In Vitro Silencing of Y-RNA: A Potential New Tool for Studying an Epigenetic Mechanism Recently Described in Allergy - European Medical Journal
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In Vitro Silencing of Y-RNA: A Potential New Tool for Studying an Epigenetic Mechanism Recently Described in Allergy

| Allergy & Immunology
Authors:
*Miguel Estravís,1,2,3 Asunción García-Sánchez,1,2,3 Alicia Landeira-Viñuela,1 Esther Moreno-Rodilla,1,2,3,4 Ignacio Dávila,1,2,3,4 Catalina Sanz,2,3,5 María Isidoro-García2,3,6,7
Disclosure:

The authors have declared no conflicts of interest.

Acknowledgements:

This work was supported by the Instituto de Salud Carlos III (ISCIII), cofounded by the European Regional Development Fund (Grant PI17/00818, PI: Ignacio Dávila), the Network for Cooperative Research in Health – RETICS ARADyAL (RD16/0006/0019, PI: Ignacio Dávila), Junta de Castilla y León Health Council (GRS1596/A/17, PI: María Isidoro), and Junta de Castilla y León Education Council (CAS086P17, PI: Ignacio Dávila).

Citation
EMJ Allergy Immunol. . ;4[1]:56-58.

Each article is made available under the terms of the Creative Commons Attribution-Non Commercial 4.0 License.

BACKGROUND

Allergic diseases are becoming a public health concern, their frequency has notably increased in the last decades, which is likely related to changes in lifestyle and environment. In recent years, the epigenetic regulation has emerged as a pivotal group of mechanisms that can help explain the allergic disease emergence and allows us to understand their molecular basis.1

Some of these mechanisms of epigenetic regulation are conducted by small non-coding RNAs (sncRNA). Small cytoplasmic RNA, or Y-RNA, are a group of sncRNA of approximately 100 bp in length and are highly conserved from the evolutionary point of view and involved in the initiation of DNA replication and RNA stability that regulate gene expression.2,3 Besides their cellular functions, their presence in the extracellular milieu, as part of ribonucleoprotein complexes or associated to extracellular vesicles, underscores their potential role in the modulation or amplification of different responses whether at local or at systemic level.4 Thus, Y-RNA have
been previously related to autoimmunity5 and cancer,6 and now to allergy. In this sense, our group has recently found differential Y-RNA expression profiles in allergic patients. In particular, the authors have shown a significant increase of certain Y-RNA in pollen allergic patients.7

The proper study of epigenetic regulation
requires the implementation of the new methods for its research. To increase the repertoire of laboratory approaches to the disease, cell line studies need to be improved with new sophisticated techniques that mimic pathological states to deepen their molecular mechanisms. To unravel the function of these small RNA in allergy, we have developed a strategy for transiently silencing Y-RNA in cell culture. This will allow us to study the physiological effects behind different expression levels of the
Y-RNA of interest in this particular cell line, as well as how it is related to signal transduction mediated by extracellular media.

METHOD

Transient silencing of different Y-RNAs was performed in the cell line Jurkat, used as a model of T lymphocyte. Dicer-substrate short interfering RNA (DsiRNA) were used; these are 27mer
duplex RNA that demonstrate increased potency in RNA interference compared to traditional,
21mer short interfering RNA8 (provided by Integrated DNA Technologies, Inc., USA) designed against the Y-RNA of interest.

Cells were plated at a density of 2.5 x105 cells/mL and transfected with DsiRNA designed for each Y-RNA or the scrambled control at a final concentration of 50 nmol/L. Briefly, DsiRNA were diluted in of Opti-MEM® (Thermo Fisher Scientific, USA) and mixed with Lipofectamine® RNAiMAX (Thermo Fisher Scientific, USA) diluted in Opti-MEM®. The complexes of DsiRNA-Lipofectamine® RNAiMAX were added directly to the cells and mixed gently. Complexes did not have to be removed following transfection. Cells were incubated at 37 °C in a CO2 incubator for 3 days post transfection before assaying for silencing gene expression.

RESULTS

With this method successful and significant silencing of the different Y-RNA was obtained. When compared to the scrambled transfected control, the levels of the specific transcript were reduced between 32% and 85% (Figure 1).

Figure 1: Changes in Y-RNA levels after transfection with Dicer-substrate short interfering RNA.
DsiRNA: Dicer-substrate short interfering RNA (***p<0.001; *p<0.05).

CONCLUSION

These findings have led to the development of an easy and affordable method to silence sncRNA in the cell line Jurkat, providing new perspectives in this cell line to study molecular mechanisms related to allergic diseases and its epigenetic in vitro control.

References
Potaczek DP et al. Epigenetics and allergy: From basic mechanisms to clinical applications. Epigenomics. 2017;9(4):539-71. Krude T. Non-coding RNAs: New players in the field of eukaryotic DNA replication. Sub-cell Biochem. 2010;50:105-18. Stein AJ et al. Structural insights into RNA quality control: The Ro autoantigen binds misfolded RNAs via its central cavity. Cell. 2005;121(4):529-39. Driedonks TAP, Nolte-‘t Hoen ENM. Circulating Y-RNAs in extracellular vesicles and ribonucleoprotein complexes; Implications for the immune system. Front Immunol. 2019;9:3164. Hendrick JP et al. Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: Further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells. Mol Cell Biol. 1981;1(12)1138-49. Haderk F et al. Tumor-derived exosomes modulate PD-L1 expression in monocytes. Sci Immunol. 2017;2(13):eaah5509. Isidoro-García M et al. YRNAs overexpression and potential implications in allergy. World Allergy Organization Journal. 2019. [Epub ahead of print]. Kim DH et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol. 2005;23(2):222-6.