Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Axmann, Markus; Karner, Andreas; Meier, Sabine M.; Stangl, Herbert; Plochberger, Birgit

doi:10.3791/59573

Cited by 10 publications

(11 citation statements)

References 11 publications

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“…Oligonucleotides have been previously reported to bind to and associate with zwitterionic lipids, potentially through a divalent cation bridge between the positively charged choline headgroup on PC and the negatively charged backbone of RNA [24,58,59]. Others have expanded on this discussion towards a model in which an unknown RNA binding protein(s) on HDL, as opposed to phospholipids, confer the association of sRNAs to HDL particles [27]. In agreement with data from Plochberger and colleagues, our study clearly showed that phospholipid vesicles and/or particles did not bind to the sRNAs we tested whereas apoA-I protein did bind to multiple sRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…phosphatidylcholine (PC) [23][24][25][26], but it is currently unknown if natural PC within HDL's shell mediate it's transport of sRNA. Results from HDL delipidation studies suggest that miRNAs may bind to HDL proteins as opposed to phospholipids [14,27]; however, to date, a candidate protein on HDL with RNA binding capacity has not been identified. Here, we tested the hypothesis that apoA-I is a novel RNA binding protein for single-stranded sRNAs, specifically tDRs.…”

Section: Introductionmentioning

confidence: 99%

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Physico-chemical principles of HDL-small RNA binding interactions

Michell

Allen

Cavnar

et al. 2021

Preprint

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Extracellular small RNAs (sRNA) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDL) of mice were enriched with multiple classes of sRNA derived from the endogenous transcriptome, but also exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and non-host species which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL bind tDRs and other single-stranded sRNAs with strong affinity, but not double-stranded RNA or DNA. Natural and synthetic RNA modifications influenced tDR binding to HDL. Reconstituted HDL bound tDRs only in the presence of apoA-I and purified apoA-I alone was sufficient for binding sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, HDL preferentially binds to single-stranded sRNAs likely through non-ionic interactions with apoA-I. These studies highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA for therapeutic approaches to prevent or treat disease.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Physico-chemical principles of HDL-small RNA binding interactions

Michell

Allen

Cavnar

et al. 2021

Preprint

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“…1414/2016). Afterward, HDL particles were isolated by serial ultracentrifugation 60 (for a detailed video, see ref ( 61 )). The apolipoprotein part of the HDL particle was covalently labeled with Alexa647-succinimidyl ester according to the protocol (Thermo Fisher) (for details, see ref ( 62 )).…”

Section: Materials and Methodsmentioning

confidence: 99%

Dual Channel Microfluidics for Mimicking the Blood–Brain Barrier

et al. 2021

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High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in in vitro models of the endothelium. In this study, we demonstrate a microfluidic system consisting of two channels horizontally separated by a cell-growth-promoting membrane. Its design allows for high-resolution (down to single-molecule level) imaging using a high numerical aperture objective with a short working distance. To reduce optical aberrations and enable single-molecule-sensitive imaging, an observation window was constructed in the membrane via laser cutting with subsequent structuring using 3D multiphoton lithography for improved cell growth. The upper channel was loaded with endothelial cells under flow conditions, which showed polarization and junction formation. A coculture of human vascular endothelial cells with pericytes was developed that mimics the blood–brain barrier. Finally, this dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles.

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“…Preparation of reconstituted HDL (rHDL) particles. Native human HDL was isolated from healthy donors using serial density ultracentrifugation as described (17,18). Reconstituted HDL particles were prepared according to the method of Jonas et al (19,20).…”

Section: Cell Lines and Culture Conditions The Murine Pancreatic Adenocarcinoma Cells Lines Panc02mentioning

confidence: 99%

The HDL particle composition determines its anti-tumor activity in pancreatic cancer

Bauer

Kuehrer

Udonta

et al. 2021

Preprint

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Pancreatic cancer is expected to become the second leading cause of cancer-related deaths in the next decade as a result of late diagnosis, a highly fibrotic tumor microenvironment and rapidly emerging resistance mechanisms. Previous research identified lipid metabolic pathways to be highly enriched in pancreatic ductal adenocarcinoma (PDAC) cells. Thereby, cholesterol uptake and synthesis promoted a growth advantage to, and chemotherapy resistance for PDAC tumor cells. We demonstrate that efficient cholesterol removal from cancer cells by high-density lipoprotein (HDL) mediated efflux, results in a significant PDAC cell growth reduction, apoptosis and a decreased PDAC tumor development in vivo. This effect is driven by an HDL particle composition-dependent interaction with major lipid flux receptors expressed on cancer cells, ABCA1 and SRB1. Eventually, we show that pancreatic cancer patient plasma samples display reduced levels of HDL-cholesterol and reduced cholesterol efflux capacity. Thus, cholesterol depletion from PDAC cells, together with interventions that shunt the import and endogenous synthesis pathways of cholesterol, might represent a promising strategy to increase the currently available treatment options for PDAC patients.

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Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Cited by 10 publications

References 11 publications

Physico-chemical principles of HDL-small RNA binding interactions

Physico-chemical principles of HDL-small RNA binding interactions

Dual Channel Microfluidics for Mimicking the Blood–Brain Barrier

The HDL particle composition determines its anti-tumor activity in pancreatic cancer

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