Barriers in systemic delivery and preclinical testing of synthetic microRNAs in animal models: an experimental study on miR-215-5p mimic.

Mus musculus is the most commonly used animal model in microRNA research; however, little is known about the endogenous miRNome of the animals used in the miRNA-targeting preclinical studies with the human xenografts. In the presented study, we evaluated the NOD/SCID gamma mouse model for the preclinical study of systemic miR-215-5p substitution with a semitelechelic poly[N-(2-hydroxypropyl)-methacrylamide]-based carrier conjugated with miR-215-5p-mimic via a reductively degradable disulfide bond. Murine mmu-miR-215-5p and human hsa-miR-215-5p have a high homology of mature sequences with only one nucleotide substitution. Due to the high homology of hsa-miR-215-5p and mmu-hsa-miR-215-5p, a similar expression in human and NOD/SCID gamma mice was expected. Expression of mmu-miR-215 in murine organs did not indicate tissue-specific expression and was highly expressed in all examined tissues. All animals included in the study showed a significantly higher concentration of miR-215-5p in the blood plasma compared to human blood plasma, where miR-215-5p is on the verge of a reliable detection limit. However, circulating mmu-miR-215-5p did not enter the human xenograft tumors generated with colorectal cancer cell lines since the levels of miR-215-5p in control tumors remained notably lower compared to those originally transfected with miR-215-5p. Finally, the systemic administration of polymer-miR-215-5p-mimic conjugate to the tail vein did not increase miR-215-5p in NOD/SCID gamma mouse blood plasma, organs, and subcutaneous tumors. It was impossible to distinguish hsa-miR-215-5p and mmu-miR-215-5p in the murine blood and organs due to the high expression of endogenous mmu-miR-215-5p. In conclusion, the examination of endogenous tissue and circulating miRNome of an experimental animal model of choice might be necessary for future miRNA studies focused on the systemic delivery of miRNA-based drugs conducted in the animal models.

Utilization of Next Generation Sequencing in Analysis of Circulating MicroRNAs as Predictive Biomarkers for Patients with Locally Advanced Rectal Carcinoma.

BACKGROUND: MicroRNAs (miRNA) are short non-coding RNAs involved in post-transcriptional regulation of gene expression. MiRNAs are essential regulators of both physiological processes as of pathogeneses of many diseases, and their dysregulation was observed in many malignancies including rectal cancer. Circulating miRNAs presented in blood plasma could be potential candidates for non-invasive predictive biomarkers of the response of patients with locally advanced rectal cancer to chemoradiotherapy. Presented study aims to evaluate the potential of next-generation sequencing in the analysis of circulating miRNAs. MATERIAL AND METHODS: MiRNA expression profiles were done using samples of RNA isolated from blood plasma collected during TNM restaging and paired samples collected before initiation of neoadjuvant chemoradiotherapy. Sequencing libraries were prepared using kit which implements universal molecular indices that help to sensitively filter biological bias during data analysis. Sequencing data were processed by multidimensional biostatistical approaches. CONCLUSION: We identified specific miRNA profile enabling to distinguish the patients accordingly to their response to chemoradiotherapy. This work was supported by the Czech Ministry of Health grant No. 16-31765A. The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 22. 2. 2019 Accepted: 27. 2. 2019.