Abstract
With the advent of next-generation sequencing technology, there is rapidly increasing interest in long noncoding RNAs (lncRNAs). The objectives of this study were to develop a novel lncRNA MALAT1 near-infrared optical probe, to evaluate the characteristics of this optical imaging probe in vitro and to determine whether it can be used for imaging MALAT1 expression in malignant tumours in vivo. Conjugation of Cy5.5 to MALAT1 ASO was accomplished using standard NHS (N-hydroxysuccinimide) ester procedures, and the labelled MALAT1 ASO was purified with a Glen-Pak DNA Purification Cartridge and reversed-phase high performance liquid chromatography (HPLC). The in vitro cellular uptake results showed that the percentage of cell binding increased with an increasing final concentration and increased with increasing incubation time for the MHCC-LM3 tumour cell flow cytometry analyses. in vivo optical imaging exhibited 5’ (Cy5.5)-MALAT1 ASO uptake in the tumour with a maximum at 30 min p.i. that slowly washed out over time. High contrast to normal tissue was gradually observed from 4 h to 48 h p.i. Tumour-to-normal ratios of fluorescence intensities were plotted as a function of time. The in vivo competition assay showed little uptake of the probe into the tumours at any time point, indicating effective competition, selectivity of probe binding and retention by tumours in vivo. Our proposed Cy5.5 labelling of MALAT1 ASO can serve as a potent optical probe for in vivo imaging of tumour expressing MALAT1. Importantly, the successful development of optical probes provides a basis for specific molecular diagnoses in the field of lncRNAs.
Highlights
With the advent of next-generation sequencing technology, there is rapidly increasing interest in long noncoding RNAs
The long noncoding RNA Malat1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1), known as MALAT-1 or nuclear-enriched abundant transcript 2 (NEAT2), is one www.impactjournals.com/oncotarget of the few biologically well-studied long noncoding RNAs (lncRNAs), located on chromosome 11q13, and it was originally found to be overexpressed in early-stage non-small cell lung cancer (NSCLC) [2]
LncRNAs are a group of RNAs that do not encode proteins, and the lengths of these molecules are greater than 200 nucleotides [16]
Summary
With the advent of next-generation sequencing technology, there is rapidly increasing interest in long noncoding RNAs (lncRNAs). Inhibition of MALAT1 expression may suppress cell proliferation, migration, invasion, and the epithelial-mesenchymal transition, and it may induce cell apoptosis and G2/M cell cycle arrest in vitro [6] These associations suggest that targeting MALAT1 may have important clinical implications because it selectively affects cancer cells or residual cancer cells. Gutschner et al [8] reported that injection of antisense oligonucleotide (ASO) into subcutaneous tumours of nude mice can effectively inhibit MALAT1 expression in vivo and block the metastasis of lung cancer cells. Based on these studies, we hypothesised that MALAT1 is a potential target for a specific molecular diagnosis
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