Changes in the repertoire of extracellular vesicles secreted by skin keratinocytes by the human protein SLURP-2

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Abstract

Extracellular vesicles secreted by keratinocytes are participants of intercellular interactions, and contain various proteins, mRNA and miRNA, lipids, due to which they can activate cell migration and proliferation. The secreted human protein SLURP-2 regulates growth and differentiation of epithelial cells, affects proliferation and migration of keratinocytes. In this work, we showed that extracellular vesicles secreted by keratinocytes in presence of SLURP-2 stimulate migration of HaCaT keratinocytes. It was also found that, expression of miRNA-96 and miRNA-183, suppressing cell migration and proliferation, is decreased in the vesicles secreted by keratinocytes in presence of SLURP-2. Thus, it has been shown that the stimulation of keratinocyte migration in presence of SLURP-2 is associated, among others, with a change in the repertoire of extracellular vesicles secreted by these cells.

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About the authors

T. Y. Gornostaeva

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Moscow Center for Advanced Studies

Email: lyukmanova_ekaterina@smbu.edu.cn
Russian Federation, Moscow; Moscow

O. V. Shlepova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: lyukmanova_ekaterina@smbu.edu.cn
Russian Federation, Moscow

I. D. Kukushkin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Moscow Center for Advanced Studies

Email: lyukmanova_ekaterina@smbu.edu.cn
Russian Federation, Moscow; Moscow

A. S. Paramonov

Moscow Center for Advanced Studies

Email: lyukmanova_ekaterina@smbu.edu.cn
Russian Federation, Moscow

М. P. Kirpichnikov

Moscow Center for Advanced Studies; Lomonosov Moscow State University

Email: lyukmanova_ekaterina@smbu.edu.cn

Interdisciplinary Scientific and Educational School of Moscow University “ Molecular Technologies of the Living Systems and Synthetic Biology”, Faculty of Biology, Academician of the RAS

Russian Federation, Moscow; Moscow

Е. N. Lyukmanova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Moscow Center for Advanced Studies; Lomonosov Moscow State University; Shenzhen MSU-BIT University

Author for correspondence.
Email: lyukmanova_ekaterina@smbu.edu.cn

Interdisciplinary Scientific and Educational School of Moscow University “ Molecular Technologies of the Living Systems and Synthetic Biology”, Faculty of Biology

Russian Federation, Moscow; Moscow; Moscow; International University Park Road 1, Dayun New Town, Longgang District, Shenzhen, Guangdong Province, 518172 PRC

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Supplementary files

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2. Fig. 1. Analysis of the effect of SLURP-2 on miRNA expression in keratinocyte extracellular vesicles. “Control” and “SLURP-2” are extracellular vesicles (i.v.) secreted by HaCaT keratinocytes without and with the addition of 100 nM SLURP-2 to the culture medium. (a) Western blotting analysis of the expression of the exosomal marker TSG101 in HaCaT keratinocyte extracellular vesicles. The observed band at 65 kDa corresponds to the ubiquitinylated form of TSG101. (b) Expression of miRNA-21, miRNA-96, miRNA-183, miRNA-203, miRNA-215, miRNA-221, and miRNA-451 in extracellular vesicles (i.v.) secreted by HaCaT keratinocytes incubated with 100 nM SLURP-2 (“SLURP-2”) and control HaCaT keratinocytes (“Control”). miRNA expression was normalized to U6 non-coding RNA expression and presented as relative miRNA expression level ± SEM (n = 3–4). * (p < 0.05) indicates statistically significant difference between data groups according to unpaired Student’s t-test.

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3. Fig. 2. Analysis of the effect of SLURP-2 on the expression of miRNA-96 and miRNA-183 in HaCaT keratinocytes. (SLURP-2) – HaCaT keratinocytes incubated in the presence of 100 nM SLURP-2, (Control) – control HaCaT keratinocytes incubated without the addition of SLURP-2. miRNA expression was normalized to the expression of non-coding RNA U6 and is presented as the relative miRNA expression level ± SEM (n = 3–4).

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4. Fig. 3. Effect of extracellular vesicles on keratinocyte viability and migration. (a) Effect of extracellular vesicles (i.v.) secreted by HaCaT keratinocytes without addition (“Control”) and with addition of 100 nM SLURP-2 (“SLURP-2”) to the culture medium on the viability of HaCaT keratinocytes. Data are presented as % of control (cells incubated in medium without exosomes, dotted line) ± SEM (n = 6) (b) Effect of extracellular vesicles (i.v.) secreted by HaCaT keratinocytes without addition (“Control”) and with addition of 100 nM SLURP-2 (“SLURP-2”) to the culture medium on the migration of HaCaT keratinocytes. Data are presented as % of control (change in wound width for cells incubated in medium without exosomes, dotted line) ± SEM (n = 4), * (p < 0.05) indicates statistically significant difference between data groups by unpaired Student's t test. (c) Representative photographs of scratch test of HaCaT cells incubated with extracellular vesicles for 0 h and 24 h.

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