Abstract
BackgroundMicroRNAs (miRs) have been shown to be closely associated with the occurrence and development of tumors and to have potential as diagnostic and therapeutic targets. The detection of miRs by noninvasive imaging technology is crucial for deeply understanding their biological functions. Our aim was to develop a novel miR-21-responsive gene reporter system for magnetic resonance imaging (MRI) visualization of the miR-21 dynamics in neuroblastoma.MethodsThe reporter gene ferritin heavy chain (FTH1) was modified by the addition of 3 copies of the sequence completely complementary to miR-21 (3xC_miR-21) to its 3’-untranslated region (3’ UTR) and transduced into SK-N-SH cells to obtain SK-N-SH/FTH1-3xC_miR-21 cells. Then, the antagomiR-21 was delivered into cells by graphene oxide functionalized with polyethylene glycol and dendrimer. Before and after antagomiR-21 delivery, FTH1 expression, MRI contrast and intracellular iron uptake were assayed in vitro and in vivo.ResultsIn the SK-N-SH/FTH1-3xC_miR-21 cells, FTH1 expression was in an “off” state due to the combination of intratumoral miR-21 with the 3’ UTR of the reporter gene. AntagomiR-21 delivered into the cells bound to miR-21 and thereby released it from the 3’ UTR of the reporter gene, thus “switching on” FTH1 expression in a dose-dependent manner. This phenomenon resulted in intracellular iron accumulation and allowed MRI detection in vitro and in vivo.ConclusionMRI based on the miR-21-responsive gene reporter may be a potential method for visualization of the endogenous miR-21 activity in neuroblastoma and its response to gene therapy.
Highlights
MicroRNAs are endogenous single-stranded smallmolecule RNAs that are approximately 20-25 nucleotides long, do not encode proteins, and have a variety of important regulatory functions in cells [1]. miRs can target and bind the 3’-untranslated regions (3’ UTRs) of specific messenger RNAs and induce their translational repression or degradation, thereby decreasing the expression of proteins encoded by the targeted mRNAs
The miR-21-responsive ferritin heavy chain 1 (FTH1) gene reporter system, namely, FTH1-3×C_miR-21, which contained 3 copies of a sequence completely complementary to miR-21 at the 3’ UTR of the FTH1 gene, was successfully constructed. This reporter gene system was transduced into SK-N-SH cells, and SK-N-SH/FTH13×C_miR-21 cells were established
To verify the activity of the reporter gene system, we evaluated FTH1 expression in the SK-N-SH/WT, SK-N-SH/ FTH1 and SK-N-SH/FTH1-3×C_miR-21 cells by qRT-polymerase chain reaction (PCR) and Western blot (WB)
Summary
MicroRNAs (miRs) are endogenous single-stranded smallmolecule RNAs that are approximately 20-25 nucleotides long, do not encode proteins, and have a variety of important regulatory functions in cells [1]. miRs can target and bind the 3’-untranslated regions (3’ UTRs) of specific messenger RNAs (mRNAs) and induce their translational repression or degradation, thereby decreasing the expression of proteins encoded by the targeted mRNAs. MiRs can target and bind the 3’-untranslated regions (3’ UTRs) of specific messenger RNAs (mRNAs) and induce their translational repression or degradation, thereby decreasing the expression of proteins encoded by the targeted mRNAs. MicroRNAs (miRs) are endogenous single-stranded smallmolecule RNAs that are approximately 20-25 nucleotides long, do not encode proteins, and have a variety of important regulatory functions in cells [1]. MiRs can target and bind the 3’-untranslated regions (3’ UTRs) of specific messenger RNAs (mRNAs) and induce their translational repression or degradation, thereby decreasing the expression of proteins encoded by the targeted mRNAs This novel mode of posttranscriptional gene regulation plays an important role in extensive biological processes, such as cell growth, differentiation, apoptosis, tumorigenicity and chemoresistance [2, 3]. Our aim was to develop a novel miR21-responsive gene reporter system for magnetic resonance imaging (MRI) visualization of the miR-21 dynamics in neuroblastoma
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