Therapeutic evaluation of microRNAs by molecular imaging

Abstract

MicroRNAs (miRNAs) function as regulatory molecules of gene expression with multifaceted activities that exhibit direct or indirect oncogenic properties, which promote cell proliferation, differentiation, and the development of different types of cancers. Because of their extensive functional involvement in many cellular processes, under both normal and pathological conditions such as various cancers, this class of molecules holds particular interest for cancer research. MiRNAs possess the ability to act as tumor suppressors or oncogenes by regulating the expression of different apoptotic proteins, kinases, oncogenes, and other molecular mechanisms that can cause the onset of tumor development. In contrast to current cancer medicines, miRNA-based therapies function by subtle repression of gene expression on a large number of oncogenic factors, and therefore are anticipated to be highly efficacious. Given their unique mechanism of action, miRNAs are likely to yield a new class of targeted therapeutics for a variety of cancers. More than thousand miRNAs have been identified to date, and their molecular mechanisms and functions are well studied. Furthermore, they are established as compelling therapeutic targets in a variety of cellular complications. However, the notion of using them as therapeutic tool was proposed only recently, given that modern imaging methods are just beginning to be deployed for miRNA research. In this review, we present a summary of various molecular imaging methods, which are instrumental in revealing the therapeutic potential of miRNAs, especially in various cancers. Imaging methods have recently been developed for monitoring the expression levels of miRNAs and their target genes by fluorescence-, bioluminescence- and chemiluminescence-based imaging techniques. Mature miRNAs bind to the untranslated regions (UTRs) of the target mRNAs and regulate target genes expressions. This concept has been used for the development of fluorescent reporter-based imaging strategies to monitor the functional status of endogenous miRNAs, or the respective miRNAs transiently co-expressed in cells. Bioluminescence-based imaging strategies have been used to investigate various stages of miRNA processing and its involvement in different cellular processes. Similarly, chemiluminsecence methods were developed for in vitro miRNA imaging such as monitoring their therapeutic roles in various cancer cell lines.

Keywords: miRNAs; molecular Imaging.

Figure 1

Schematic representation of miRNA processing and various modes of Imaging.

Figure 2

In vivo visualization of expression of pri-miR-23a transcripts and activity of mature forms of miR-23a in nude mice. (A) HeLa cells, (B) 293 cells, and (C) undifferentiated P19 cells were transfected with miR23P639/Fluc and CMV/Gluc/3xPT_mir23, and were grafted (1 Х 10⁷ cells) to right side of mice. The same cells transfected with pGL3_basic and CMV/Gluc were grafted to left side as controls. Quantitative data from imaging in vivo were obtained by ROI analysis and are shown next to each small-animal picture. Gluc images were acquired first with coelenterazine, and then Fluc images were then acquired with D-luciferin. When viewing Fluc images, pay attention to increased bioluminescence on right side of grafted cells expressing pri-miR-23a transcripts. When viewing Gluc images, note the decreased bioluminescence on right side compared with luciferase activity on left-sided CMV/Gluc controls, disclosing mature miR-23a activities. Fluc activities increased in all animals on right side, and Gluc activities decreased in HeLa and P19 cells, but decreased the least in 293 cells transfected with CMV/Gluc/3xPT_mir23 on right side.

Figure 3

Schematic of the reverse complementary multimodal imaging system.

Figure 4

A. Schematic of antagomiR-21 encapsulated PLGA nanoparticles (500µg: 2.5µg RNA) systemically injected in mice bearing SUM159 subcutaneous tumor, by tail vein. B. Fluorescent microscope image of tumor slices of animals sacrificed 24 hrs after the injection of PLGA-NP-Cy5-antagomiR-21 for released Cy5-antagomiR-21. C. Fluorescent microscope image of liver and kidney slices of animals sacrificed 30 mins and 24 hrs after the injection of PLGA-NP-Cy5- antagomiR-21 for released Cy5-antagomiR-21 and corresponding H&E stained slices. D. Optical fluorescence imaging of animals implanted with SUM159 cells pre-exposed with PLGA-NP-Cy5-antagomiR-21 for 24 hrs. The animals were periodically imaged for Cy5- antagomiR-21 signal. The images shown here are animals imaged on day2 and day 10. The images are adjusted in the same scale for comparison.