With the advent of next-generation sequencing technology, there is rapidly increasing interest in long noncoding RNAs (lncRNAs). Researchers from Zhejiang University set out 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. This 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.
In vivo dynamic imaging study of the targeting specificity of 5’ (Cy5.5)-MALAT1
In vivo dynamic imaging study of the targeting specificity of 5’ (Cy5.5)-MALAT1 ASO to MHCC-LM3 cell in the right axilla in xenograft mice at 10 min (A), 30 min (B), 1 h (C), 2 h (D), 4 h (E), 8 h (F), 24 h (G), and 48 h (H) after intravenous administration. Quantification and kinetics of the in vivo targeting characteristics of 5’(Cy5.5)-MALAT1 ASO (I). C. Tumour/non-tumour ratios at 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h (J).