Name: Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression
Uploaded: 2023-10-06T12:00:00
Description: Watch this Scientific Journal Video about Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression at JoVE.com

We acknowledge support and advice from Dr. Jill Hutchcroft, Director of the Flow Cytometry Core Facility at Purdue University. This work was supported by R01CA226259 and R01CA205420 to A.L.K., an American Lung Association Innovation Award (ANALA2023) IA-1059916 to A.L.K., a Purdue Shared resource facility grant P30CA023168, and a flagship Fulbright Doctoral Scholarship awarded by the Department of the State, USA to H.H.

NOTE: As a prerequisite to using this technique, identification and validation of selectively exported miRNAs is required. Since different cell lines vary based on the miRNA cargo sorted into their EVs, it is recommended that the cell line of interest and associated EVs be evaluated for miRNAs prior to use. Additionally, lipofectamine-based transfection is one of the critical steps in the protocol; predetermining transfection efficiency prior to setting up the experiment is recommended.
...

A Flow Cytometry Protocol for Understanding Export Mechanisms

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+en+route+to+the+clinic:+Progress+in+validating+and+targeting+microRNAs+for+cancer+therapy.">MicroRNAs en route to the clinic: Progress in validating and targeting microRNAs for cancer therapy.</a> Nature Reviews Cancer. 11 (12), 849-864 (2011).</li><li>Treiber, T., Treiber, N., Meister, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+microRNA+biogenesis+and+its+crosstalk+with+other+cellular+pathways.">Regulation of microRNA biogenesis and its crosstalk with other cellular pathways.</a> Nature Reviews Molecular Cell Biology. 20 (1), 5-20 (2019).</li><li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiles+classify+human+cancers.">MicroRNA expression profiles classify human cancers.</a> Nature. 435 (7043), 834-838 (2005).</li><li>Orellana, E., Tenneti, S., Rangasamy, L., Lyle, L., Low, P., Kasinski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FolamiRs:+Ligand-targeted,+vehicle-free+delivery+of+microRNAs+for+the+treatment+of+cancer.">FolamiRs: Ligand-targeted, vehicle-free delivery of microRNAs for the treatment of cancer.</a> Science Translational Medicine. 9 (401), eaam9327 (2017).</li><li>Orellana, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+microRNA+activity+through+increased+endosomal+release+mediated+by+nigericin.">Enhancing microRNA activity through increased endosomal release mediated by nigericin.</a> Molecular Therapy- Nucleic Acids. 16, 505-518 (2019).</li><li>Abdelaal, A. 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The newly established protocol enables capturing kinetics of miRNA release into EVs post-transfection of EV-miRNAs. The approach allows simultaneous analysis of multiple EV-miRNAs and cell-miRNAs, subject to the capabilities of the cytometer. Moreover, while flow cytometry analysis can provide valuable insights into EV miRNA biology, it is not without limitations. Albeit some of the limitations can be overcome when used in conjunction with other techniques for a more comprehensive understanding. 
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MicroRNAs (miRNAs) are one of the best-characterized subsets of small noncoding RNAs, which are known for their critical role in post-transcriptional gene regulation. The expression and biogenesis of most miRNAs follow a coordinated series of events that begins with transcription of the primary miRNAs (pri-miRNAs) in the nucleus. Following nuclear processing by the microprocessor complex into precursor-miRNAs (pre-miRNAs), the pre-miRNAs are exported to the cytoplasm, where they undergo further processing by the RNase III endonuclease, dicer into 21-23 nucleotide mature miRNA duplexes1. One of the strands of the processed mature miRNA binds to target messenger RNAs (mRNAs), leading to degradation or translational repression of the targets2. Based on the pleiotropic role of miRNAs in simultaneously regulating multiple diverse target mRNAs, it is not surprising that miRNA expression is tightly regulated3. Indeed, inappropriate expression contributes to various disease states, especially in cancer. The aberrant expression of miRNAs not only represents a disease-specific signature but has also emerged as a target for prognostic and therapeutic potential4,5,6. In addition to their intracellular roles, miRNAs also have non-autonomous roles. For example, miRNAs can be selectively packaged into EVs by donor cells and exported to recipient sites where they elicit diverse phenotypic responses in normal and disease physiology7,8,9.
Despite clear evidence that EV-associated miRNAs are functional biomolecules, it is not completely understood how specific subsets of miRNAs are dysregulated in disease states such as cancer and how cellular machinery selects and sorts miRNAs into Evs10,11. Given the potential role of EV-miRNAs in modulating the microenvironment, it is critical to identify the mechanisms involved in the export of select miRNAs to fully elucidate the role of EVs in intercellular communication and disease pathogenesis. Understanding the process of miRNA release into EVs will not only highlight important mediators of miRNA export but can also provide insights into potential therapeutics. To achieve this level of knowledge, tools need to be adapted to faithfully address these new experimental questions. Indeed, studies that evaluate EVs are growing exponentially due to the introduction of new techniques and controls12,13. In relation to evaluating the abundance of exported miRNAs into EVs, next-generation sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) have been the current standard tools. While these tools are useful for evaluating the abundance of miRNAs in EVs, there are limitations to their sensitivity and specificity, and using these static approaches for evaluating dynamics is insufficient. Delineating the dynamics of miRNA export into EVs requires a combination of specialized tools. Here, we present a comprehensive protocol using fluorescently conjugated miRNAs, to analyze the dynamics of miRNA release from donor cells and incorporation into EVs. We also discuss the advantages and limitations of the method and provide recommendations for optimal use. The versatile method presented in this paper will be useful to researchers interested in studying miRNA release into EVs and their potential roles in cellular communication and disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon tubes</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well clear flat bottom surface treated tissue culture plates</td>
			<td>Fisher Scientific</td>
			<td>FB012927</td>
			<td></td>
		</tr>
		<tr>
			<td>ApogeeMix 25 mL, (PS80/110/500 &#38; Si180/240/300/590/880/1300 nm)&#160;</td>
			<td>Apogee Flow Systems</td>
			<td>1527</td>
			<td>To set up gating and parameters for particle detection&#160;</td>
		</tr>
		<tr>
			<td>Attune Nxt Flow Cytometer</td>
			<td>ThermoFisher Scientific</td>
			<td>N/A</td>
			<td>For fluorescence analysis in EVs</td>
		</tr>
		<tr>
			<td>BD Fortessa Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>For fluorescence analysis in cells&#160;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Maintaining temperature of 37&#160;&#176;C and 5% CO2</td>
		</tr>
		<tr>
			<td>Cell Culture medium</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>specific for the cell-line of interest</td>
		</tr>
		<tr>
			<td>Cell line of interest&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any cell line tested and evaluated for EV and cellular abundance of miRNAs on interest.</td>
		</tr>
		<tr>
			<td>Cell-miRNA (miRIDIAN microRNA Mimic Red Transfection Control)</td>
			<td>Horizon discovery</td>
			<td>CP-004500-01-05</td>
			<td>miRNAs predetermined to be retained by the cell-line of interest and not selectively exported out into EVs</td>
		</tr>
		<tr>
			<td>EV-miRNA (Fluorophore conjugated miRNAs)</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td>miRNAs predetermined to be selectively sorted into EVs&#160;</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Opti-MEM Reduced Serum Medium</td>
			<td>Fisher Scientific</td>
			<td>31-985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Hausser Scientific Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone 1x PBS (for cell culture)</td>
			<td>Fisher</td>
			<td>SH30256FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAimax</td>
			<td>Fisher Scientific</td>
			<td>13-778-150</td>
			<td></td>
		</tr>
		<tr>
			<td>miRCURY LNA Reverse Transcriptase</td>
			<td>Qiagen</td>
			<td>339340</td>
			<td></td>
		</tr>
		<tr>
			<td>miRCURY LNA SYBR Green PCR</td>
			<td>Qiagen&#160;</td>
			<td>339347</td>
			<td></td>
		</tr>
		<tr>
			<td>mirVana RNA Isolation</td>
			<td>Qiagen</td>
			<td>AM1561</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Fisher Scientific</td>
			<td>4387936</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 4% in PBS</td>
			<td>Fisher</td>
			<td>AAJ61899AK</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoir nonsterile</td>
			<td>VWR</td>
			<td>89094-684</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo ABI QuantiStudio DX Real-Time PCR</td>
			<td>ThermoFisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Fisher&#160;</td>
			<td>SH3004201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking mirna release into extracellular vesicles using flow cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, microRNAs, and other noncoding RNAs), from one cell to another, leading to phenotypic consequences in the recipient cells. Of all the various EV cargo, microRNAs (miRNAs) have garnered a great deal of attention for their role in shaping the microenvironment and in educating recipient cells because of their clear dysregulation and abundance in EVs. Additional data indicates that many miRNAs are actively loaded into EVs. Despite this clear evidence, research on the dynamics of export and mechanisms of miRNA sorting is limited. Here, we provide a protocol using flow cytometry analysis of EV-miRNA that can be used to understand the dynamics of EV-miRNA loading and identify the machinery involved in miRNA export. In this protocol, miRNAs predetermined to be enriched in EVs and depleted from donor cells are conjugated to a fluorophore and transfected into the donor cells. The fluorescently tagged miRNAs are then verified for loading into EVs and depletion from cells using qRT-PCR. As both a transfection control and a tool for gating the transfected population of cells, a fluorescently labeled cellular RNA (cell-retained and EV-depleted) is included. Cells transfected with both the EV-miRNA and cell-retained-miRNA are evaluated for fluorescent signals over the course of 72 h. The fluorescence signal intensity specific for the EV-miRNAs diminishes rapidly compared to the cell-retained miRNA. Using this straightforward protocol, one could now assess the dynamics of miRNA loading and identify various factors responsible for loading miRNAs into EVs.

Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression 

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

My lab works on studying microRNAs, and we have known for decades that microRNAs are depleted in cancer. However, the mechanism as to how these microRNAs are lost has not been actively understood. Our recent work on extracellular vesicles suggests that microRNA that are depleted in cancer are being actively loaded into these extracellular vesicles.Our goal now is to really get to the mechanism as to how these RNAs are actively loaded into these extracellular vesicles in the process of tumor genesis. The protocol helps understand and capture the dynamic process of microRNAs release into extracellular vesicles, which is one of the challenging areas in studying extracellular vesicle biology. Our findings provide a tool to advance the study of pathways that regulate depletion of microRNAs in cancer.We plan on identifying functional and phenotypic consequences of the loss of specific subsets of microRNA via extracellular vesicles in cancer. Once we identify the factors that are involved in loading these microRNAs into extracellular vesicles, our goal is to down-regulate these factors and evaluate the phenotypic consequence. Moreover, because we identified a motif that's contained in these small RNAs, we're curious to know whether this motif is both necessary and sufficient for loading these RNAs into the extracellular vesicles.

Here, we utilize flow cytometry as a powerful tool to investigate the release of miRNA from the cells into EVs. Using this protocol, flow cytometry analysis of cells transfected with cell-miRNA and EV-miRNA revealed a sequential decrease of fluorescence signal corresponding to EV-miRNA, while the signal corresponding to the cell-miRNA was retained in the cells. To ensure that fluorophore conjugation does not interfere with miRNA release into EVs, miR-451a was conjugated to two separate fluorophores (i.e., Alexa fluor 488...

Watch this Scientific Journal Video about Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression at JoVE.com

Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...