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. Author manuscript; available in PMC: 2018 Aug 1.
Published in final edited form as: Virology. 2017 May 10;508:63–69. doi: 10.1016/j.virol.2017.05.005

Human Papillomavirus 16 E6 and E7 oncoprotein expression alters microRNA expression in extracellular vesicles

Mallory E Harden 1,2, Karl Munger 2,*
PMCID: PMC5506845  NIHMSID: NIHMS875751  PMID: 28500882

Abstract

Extracellular vesicles released by cancer cells are mediators of intercellular communication that have been reported to contribute to carcinogenesis. Since they are readily detected in bodily fluids, they may also be used as cancer biomarkers. The E6/E7 oncoproteins drive human papillomavirus (HPV)-associated cancers, which account for approximately 5% of all human cancers worldwide. Here, we investigate how HPV16 E6/E7 oncogene expression in primary human epithelial cells alters miR expression in extracellular vesicles and compare these to changes in intracellular miR expression. Examining a panel of 68 cancer related miRs revealed that many miRs had similar expression patterns in cells and in extracellular vesicles, whereas some other miRs had different expression patterns and may be selectively packaged into extracellular vesicles. Interestingly, the set of miRs that may be selectively packaged in HPV16 E6/E7 extracellular vesicles is predicted to inhibit necrosis and apoptosis.

Keywords: microRNA, cervical cancer, exosomes, human papillomaviruses

INTRODUCTION

Extracellular vesicles (EVs) are lipid bilayer surrounded structures, ranging from 40 nm to several μm in size, that are released by a variety of cells, including tumor cells. There are multiple types of EVs, which differ in size, biogenesis and molecular composition. EVs are classified into three main groups based on their biogenesis and size: membrane shedding-, multivesicular body- and apoptotic-derived (Kim et al., 2017). Membrane shedding EVs are derived from budding of the plasma membrane, range from 0.2 to 1 μm in diameter and are referred to as microvesicles or microparticles (Muralidharan-Chari et al., 2010; Ratajczak et al., 2006). Exosomes are small EVs (40 to 150 nm) that originate from the late endosomal trafficking machinery (Yanez-Mo et al., 2015). Their biogenesis involves accumulation in multivesicular bodies and release through fusion with the plasma membrane (Pan et al., 1985). Apoptotic derived EVs, or apoptotic bodies, have diameters ranging from 0.5 to 2 μm, and are released via blebbing of the plasma membrane of apoptotic cells (Hristov et al., 2004; Kranich et al., 2008).

Exosomes have recently been the focus of intense interest in cancer research. Exosomes released from cancer cells can promote tumorigenesis through multiple mechanisms including influencing the tumor microenvironment, providing immune system regulation and stimulating angiogenesis (reviewed in (Ciardiello et al., 2016)). In fact, the cargo of cancer-derived exosomes, comprised of functional proteins, microRNAs (miRs), DNA and/or mutated mRNA, can have oncogenic or tumor suppressive activities. Furthermore, the ability of exosomes to condition the pre-metastatic niche has been shown in vivo (Peinado et al., 2012). Some miRs packaged in exosomes may regulate the expression of target RNAs in recipient cells but other functions have also been uncovered. For example, exosome-associated miRs can be ligands for Toll-like receptors (TLRs), resulting in induction of the immune response or inhibition of macrophage activation through suppression of TLR signaling (Alexander et al., 2015; Chen et al., 2013; Fabbri et al., 2012; Phinney et al., 2015).

The first report investigating exosomes in HPV18 positive HeLa cervical carcinoma cells showed that silencing of E6/E7 expression in HeLa cells led to reduced Survivin levels in exosomes and an increase in the overall amount of exosomes released from HeLa cells (Honegger et al., 2013). The E6 and E7 proteins were not detected in HeLa exosomes by this group (Honegger et al., 2013) but a more recent study detected E6 and E7 mRNAs in exosomes released from HPV16 E6/E7 expressing primary human foreskin keratinocytes (HFKs) (Chiantore et al., 2016).

Just these few studies have examined miRs in exosomes released from HPV containing cells. An RT-PCR based study with HPV16 E6/E7 expressing HFKs detected only 8 of the 384 miRs that were included in their assay, with miR-222 being the most highly abundant (Chiantore et al., 2016). In another study, the authors silenced HPV18 E6/E7 expression in HeLa cells and showed that expression of HPV18 E6/E7 determined expression of seven exosomal miRs, and that these miRs possess pro-proliferative or anti-apoptotic potential (Honegger et al., 2015). Silencing E6/E7 expression in the HPV16 positive SiHa cervical cancer line identified a similar set of HPV16 E6/E7 regulated miRs in exosomes (Honegger et al., 2015). Additionally, determination of exosomal miRs in cervicovaginal lavage specimens of cervical cancer patients showed that miR-21 and miR-146a levels were significantly higher in vesicles from HPV positive cervical cancer patients (Liu et al., 2014).

Understanding the results of some studies and comparisons between studies has been difficult since various methods have been used for exosome isolation. It is now clear that all the existing isolation methods for exosomes also yield various amounts of other EVs (Choi et al., 2012; Haqqani et al., 2013; Muralidharan-Chari et al., 2009) and it may be prudent to refer to these preparations as exosome-enriched EVs (Exo-EVs).

Here, we investigated expression of a panel of 68 cancer-related miRs in cells, in exosome-enriched EVs (Exo-EVs) released by HPV16 E6/E7 expressing HFKs, and matched control vector transduced HFKs. We show that most miRs analyzed are similarly regulated by E6/E7 expression in cells and in Exo-EVs. Some miRs, however, are expressed differently in cells and in Exo-EVs, suggesting that several miRs may be selectively packaged in Exo-EVs secreted by HPV16 E6/E7 expressing cells. Interestingly, these selectively packaged miRs are predicted to inhibit apoptosis and necrosis. Our results, therefore, agree with and extend previous studies (Honegger et al., 2015) that suggest expression of the high-risk HPV oncoproteins alters the expression of miRs secreted in EVs.

MATERIALS AND METHODS

Cell Culture

HFKs were isolated and maintained in keratinocyte-serum-free media (KSFM) as previously described (Harden et al., 2017). HFKs were transduced with LXSN based recombinant retroviruses encoding both HPV16 E6 and E7 (Halbert et al., 1991) or a control LXSN vector as previously described (Harden et al., 2017). Retroviral transduction of HFKs was validated by immunoblotting and RT-qPCR to assess the protein and RNA levels of HPV16 E6 and E7, respectively. HFKs were grown to 80% confluence prior to passaging and only passaged up to 8 times. In all experiments, donor and passage matched HFK populations were used.

Isolation of Exosome-enriched Extracellular Vesicles

For the isolation of Exo-EVs, HFK media was cleared of endogenous exosomes present in bovine pituitary extract (Riches et al., 2014), which is a component of KSFM, by ultracentrifugation at 100,000 x g for 24 hours at 4°C. The cleared media was then added to HPV16 E6/E7 expressing and matched control vector transduced HFKs for 24 hours and then used for Exo-EV isolation using Invitrogen’s Total Exosome Isolation Reagent (from cell culture media) according to the manufacturer’s instructions.

RNA methods

Total RNA was harvested from cells and Exo-EVs using the mirVana miR Isolation Kit (Ambion) according to the manufacturer’s instructions. Cellular RNA sample concentrations were determined using a NanoDrop2000c spectrophotometer (Thermo Fisher Scientific). The quantity and quality of RNA from Exo-EVs was determined by Agilent Bioanalyzer 2100 with a Small RNA Chip.

For miR RT-qPCR, total RNA was reverse transcribed with the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Life Technologies) as described in the manufacturer’s protocol, utilizing miR-specific, stem loop primers (Applied Biosystems, Life Technologies). TaqMan® MicroRNA Assays (Applied Biosystems, Life Technologies) were employed to detect miR-16-5p and miR-34a-5p using the comparative Ct method with the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Assay IDs 000391 and 000426 were used to quantify miR-16-5p and miR-34a-5p, respectively. RT-qPCR assays were performed in triplicate and the non-coding small nuclear RNA (snRNA) U6 (Assay ID: 001973) was utilized as an endogenous, small RNA control.

Protein methods

Protein lysates from cells and Exo-EVs were prepared in ML buffer (300 mM NaCl, 0.5% Nonidet P-40 [NP-40], 20 mM Tris-HCl [pH 8.0], 1 mM EDTA) supplemented with one Complete EDTA-free Protease Inhibitor Cocktail tablet (Roche) per 50 ml lysis buffer. Protein concentrations were determined via the Bradford method (Bradford, 1976). Samples from cells and Exo-EVs containing 60 μg of protein were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Primary antibodies were as follows: RIG-I, AG-20B-0009-C100 (Adipogen), 1:1,000; HSC70, sc-7298 (Santa Cruz Biotechnology), 1:200; CD9, C9993 (Sigma-Aldrich), 1:500; Rab5B, sc-598 (Santa Cruz Biotechnology) 1:100; Histone H3, 17-10046 (EMD Millipore), 1:1,000; actin, MAB1501 (Millipore) 1:1,000. Secondary anti-mouse IgG and anti-rabbit IgG horseradish peroxidase-conjugated antibodies (Amersham) were used at 1:10,000 dilutions. Proteins were visualized by enhanced chemiluminescence (Luminata™ Crescendo Western HRP Substrate; Millipore) and electronically acquired with a Syngene G:BOX image station (Syngene) equipped with GeneSys software, v1.5.6.0.

Nanoparticle Tracking Analysis (NTA)

Determination of Exo-EV concentration and size was performed using a NanoSight NS300 with a high sensitivity sCMOS camera and a green 532 nm laser (Malvern). NTA allows the visualization and analysis of extracellular particles by relating Brownian motion to particle size. Exo-EV samples were analyzed at 1:1,000 dilutions to achieve the optimal particle number (20–100 particles) in the field of view.

FirePlex® miR profiling

To examine the expression of miRs in Exo-EVs, we utilized the FirePlex® miR oncology assay to detect a panel of 68 cancer-related miRs. This assay utilizes three-dimensional hydrogel particles encoded with unique miR “barcodes” made by optical liquid stamping (Chapin et al., 2011; Chapin and Doyle, 2011). RNAs isolated from Exo-EVs released from two, independent passage and donor matched populations of HPV16 E6/E7 expressing and control vector transduced HFKs were submitted for FirePlex® miR profiling. Data analysis was performed using the Firefly® Analysis Workbench software (http://www.abcam.com/kits/firefly-analysis-workbench-software-for-multiplex-miR-assays). Unpaired, two-tailed t-tests with a 95% confidence level were performed to determine statistical significance between cell and Exo-EV samples.

RESULTS

Characterization of exosome-enriched extracellular vesicles released from human foreskin keratinocytes

To validate our Exo-EV preparations, we examined several marker proteins. Our preparations scored positive for the exosome markers HSC70, CD9 and RAB5. In contrast, RIG-I, Actin and Histone H3, which should not be present in exosomes, were not detected in our EV preparations (Figure 1A). These results are consistent with the isolation of exosomes. However, since we cannot rule out that other EVs may be also present in our samples, we refer to these preparations as exosome-enriched EVs (Exo-EVs).

Figure 1.

Figure 1

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Biochemical and biophysical characterization of extracellular vesicles isolated from HPV16 E6/E7 expressing primary human foreskin keratinocytes. (A) Immunoblot analysis of exosome markers HSC70, CD9 and RAB5 and non-exosome associated proteins RIG-I, actin and histone H3. (B) Size determination and quantification of extracellular vesicles by Nanoparticle Tracking Analysis. See text for details.

Analysis of the size and concentration of exosome-enriched extracellular vesicles released from human foreskin keratinocytes

To analyze the size and concentration of Exo-EVs released from HFKs, we performed nanoparticle tracking analysis (NTA). NTA tracks the movement of single particles, as small as 10 nm in diameter, in a suspension under Brownian motion. The particles scatter light when illuminated by a laser, which is captured by a high sensitivity camera. Software is then used to track the motion of each particle from frame to frame and the rate of particle movement is related to a sphere equivalent hydrodynamic radius as calculated through the Stokes-Einstein equation (Dragovic et al., 2011; Hole et al., 2013). A representative size distribution profile of Exo-EVs released from HPV16 E6/E7 expressing HFKs as determined by NTA in shown in Figure 1B. The representative size distribution profile shows several peaks, one at 67 nm, one at 89 nm and one at 121 nm. This analysis shows that HPV16 E6/E7 expressing HFKs release EVs in the size range of exosomes and allowed us to determine the concentration of Exo-EVs in each sample for downstream analysis.

Validation of the FirePlex® miR Assay

We compared intracellular expression of miRNA-16-5p and miRNA-34a-5p by miR sequencing, quantitative RT-PCR and FirePlex® miR Assay (Figure 2). These analyses show that the FirePlex® miR Assay yields expression data that are consistent with other detection methods. In addition, there was excellent agreement of intracellular miR expression in control and HPV16 E6/E7 expressing HFKs with our previously published miR sequencing data (Harden et al., 2017).

Figure 2.

Figure 2

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Quantification of intracellular miR-16-5p (top) and miR-34a-5p (bottom) expression in different populations of HPV16 E6/E7 expressing primary human foreskin keratinocytes (black bars) and matched control vector transduced primary human foreskin keratinocytes (white bars) by microRNA sequencing (miR-seq) (Harden et al 2017), reverse transcription quantitative PCR (RT-qPCR) and Fireplex® miR assays. Y-axis values are relative to matched control vector transduced primary human foreskin keratinocytes.

Expression of HPV16 E6/E7 alters the expression of miRs in exosome-enriched extracellular vesicles released from human foreskin keratinocytes

Total RNA was harvested from cells and Exo-EVs released from two, independent populations of HPV16 E6/E7 expressing HFKs and matched control HFKs. We used the FirePlex® miR oncology assay to assess levels of a panel of 68 cancer related miRs. As a control, we also utilized the same method to analyze intracellular miR expression in total RNA from the same HFK samples. The Firefly® Analysis Workbench was used to analyze the expression of cellular and Exo-EV-associated miRs. Unfiltered expression data for all 68 miRs examined in the oncology panel are shown in Supplemental Table 1. Each of the two matched keratinocyte populations is prepared from three or more foreskin samples each from a different donor and it is not unusual to observe considerable variation between different keratinocyte preparations. Our analysis included all miRs with expression above the limit of detection of the assay and those that showed consistent results in expression (HPV16 E6E7/C) in both HFK populations tested. This resulted in 31 differentially expressed miRs in Exo-EVs and 48 intracellular miRs above the limit of detection of the assay with consistent results in expression in both HFK populations tested (Table 1). Of the differentially expressed miRs in Exo-EVs, 19 were upregulated and 12 were downregulated and, of the differentially expressed intracellular miRs, 31 were upregulated and 17 were downregulated (Table 1). Overall, these results show that HPV16 E6/E7 expression in HFKs alters the expression of miRs in Exo-EVs released from these cells compared to control vector transduced HFKs.

Table 1.

Expression of miRs in two independently derived populations of HPV16 E6/E7 expressing HFKs (Cells) and in exosome-enriched extracellular vesicles (Exo-EV) as compared to control HFKs. FC, Fold Change; SD, Standard Deviation. Only miRs that showed the same trend in expression in both populations are listed

FC
(E6E7/C)
Cells		 SD
(E6E7/C)
Cells		 FC
(E6E7/C)
Exo-EVs		 SD
(E6E7/C)
Exo-EVs
let-7d-5p 1.31 0.091 – –
let-7g-5p 1.52 0.244 – –
let-7i-5p – – 0.47 0.225
miR-9-5p 2.13 0.076 – –
miR-10b-5p 1.22 0.212 – –
miR-15b-5p 1.28 0.176 – –
miR-16-5p 1.37 0.02 1.04 0.037
miR-17-5p 1.15 0.201 – –
miR-18a-5p – – 2.24 1.089
miR-19a-3p 1.77 0.195 1.84 1.068
miR-20a-5p 1.35 0.081 – –
miR-21-5p 0.91 0.097 0.58 0.106
miR-22-3p – – 0.44 0.158
miR-25-3p 2.21 0.647 3.2 2.335
miR-29a-3p 0.78 0.059 – –
miR-29b-3p 0.76 0.097 – –
miR-29c-3p 0.96 0.017 – –
miR-34a-5p 0.24 0.074 0.37 0.081
miR-92a-3p 1.1 0.029 – –
miR-93-5p 1.29 0.098 1.68 0.624
miR-103a-3p – – 1.6 1.635
miR-106a-5p 1.28 0.126 – –
miR-106b-5p 1.85 1.113 2.35 1.635
miR-107 0.89 0.029 1.51 0.634
miR-125b-5p 0.95 0.065 – –
miR-127-3p 0.64 0.171 – –
miR-130a-3p – – 1.54 0.59
miR-141-3p 0.94 0.008 – –
miR-146a-5p 0.48 0.202 – –
miR-148a-3p 1.34 0.271 – –
miR-148b-3p 1.47 0.274 1.51 0.311
miR-150-5p 0.79 0.224 – –
miR-151a-3p – – 0.76 0.158
miR-155-5p 2.84 0.814 2.31 1.643
miR-181a-5p 1.21 0.084 – –
miR-182-5p 0.91 0.08 4.23 4.179
miR-187-3p 0.81 0.239 – –
miR-192-5p – – 0.84 0.145
miR-195-5p 1.71 0.994 1.3 0.125
miR199a-3p 1.46 0.481 – –
miR-199a-5p 3.07 2.107 – –
miR-200b-3p 1.39 0.2 0.92 0.053
miR-200c-3p 0.96 0.026 – –
miR-205-5p 1.14 0.098 0.71 0.193
miR-210-3p 0.8 0.045 – –
miR-218-5p 1.45 0.402 2.04 1.36
miR-221-3p – – 0.63 0.368
miR-222-3p 0.85 0.004 1.65 0.362
miR-320a 1.47 0.03 0.47 0.284
miR-335-5p 2.44 0.239 2.87 1.939
miR-375 1.41 0.04 2.96 1.485
miR-376c-3p 0.56 0.295 0.71 0.137
miR-378a-3p 1.68 0.213 0.61 0.307
miR-574-3p 1.81 0.633 – –
miR-625-3p 1.43 0.46 2.59 0.705
miR-652-3p 1.2 0.225 1.68 0.139
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Comparing intracellular miR expression and miRs in exosome-enriched extracellular vesicles

We then compared the expression of miRs in cells and in Exo-EVs. As before, we only included miRs with expression above the limit of detection of the assay and those that showed consistent modulation of expression (HPV16 E6E7/C) in both HFK populations tested. There were 23 miRs that met these criteria, both intracellularly and in Exo-EVs (Table 2). To determine miRs with expression patterns that were statistically different between cells and Exo-EVs, we utilized unpaired, two-tailed t-tests with a 95% confidence level, comparing the expression of each miR in cells to expression of the same miR in Exo-EVs. We found that 16 miRs are similarly regulated intracellularly and in Exo-EVs, with most miRs upregulated intracellularly and in Exo-EVs and only a few miRs downregulated intracellularly and in Exo-EVs. In contrast, seven miRs showed a different abundance in Exo-EVs than intracellularly. Together, our data show that HPV16 E6/E7 expression alters the expression of many miRs in a similar manner intracellularly and in Exo-EVs. However, the expression of some miRs is different intracellularly as compared to Exo-EVs.

Table 2.

Expression of miRs detected in HPV16 E6/E7 expressing HFKs (Cells) and the corresponding exosome-enriched extracellular vesicles (Exo-EVs) as compared to control HFKs. FC, Fold Change; SD, Standard Deviation; ns, not statistically significant. Only miRs above the limit of detection of the assay, that showed the same trend in expression in both HFK populations tested and with data from cells and Exo-EVs are listed.

FC
(E6E7/C)
Cells		 SD
(E6E7/C)
Cells		 FC
(E6E7/C)
Exo-EVs		 SD
(E6E7/C)
Exo-EVs		 P value FC
Cells vs
Exo-EVs
miR-16-5p 1.37 0.02 1.04 0.037 0.0002
miR-19a-3p 1.77 0.195 1.84 1.068 ns
miR-21-5p 0.91 0.097 0.58 0.106 0.0176
miR-25-3p 2.21 0.647 3.2 2.335 ns
miR-34a-5p 0.24 0.074 0.37 0.081 ns
miR-93-5p 1.29 0.098 1.68 0.624 ns
miR-106b-5p 1.85 1.113 2.35 1.635 ns
miR-107 0.89 0.029 1.51 0.634 ns
miR-148b-3p 1.47 0.274 1.51 0.311 ns
miR-155-5p 2.84 0.814 2.31 1.643 ns
miR-182-5p 0.91 0.08 4.23 4.179 ns
miR-195-5p 1.71 0.994 1.3 0.125 ns
miR-200b-3p 1.39 0.2 0.92 0.053 0.0159
miR-205-5p 1.14 0.098 0.71 0.193 0.0255
miR-218-5p 1.45 0.402 2.04 1.36 ns
miR-222-3p 0.85 0.004 1.65 0.362 0.0191
miR-320a 1.47 0.03 0.47 0.284 0.0038
miR-335-5p 2.44 0.239 2.87 1.939 ns
miR-375 1.41 0.04 2.96 1.485 ns
miR-376c-3p 0.56 0.295 0.71 0.137 ns
miR-378a-3p 1.68 0.213 0.61 0.307 0.0077
miR-625-3p 1.43 0.46 2.59 0.705 ns
miR-652-3p 1.2 0.225 1.1 0.139 ns
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Analysis of differentially expressed miRs in Exo-EVs released from HPV16 E6/E7 expressing human foreskin keratinocytes

To uncover potential functions of miRs in Exo-EVs, we utilized the core analysis function of Ingenuity Pathway Analysis (Qiagen), which identifies relationships, mechanisms, functions and pathways of relevance to a particular dataset. Specifically, we compared core analyses of miRs that were expressed similarly intracellularly and in Exo-EVs and those miRs that were differentially expressed intracellularly and in Exo-EVs.

The molecular/cellular functions the two sets of miRs had in common were “cell cycle, development and cell to cell signaling.” For the miRs that were similarly regulated by expression of HPV16 E6/E7 intracellularly and in Exo-EVs, the top molecular and cellular functions that were unique to that dataset were “cell growth and proliferation” and “cell movement”. In contrast, for the miRs regulated differently by HPV16 E6/E7 intracellularly and in Exo-EVs, distinct molecular and cellular functions were “cell death and survival” and “cellular compromise.” “Cellular compromise” refers to any process that may compromise the function of the cell, as well as functions associated with damage or degeneration of cells, including cellular atrophy, damage, disruption and swelling. When we examined the miRs regulated differently by HPV16 E6/E7 intracellularly and in Exo-EVs more closely, we found that within the “cell death and survival” category, “necrosis” and “apoptosis” were predicted to be inhibited by this group of miRs, although the Z-scores were not significant (−1.170 and −1.053, respectively) (Table 3). Nonetheless, these results suggest that miRs that are selectively packaged into Exo-EVs of HPV16 E6/E7 expressing HFKs may inhibit cell death.

Table 3.

miRs that are differentially expressed (16E6E7/C) intracellularly and in exosome-enriched extracellular vesicles are predicted to inhibit cell death and promote survival

Cell Death/Survival Activation Z-score P-value Associated miRs
necrosis −1.17 0.00699 miR-16-5p, miR-200b-3p, miR-222-3p, miR-320a, miR-378a-3p,
apoptosis −1.053 0.00673 miR-16-5p, miR-200b-3p, miR-222-3p, miR-320a, miR-378a-3p
cell viability 0.262 0.00328 miR-16-5p, miR-200b-3p, miR-222-3p, miR-378a-3p
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DISCUSSION

Our study examined how EV-associated miRs are altered by expression of the high-risk HPV E6 and E7 oncoproteins, the major drivers of HPV-associated cancers. As there is no consensus on a gold standard method for exosome isolation, it cannot be claimed that there is an optimal method that should be used and the commercial kit we utilized in this study has been thoroughly compared to other methods for the isolation of exosomes (Lotvall et al., 2014).

To biochemically analyze our exosome preparations, we followed recommendations from the International Society for Extracellular Vesicles (Lotvall et al., 2014). While there are no true exosomes-specific markers, there are proteins that are exosomes-enriched, and it is recommended to examine three or more of these proteins from the following categories: 1) transmembrane/lipid-bound extracellular proteins; 2) cytosolic proteins; 3) intracellular proteins (Lotvall et al., 2014). We analyzed six different proteins: RIG-I, HSC70, actin, CD9, RAB5 and H3. RIG-I is a cytosolic RNA sensor of the innate immune system, is not packaged into exosomes (Boelens et al., 2014) and we did not observe this protein in our samples. HSC70 is a chaperone protein found in exosomes from most cell types (Geminard et al., 2004; Thery et al., 2001) and we detected HSC70 in our samples. We did not detect actin in our samples, indicating that our preparations were free of cellular debris (Angeloni et al., 2016). The CD9 tetraspanin is expected to be present in exosomes (Lotvall et al., 2014) and we detected this protein in our samples. Endosome or membrane binding proteins, such as RAB5, are expected to be present in exosomes (Lotvall et al., 2014), and we detected RAB5 in our samples. Nuclear histones, such as H3, should be absent in exosomes, but are present in some other EV types (Lotvall et al., 2014), and we did not observe H3 in our preparations. Overall, our biochemical characterization of our Exo-EV preparations meets and exceeds the requirements recommended by the International Society for Extracellular Vesicles for the characterization of exosomes.

We also utilized NTA to assess the size of Exo-EVs, and to determine the concentration of Exo-EVs in our samples. We found that HPV16 E6/E7-expressing HFKs release three distinct populations of Exo-EVs of 67, 89 and 121 nm in diameter. This might seem surprising, as it is generally assumed that exosomes are a homogeneous population. However, a recent study revealed that multiple cell types release more than one subpopulation of exosomes (Willms et al., 2016). These subpopulations were shown to carry different protein and RNA cargoes, suggesting that they may have distinct biological activities on recipient cells (Willms et al., 2016). Hence, HPV16 E6/E7 expressing HFKs release may also release several Exo-EV subpopulations and it will be interesting to determine whether they each carry unique cargoes, and have different activities on neighboring cells. Due to the amount of sample required for NTA, we were only able to investigate Exo-EV size distributions from HPV16 E6/E7 expressing HFKs and it will be interesting to determine whether HPV16 E6/E7 expressing cells induces alterations in the abundance of specific Exo-EV populations.

There are only a few studies of miRs contained in exosomes secreted from HPV expressing cells and clinical lesions (Chiantore et al., 2016; Honegger et al., 2013; Honegger et al., 2015). One previous study found miR-222-3p to be significantly expressed in exosomes from HPV16 expressing HFKs (Chiantore et al., 2016). We also found miR-222-3p to be upregulated in our HPV16 E6/E7 Exo-EV samples. Interestingly, this miR is downregulated in HPV16 E6/E7 expressing HFKs but upregulated in Exo-EVs released from the same cells. The same study (Chiantore et al., 2016) also detected miR-320a in exosomes and we found miR-320a to be expressed in exosomes released from HPV16 E6/E7 expressing HFKs as well. This miR was upregulated in HPV16 E6/E7 HFKs but downregulated in exosomes released from these cells.

We also observed some overlap with another study, which utilized deep sequencing to examine exosomes released from HeLa cells in which expression of HPV18 E6/E7 was silenced (Honegger et al., 2015). Consistent with this study we also detected miR-21-5p, -222-3p, -320a and -378a-3p in our Exo-EVs. Two of these miRs, miR-378a-3p and miR-21-5p, are part of the seven miR signature associated with HPV18 E6/E7 oncogene expression that was identified in this study (Honegger et al., 2015). An important finding from our analysis of miR expression in Exo-EVs released by HPV16 E6/E7 expressing HFKs was that some miRs are expressed similarly in Exo-EVs and intracellularly whereas others are not. This is consistent with what has been previously reported for cancer-associated EVs. In some cases, the miR content of exosomes mirrors miR expression in the tumor (Rabinowits et al., 2009; Taylor and Gercel-Taylor, 2008). However, in other cases, some miRs are much more highly abundant in exosomes than within tumor cells, suggesting that these miRs are preferentially packaged in exosomes (Jaiswal et al., 2012; Pigati et al., 2010). Any miRs expressed at lower or higher levels in exosomes released from HPV16 E6/E7 expressing cells compared to control HFKs (Table 1) may potentially serve as biomarkers for HPV-associated diseases and cancers. Candidates include miR-21-5p, identified in our study and one other (Honegger et al., 2015), as well as miR-222-3p, -320a and -378a-3p that were observed in our experiments and two other studies (Chiantore et al., 2016; Honegger et al., 2015). Pathway analysis of the miRs that are differentially expressed in HPV16 E6/E7 HFK secreted Exo-EVs than intracellularly suggest that these miRs inhibit necrosis and apoptosis. While the z-scores associated with necrosis and apoptosis were not significant, this is likely due to the small list of miR analyzed. The transfer of exosomes by other cell types to recipient cells has been previously linked to effects on apoptosis (Rivoltini et al., 2016; Yang et al., 2015) and necrosis (Nong et al., 2016) and our data suggest that expression of HPV16 E6/E7 inhibits apoptosis and necrosis in neighboring normal cells through miRs secreted in Exo-EVs. Supporting this finding, one of the few previous studies of HPV-associated miRs in exosomes reported that several of E6/E7-dependent exosomal miRs were linked to control of cell proliferation and apoptosis (Honegger et al., 2015).

Supplementary Material

supplement

Highlights.

  • Human papillomavirus E6/E7 expression causes changes in microRNAs excreted in exosomes

  • The microRNA changes mostly parallel those observed intracellularly

  • Some microRNAs are differentially modulated intracellularly and in exosomes

Acknowledgments

We thank Drs. Jessica D. Tytell and Jonathan G. Mehtala for their help with the Fireplex® miR assay and Nanoparticle Tracking Analysis, respectively. We also thank Jessica J. Chiang in the laboratory of Dr. Michaela Gack for the RIG-I antibody and Dr. Katharina Bernhardt for helpful discussions on exosome isolation and analysis. Supported by PHS grants F31CA180516 (MEH) and R01CA066980 (KM).

Footnotes

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