Exosomes-mediated synthetic Dicer substrates delivery for intracellular Dicer imaging detection

Highlights

• A fluorescent labeling probe was designed with high selectivity for intracellular Dicer detection.

• Exosomes were used as nanocarriers for gene probe delivery.

• After packaging the reliable probe into exosomes, the assembled nanosystem could detect intracellular Dicer.

Abstract

Ribonuclease Dicer initiates gene-silencing process by cleaving exogenously long RNA duplexes into small interfering RNA (siRNA) or endogenous precursor microRNAs (pre-miRNAs) into mature miRNAs. It holds great promise in cancer diagnosis and therapeutics due to its molecular ruler role. However, the intracellular Dicer detection remains a key challenge and Dicer related gene therapy has never been explored. In this study, we design a fluorescent labeling Dicer substrate and effectively deliver it into cell by exosomes derived from the target parent cells for intracellular Dicer expression level monitor and gene therapy. Using pre-miRNA let-7a as a model, the Dicer substrates with two terminals labeled with fluorescent and quencher group respectively was obtained by T4 RNA mediated ligase reaction from two short RNA sequences. Then, the substrate was packaged into exosomes by electroporation and delivered to target cells for intracellular dicer imaging detection. After packaging substrates into exosomes with little immunogenicity and good innate biocompatibility by electroporation and delivered to target cells, the Dicer mediated substrate cleavage was effectively monitored by the fluorescence recovery, providing a powerful tool for Dicer analysis. Importantly, the cleaved product exhibited significant suppression toward tumor cell growth and regulated cancer cells cycle. This work might open a new avenue for Dicer analysis and Dicer-related clinical application.

Introduction

Dicer is an endoribonuclease in the RNase III family, mainly exists in cytoplasm, and highly conserved in evolution (Lee et al., 2004; Park et al., 2011). It cleaves the precursor microRNAs (pre-miRNAs) to mature miRNAs and exogenously long RNA duplexes into small interfering RNA (siRNA) to mediate gene silence (Hoehener et al., 2018; Hyun et al., 2014; Macrae et al., 2006). Thus, it plays key regulatory roles in a variety of biological processes and pathological processes. Especially, impaired Dicer has been confirmed in many kinds of tumors and it could act as an important tumor inhibitor (Kumar et al., 2009; Pampalakis et al., 2010; Prodromaki et al., 2015). As a cleaved product of Dicer enzyme, miRNAs are small, endogenous and noncoding regulatory RNAs that regulate gene expression at the post-transcriptional level to induce either translational repression or mRNA degradation or sequestration of mRNA from translational machinery (Ambros, 2004; Bartel, 2004; Dong et al., 2013). Recent studies have shown that impaired Dicer function induced a decline in mature miRNAs expressional level, and indirectly influenced the process of tumor occurrence, development and metastasis (Iliou et al., 2014; Kumar et al., 2007). Traditionally, the Dicer detection is involved of complex cell lysis and detection by Western blot, real time PCR, ELISA or electrochemistry, and they suffered from limited sensitivity, complicated operation process and quantitative detection (Perron et al., 2011; Passon et al., 2012; Zhao et al., 2017; Prodromaki et al., 2015). The expression levels of the Dicer related miRNAs remains unexplored. Therefore, efficient intracellular real-time monitor probe of Dicer is urgently needed for cancer early clinical diagnosis or prognosis, and the Dicer substrates of pre-miRNAs hold great promise for probe design.

Besides detection probe, the other significant biological barrier for intracellular Dicer detection is the natural cell membrane, which required efficient gene vector due to the low transfection efficiency of the free gene probe. Commercial liposomes are artificial lipoid bodies prepared by arrayed phospholipid biomolecules and widely used as carriers in the gene transfection (Felgner and Ringold, 1989; Malone et al., 1989). However, their potential cytotoxicity would seriously influence the cell status and induce false information, which has attracted intense attention (Lv et al., 2006). Recently, exosomes derived from the membranous vesicle of late endocytosis in living cells cytoplasm have emerged as promising drug and gene delivery vectors for diagnostic and therapeutic (Alvarez-Erviti et al., 2011; Oh et al., 2019; Xitong and Xiaorong, 2016). It is nano-sized membrane vesicles secreted by virtually all cells with a mean diameter of about 40–100 nm for intercellular communication (An et al., 2019; Mathieu et al., 2019; Milane et al., 2015; Yang et al., 2019). In comparison with traditional commercial liposomes, it exhibits multiple advantages such as little immunogenicity, good cell fusion ability, high abundance in biological fluids and especially innate biocompatibility (Heusermann et al., 2016; Tian et al., 2013). Thus, the natural exosomes are promising tools for gene delivery across membrane barriers (Alvarez-Erviti et al., 2011; Pitt et al., 2016; Usman et al., 2018; Wang et al., 2017).

Herein, we design a Dicer substrate of pre-miRNA let-7a, a hairpin sequence with two terminals labeled with fluorescent dye and quencher, respectively, and package it into exosomes for intracellular Dicer detection and gene therapy. As shown in Scheme 1 and Fig. S1, the synthetic Dicer substrate was originally in native hairpin formation, and the 5′ terminal fluorophore was in close proximity to the 3’ terminal quencher, leading to fluorescence quenching. The Dicer substrate was packaged into exosomes through electroporation, and it promoted the cell internalization of the Dicer substrate due to the good cell fusing capability. After the Dicer substrate was recognized and cleaved by intracellular Dicer, the fluorophore separated from the quencher to produce strong fluorescence for Dicer detection. Notably, the cleavage products maintain the intrinsic gene silence ability to regulate the cell proliferation and cycle. To our best of knowledge, this is the first report about the intracellular Dicer detection and biomedical applications, and it may open a new avenue for Dicer analysis and Dicer-based clinical diagnosis and therapeutics.