RNAi Transfection Results in Lipidome Changes

Özbalci, Cagakan; Storck, Elisabeth M.; Eggert, Ulrike

doi:10.1002/pmic.201800298

Cited by 6 publications

(6 citation statements)

References 19 publications

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“…This is because cellular lipid metabolism is extremely complex and redundant and many of the “building blocks” of complex lipid species are shared across multiple lipid classes ( 18 ). To accurately define the lipid species involved in regulating differentiation, we performed a comprehensive lipidomic analysis of cells transfected with siRNA targeting ELOVL1 or SLC27A1 or a nontargeting siRNA to control for the potential influence of transfection reagents ( 31 ). We collected samples at 24, 48, or 72 h posttransfection and performed lipidomics analysis on them ( Dataset S3 ).…”

Section: Resultsmentioning

confidence: 99%

Human epidermal stem cell differentiation is modulated by specific lipid subspecies

Rudan

Mishra

Klose

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: ELOVL1, encoding elongation of very long-chain fatty acids protein 1, and SLC27A1, encoding fatty acid transport protein 1. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Several of these—ceramides and glucosylceramides—induced differentiation when added to primary keratinocytes in culture. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment.

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Section: Resultsmentioning

confidence: 99%

Human epidermal stem cell differentiation is modulated by specific lipid subspecies

Rudan

Mishra

Klose

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…We tested this hypothesis using global lipidomic analysis by liquid chromatography–mass spectrometry (LC–MS) and compared the lipidomes of cells treated with nontargeting control siRNA and cells where stomatin had been depleted by RNAi. This comparison is necessary to rule out lipid changes induced by transfection reagents . Due to the chemical diversity of lipids, fragmentation by tandem MS followed by analysis using lipidomics databases is required to identify each lipid species.…”

Section: Resultsmentioning

confidence: 99%

Removal of Stomatin, a Membrane-Associated Cell Division Protein, Results in Specific Cellular Lipid Changes

et al. 2022

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Lipids are key constituents of all cells, which express thousands of different lipid species. In most cases, it is not known why cells synthesize such diverse lipidomes, nor what regulates their metabolism. Although it is known that dividing cells specifically regulate their lipid content and that the correct lipid complement is required for successful division, it is unclear how lipids connect with the cell division machinery. Here, we report that the membrane protein stomatin is involved in the cytokinesis step of cell division. Although it is not a lipid biosynthetic enzyme, depletion of stomatin causes cells to change their lipidomes. These changes include specific lipid species, like ether lipids, and lipid families like phosphatidylcholines. Addition of exogenous phosphatidylcholines rescues stomatin-induced defects. These data suggest that stomatin interfaces with lipid metabolism. Stomatin has multiple contacts with the plasma membrane and we identify which sites are required for its role in cell division, as well as associated lipid shifts. We also show that stomatin’s mobility on the plasma membrane changes during division, further supporting the requirement for a highly regulated physical interaction between membrane lipids and this newly identified cell division protein.

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“…Only one out of the four reagents we tested showed none toxicity as recorded for the WRAP peptides. Therefore, researcher should be aware of the interpretation of their protein silencing due to potent long-term cytotoxic effect or other side effects such as alterations of the lipidome of hepatocytes [ 36 ] and of Hela cells [ 37 ] or markedly inhibit cholesterol biosynthesis [ 38 ] by some of the lipid-based transfection reagents.…”

Section: Discussionmentioning

confidence: 99%

WRAP-based nanoparticles for siRNA delivery: a SAR study and a comparison with lipid-based transfection reagents

et al. 2021

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Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure–activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.

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RNAi Transfection Results in Lipidome Changes

Cited by 6 publications

References 19 publications

Human epidermal stem cell differentiation is modulated by specific lipid subspecies

Human epidermal stem cell differentiation is modulated by specific lipid subspecies

Removal of Stomatin, a Membrane-Associated Cell Division Protein, Results in Specific Cellular Lipid Changes

WRAP-based nanoparticles for siRNA delivery: a SAR study and a comparison with lipid-based transfection reagents

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