Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype

Loss of functionality during aging of cells and organisms is caused and accompanied by altered cell-to-cell communication and signalling. One factor thereby is the chronic accumulation of senescent cells and the concomitant senescence-associated secretory phenotype (SASP) that contributes to microenvironment remodelling and a pro-inflammatory status. While protein based SASP factors have been well characterized, little is known about small extracellular vesicles (sEVs) and their miRNA cargo. Therefore, we analysed secretion of sEVs from senescent human dermal fibroblasts and catalogued the therein contained miRNAs. We observed a four-fold increase of sEVs, with a concomitant increase of >80% of all cargo miRNAs. The most abundantly secreted miRNAs were predicted to collectively target mRNAs of pro-apoptotic proteins, and indeed, senescent cell derived sEVs exerted anti-apoptotic activity. In addition, we identified senescence-specific differences in miRNA composition of sEVs, with an increase of miR-23a-5p and miR-137 and a decrease of miR-625-3p, miR-766-3p, miR-199b-5p, miR-381-3p, miR-17-3p. By correlating intracellular and sEV-miRNAs, we identified miRNAs selectively retained in senescent cells (miR-21-3p and miR-17-3p) or packaged specifically into senescent cell derived sEVs (miR-15b-5p and miR-30a-3p). Therefore, we suggest sEVs and their miRNA cargo to be novel, members of the SASP that are selectively secreted or retained in cellular senescence.

. Scheme of experimental workflow. (A) SIPS was triggered in three donors of primary human dermal fibroblasts' (HDF) by chronic low doses of H 2 O 2 . Seven days (D7) and 21 days (D21) after the last H 2 O 2 pulse, intracellular RNA was harvested and cDNA library for small RNA NGS was synthesized. Correspondingly, sncRNA from small extracellular vesicles (sEVs) was isolated from conditioned supernatants by differential centrifugation. SEV-RNA was prepared for qPCR panels to identify senescence-associated sEV-miRNAs. synthetic RNA-spike-in confirms technical coverage (ΔCt r values below 1) of screening comprising 12 samples of vesicular RNA from three different donors and two different time points (D7 and D21). Each qPCR plate contained primer for synthetic spike in RNAs that were added during RNA isolation (Unisp2, Unisp4, Unisp5) and cDNA synthesis (Unisp6, cel-miR-39). Additionally, each panel included two interplate calibrator (IPC) and an empty negative control. (B) Bar chart of significantly higher secreted miRNAs of SIPS HDF on D7 after the treatment. Log2FC values from three biological triplicates were calculated and plotted on y-axis. Bars plotted on y-axis show all miRNAs reaching an adjusted p-value < 0.05 after applying the BH method for FDR. On D7, 221 EV-miRNAs passed the adjusted p-value. Dotted lines represent log2FC = 1. (C) Bar chart of significantly higher secreted sEV-miRNAs of SIPS HDF on D21 after the treatment. Log2FC values from three biological triplicates were calculated and plotted on y-axis. Bars plotted on y-axis show all miRNAs reaching an adjusted p-value < 0.05 after applying the Benjamini Hochberg method for FDR. On D21, 321 EV-miRNAs passed the adjusted p-value. Dotted lines represent log2FC = 1. Figure S4. Calculation of Global means and changes in miRNA abundances over time in vesicles and intracellularly. (A) Global mean used for normalization. Averages of three different HDF strains +/-STDEV is shown. 2-way ANOVA was used to test for condition (p = 0.73) and day (p = 0.11); (n.s) p > 0.05. (B) 21 sEV-miRNAs of SIPS cells change their composition over time. Global mean-normalized Ct-values from biological triplicates were averaged and log2FC relative to day 7 recovery were calculated (p-value < 0.05). (C) 3 miRNAs are differentially transcribed in SIPS cells over time. Intracellular miRNA transcription relative to day 7 was calculated from NGS data. miRNAs with an adj. p-value < 0.05 were taken into account. www.aging-us.com

Senescence associated (SA) ß-Gal staining
SIPS HDF and sub-confluent HDF at the middle of their replicative lifespan were stained according to the standard protocol described by Dimri et al. 1995 [1]. 15 pictures per well were taken at 100 x magnification and after randomization and blinding, SA-ß-Gal positive and negative cells were counted.

Nanoparticle tracking analysis
Experiments related to sEV Isolation were performed according to standards recommended from the international society for extracellular vesicles (ISEV) [2].
For determination of size and concentration of vesicles,

Electron microscopy
SEVs for Transmission Electron microscopy (TEM) were freshly prepared. Solutions used for the staining procedure were pre-filtered using 0.22 µm filter units (Millipore, Germany, SCGPU05RE). SEVs were adhered on Athene Old 300 mesh copper grids (Agar Scientific, Stansted, Essex, UK) and fixed with 1% glutaraldehyde. After washing three times with nuclease free water, vesicles were stained for 5 min with 2% phosphotungstic acid hydrate (Carl Roth, Karlsruhe, Germany). The grids were left to dry and the specimens were visualized using TEM (FEI Tecnai T20, FEI Eindhoven, Netherlands) operated at 160 kV.

Protein quantification, western blot and antibodies
Vesicles and corresponding cells were lysed in 1 x TNE buffer (2 x TNE: 100 mM Tris/HCl, pH 8.0, 300 mM www.aging-us.com