miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential retroviral restriction factor in macrophages

The goal of this study was to characterize extracellular vesicles (EVs) and miRNAs of primate cervicovaginal lavage (CVL) during the menstrual cycle and simian immunodeficiency virus (SIV) infection, and to determine if differentially regulated CVL miRNAs might influence retrovirus replication. CVL and peripheral blood were collected from SIV-infected and uninfected macaques. EVs were enriched by stepped ultracentrifugation and characterized thoroughly. miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform and validated by single qPCR assays. Hormone cycling was abnormal in infected subjects, but EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs were found to vary during the menstrual cycle or SIV infection. Among them was miR-186-5p, which was depleted in retroviral infection. In experiments with infected macrophages in vitro, this miRNA inhibited HIV replication. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract.


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The cervicovaginal canal is a potential source of biological markers for forensics investigations Total RNA was eluted with 50 µL RNase-free water and stored at -80°C. As quality control, 5 A custom 48-feature TaqMan low-density array (TLDA) was ordered from Thermo Fisher, with 178 features chosen based on results of a human CVL pilot study (GVH and KWW, unpublished data).

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Stem-loop primer reverse transcription and pre-amplification steps were conducted using the 180 manufacturer's reagents as previously described 46  process for these invariant miRNAs was to 1) rank miRNAs by coefficient of variation; 2) remove 186 miRNAs with high average Cq (>30), non-miRNAs, and those with low amplification score; 3) select 187 the lowest-CV member of miRNA families (e.g., the 17/92 clusters); and 4) pick the top 10 remaining 188 candidates by 189 and -320a-3p.

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BC, Canada Cat #: 15450 Lot #: 06102016) and centrifuged for 10 minutes at 1200 × g. Plasma and 209 PBMC fractions were removed, washed in PBS+ 2% FBS, and pelleted at 300 × g for 8 minutes. Pellets 210 from 5 tubes were combined by resuspension in 10 mL RBC lysis buffer (4.15 g NH # Cl, 0.5 g KHCO ( , 211 0.15 g EDTA in 450 mL H ) O; pH adjusted to 7.2-7.3; volume adjusted to 500 mL and filter-sterilized); 212 total volume was brought to 40 mL with RBC lysis buffer. After incubation at 37 °C for 5 mins, the 213 suspension was centrifuged at 400 × g for 6 mins at room temperature. The cell pellet was 214 resuspended in Macrophage Differentiation Medium with 20% FBS (MDM20) to a final concentration 215 of 2´10 , cells/mL. PBMCs were plated at 4´10 , cells per well in 12-well plates and cultured in 216 MDM20 for 7 days. One half of the total volume of medium was replaced on day 3. On day 7, cells 217 were washed 3 times with PBS to remove non-adherent cells.   Figure 1B). The 100,000 x g pellet included apparent EVs up to 200 nm in diameter ( Figure 1C).

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EV markers (shown: CD63, CD81, and TSG101) were confirmed by Western blot ( Figure 1D). The 288 nuclear marker nucleoporin was detected only in tissue samples ( Figure 1D). The relative EV 289 tetraspanin profiles of both CVL and control EV samples were corroborated with single particle 290 interferometric reflectance imaging: CVL EVs had a higher CD63 expression and dendritic cell EVs

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Individual stem loop RT/hydrolysis probe qPCR assays were used to verify TLDA results for 341 eleven selected miRNAs plus miR-375-3p (not included on the array), which was also measured 342 because of a reported association with goblet cells 49 . Some miRNAs were chosen due to high 343 expression levels. miR-181a-5p was measured due to its association with endometrial cells 50,51 . miR-344 125b-5p has been reported as a diagnostic marker of endometriosis 52 . Other miRNAs (miRs-186-5p, -451a-5p, -200c-3p, -222-3p, -193b-3p) were selected based on our previous experience and results 346 from other studies evaluating miRNAs in the context of HIV-1 and SIV infections. Results of qPCR 347 assays, adjusted by miR-16-5p for each sample (since we found relatively low qPCR variation of miR-348 16-5p, a commonly used normalizer 53 ), are shown in Figure 3A. Figure 3B compares miRNA ranks

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(1-11) by TLDA and individual qPCR, which are generally in concordance. Note that expression of 350 red blood cell miRNA miR-451a-5p was low, suggesting minimal contamination from blood for most 351 samples.

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which increased miR-186-5p levels from around 5-fold to nearly 500-fold, respectively. Strikingly, 417 the miR-186-5p level was inversely correlated with released p24 across these five donors. It should 418 be noted that miR-186-5p antisense inhibitors were also introduced in these experiments. While they did not significantly increase HIV p24 release (Figure 7), they also did not achieve a consistent 420 knockdown of native miR-186-5p ( Figure 8A).