter (hNIS)/firefly luciferase (Fluc) fusion gene to obtain a control construct
Establishment of gastric cancer cell lines stably expressing hNIS and Fluc genes To generate a fusion reporter gene (GV260-hNIS/Fluc, referred to NF-empty), the hNIS cDNA was cloned into a lentivirus vector (GV260-Fluc-puro) encoding a Fluc gene to generate a fusion protein, which was under the control of ubiquitin promoter

Summary

Introduction

Gastric cancer remains the first leading cause of cancer death in China and the fourth most common malignancy worldwide despite a dramatic decrease in its mortality and morbidity over the past three decades [1]. The MDR has been considered as a multifactorial phenomenon involving several main mechanisms, including increased metabolism of drugs, decreased uptake of water-soluble drugs, altered drug targets, reduced intracellular drug concentration by efflux pumps, altered cell cycle checkpoints and induced emergency response genes to impair apoptotic pathways, etc [5]. It was reported that two miRNAs, miRNA-15b and miRNA-16, were differentially expressed in a multidrug-resistant human gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901 [11]. It was conducted only at the in vitro level and could not reflect the in vivo information on the functions of miRNAs in the anticancer drug resistance

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Results

Conclusion