Name: 형광단 접합 EV-miRNA로 transfection된 Calu6 세포에서 분리된 EV의 세포-miRNA 및 EV-miRNA 신호의 유세포 분석
Uploaded: 2023-10-06T12:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in EVs Isolated from Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA at JoVE.com

flow cytometry analysis of cell-mirna and ev-mirna signal in evs isolated from calu6 cells transfected with fluorophore-conjugated ev-mirna

Export Mechanisms를 이해하기 위한 유세포 분석 프로토콜

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.

저자 스포트라이트: 암 진행에서 세포외 소포체에 MicroRNA 로딩이 들어가는 메커니즘 탐구 

유세포 분석을 사용하여 세포외 소포체로의 miRNA 방출 추적

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.

Watch this Scientific Journal Video about Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in EVs Isolated from Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA at JoVE.com

형광단 접합 EV-miRNA로 transfection된 Calu6 세포에서 분리된 EV의 세포-miRNA 및 EV-miRNA 신호의 유세포 분석

먼저 Calu6 세포에서 분리한 EV를 형광단 접합 EV-마이크로 RNA 및 세포-마이크로 RNA로 transfection합니다. EV를 적절한 양의 초미세 여과된 PBS에 희석하여 유세포 분석을 위한 세포외 소포체 현탁액을 준비합니다. 나노 유세포 분석기를 사용하여 나노 크기의 입자를 분석합니다.소프트웨어를 실행하고 권장 교정 지침에 따라 기기를 구성합니다. 성능 나노 비드를 사용하여 성능 테스트를 수행합니다. 기기의 성능은 나노 비드가 표준 크기 범위 내에서 검출되는 경우 최적의 것으로 간주됩니다.EV 입자 감지에 적합한 유량을 결정합니다. 이제 초순수 여과수를 사용하여 품질 관리 검사를 수행하여 기기 유체를 검증하고 검출기 및 레이저의 성능을 평가합니다. 제조업체의 프로토콜에 따라 비드 혼합물에 있는 다양한 모집단의 민감도와 분리능을 평가하도록 매개변수를 구성합니다.그런 다음 적절한 게이팅을 적용하여 비드 모집단을 기기 노이즈와 구별합니다. 다음으로, 분석을 위해 EV를 로드하기 전에 EV의 균일한 분포를 보장하기 위해 샘플을 철저히 소용돌이칩니다. 유세포 분석 데이터 분석 소프트웨어를 실행하고 초미세 여과 PBS를 도입하십시오.초미세 필터링된 PBS를 사용하여 EV 입자에 대한 게이팅을 설정하여 게이팅이 보정에 따라 적절한 입자 크기에 해당하도록 합니다. 조율된 호환 튜브에서 전송 및 볼텍싱된 EV를 가져와 튜브를 기기에 로드합니다. x축에 FSC를 플로팅하고 y축에 SSC를 플로팅하면서 EV 신호를 캡처합니다.x축의 cell micro RNA 검출의 경우 형광 신호 1을 선택하고 y축의 EV micro RNA 검출의 경우 형광 신호 2를 선택합니다. 단일 마이크로 RNA로 transfection된 세포에서 분리한 EV 샘플 사용. fluorophores에 대한 보정 매개변수를 설정했습니다.유세포 분석은 효율적인 패키징 및 방출을 지원하는 EV에서 miR-451a, Alexa fluor-488의 시간 의존적 축적을 보여주었습니다. 대조적으로, miR-67 셀의 형광은 EV에서 관찰되지 않았습니다.

Research

JoVE Journal

Biology

Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in EVs Isolated from Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA

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 ...