The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research, but has left many unanswered questions about how this regulation works and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in blood plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations; and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication^[1-5]^. Such a system would be expected to export specific miRNAs from cells in response to specific biological stimuli. We report here that after serum deprivation several human cell lines tested do export a spectrum of miRNAs into the culture medium. The export response is substantial and prompt. The exported miRNAs are found both within and outside of microvesicles and exosomes. We have identified some candidate protein components of this system outside the cell, and found one exported protein that plays a role in protecting miRNA from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that may involve cell-cell communication.
miRNA export from mammalian cells8/29/09Mammalian cells in culture actively export specific microRNAsKai Wang,♦ Shile Zhang, Jessica Weber, David Baxter and David J. Galas∗ Institute for Systems Biology 1441 N. 34th Street Seattle, WA 98103 The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research, but has left many unanswered questions about how this regulation works and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in blood plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations; and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication [1-5]. Such a system would be expected to export specific miRNAs from cells in response to specific biological stimuli. We report here that after serumdeprivation several human cell lines tested do export a spectrum of miRNAs into the culture medium. The export response is substantial and prompt. Theexported miRNAs are found both within and outside of microvesicles and exosomes. We have identified some candidate protein components of this system outside the cell, and found one exported protein that plays a role in protecting miRNA from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that may involve cell-cell communication. [234 words]♦ ∗firstname.lastname@example.org email@example.com , correspondence should be addressed to this author.1*miRNA export from mammalian cells8/29/09One of the hypotheses advanced to explain the biological significance of the presence of miRNA in the plasma is that they are part of a cell-cell communication system. This biological function would require that the miRNAs convey specific information, and therefore that only certain miRNAs are exported from cells in response to biological stimuli. This export mechanism would have to be able to select specific miRNAs for export in response to the specific cellular state of the exporting cell, and transport these to the extra-cellular space in a form suitable to be taken up by another cell. knowledge no such mechanism has yet been described or even hypothesized. To ourFor cells in culture, we report here observations that indicate the existence of such a system. Specifically, we asked whether there is a significant difference between the miRNA spectrum within growing cells and the spectrum of miRNAs found in the external medium. We examined this question using miRNA array profiling for several different cell lines. The results, shown in figure 1a, are that the several different cell types examined all exhibited striking differences between internal and external spectra(groups marked as “out” or “in” on the figure for HepG2). While there could be several possible explanations for this, including preferential stability inside or outside the cell, these results are consistent with the specific export hypothesis. We carefully looked for evidence of cell lysis in all these cultures (by measuring the levels of LDH in the medium at several time points, and examining the cells and culture media microscopically). The LDH levels in serum free media showed no significant changes during the experimental period, up to 48 hours of serum depletion (data not shown.) Finding no detectable evidence of lysis we concluded that the external miRNAs are exported from intact cells, consistent with the observed striking difference between intracellular and extracellular miRNA spectra (see below.)A strong biological stimulus to cells growing in culture is caused by depriving them of the various factors present in culture medium supplemented with serum. Cells must alter their internal states significantly when so stimulated, including cell cycle arrest and in some cases cell death during extended periods of serum depletion . Thus, a testablehypothesis is that when deprived of serum cells respond to their change of state in part by2*miRNA export from mammalian cells8/29/09exporting miRNAs. To test this, we carefully characterized the miRNA spectra of two different cell lines long established in culture, A549 and HepG2, which were derived from different sources. HepG2 is derived from a human hepatocellular carcinoma, and A549 from lung epithelial carcinoma. The absence of miRNAs present in bovine serum is a distinct advantage of using serum depletion to study extracellular miRNA as it removes a potential source of interference. When these cells are stimulated by serum deprivation, several notable changes of miRNA levels occur. Validation and crosschecking of the array results, shown in figure 1a, was performed for HepG2 and A549 by Q-PCR on selected miRNAs, as indicated in figure 1b and c. There are 499 and 578 observable miRNA species in A549 cells and HepG2 cells, respectively. Among these there are 476 miRNAs in common between the two cell lines. Fewer than 5% of the miRNAs observed in A549 cells are not observed in HepG2, and fewer than 18% of the miRNAs observed in HepG2 are not observed in A549. In the culture media, thepercentages are similar (2% of the miRNAs observed from A549 are not observed from HepG2, and 15% of the miRNAs observed from HepG2 are not observed from A549). Based on the global miRNA profiling results, we selected a set of 30 miRNAs that showed good signals and consistent measurement profiles with Q-PCR to study in more detail.In order to track the cellular responses carefully we carried out a time series of measurements from just before serum deprivation (SD) to 48 hours after SD. Thisrevealed some dramatic differences in the profile of external miRNAs in time, not only among specific species, but also some differences between cell lines. If we compare the changes in levels of certain miRNAs inside and outside the cells, before and after serum deprivation, the rate of export for most of the miRNAs examined increases dramatically during the first 2 hours after SD and declines thereafter (figure 2.)One observation from these data was that some miRNAs exhibited a modest decline in concentration in the medium after increasing strongly in the first few hours (e.g. miR135a, 133b). To determine whether these miRNAs were specifically being degraded in the serum-free culture medium, we carried out extensive time point measurements of the3*miRNA export from mammalian cells8/29/09decay of miRNA in media from which the cells were removed.The data clearly show(figure 4c, d) that different miRNAs decay at different rates. As shown in Figure 3 and in the supplementary material (S1), the decay of miRNAs in the medium over many hours is readily detectable, but not severe for most miRNA species. The overall decay rate doesn’t appear to be greater than 0.02/hr (this decay constant is defined in Figure 3 as the coefficient in the exponent of 2) in any single case.The time series data, taken together with the miRNA decay measurements, enables us to estimate the quantitative kinetics of the export of miRNA from cells after SD. Since the levels rise rapidly in the first 2 hours or so after SD and then remain roughly constant or decay somewhat, we conclude that for these miRNAs there is a strong pulse of exported miRNA that rapidly subsides, accompanied by the steady, but relatively slow, decay of the miRNA outside the cell. Modeling of simple kinetics of this kind shows that most of our data is consistent with the general scheme of kinetics illustrated in Figure 3c. This kinetic profile, with a rapid rise in export rate, raises the question of whether or not the miRNA exported is pre-synthesized and prepackaged.We measured the intracellular levels as well as the exported miRNA levels in the medium after SD for a large set of miRNAs from both cell lines. We illustrate our general observations in figure 3d: an initial intracellular drop in miRNA levels simultaneous with a strong increase in external levels. The Q-PCR data shown here for 3 representative miRNAs also indicates a recovery of intracellular levels after a few hours (2-6 hrs). In order to see the overall behavior of intracellular miRNA dynamics, we averaged all 24 of the exported miRNA that were measured at each time point. These 24 miRNAs (those that exhibited standard deviation of the 3 replicates of less than 1.4) provide us with strong qualitative view of the dynamics. The corresponding average dynamics of exported miRNA concentration in the medium during a period of 30 hours after SD are shown in figure 3e. An expanded view of the behavior of these miRNA averaged miRNA dynamical levels during the first 2 hours after SD, inside and outside the cell, shown in figure 3f, illustrates the precise behavior expected from the kinetic model we propose. In addition, it seems that the levels of intracellular miRNA overshoot then tend4*miRNA export from mammalian cells8/29/09to relax back to their original levels. They are notably closer to the pre-SD levels after 24 hours (Figure 3e). The relatively short export response time and this analysis stronglysuggest that most of the exported miRNAs are initially from a pre-synthesized pool since the substantial export in the first hour is unlikely to come from newly transcribed and processed miRNA. The initial reduction in intracellular levels is consistent with this view. This hypothesis will be carefully investigated in the future.Up to now, observations of miRNA outside cells have been focused on known cellderived vesicles, like micro-vesicles and exosomes [1,2]. In the serum deprivationsystem used here, we were able to examine the question of how the exported miRNA are contained or packaged in more detail. We fractionated the medium containing theexported miRNAs and determined the miRNA content of each fraction. The absence of interfering bovine miRNAs, proteins, and other bovine serum derived biological products in the serum-free medium enables quantitative measurements that are more accurate and reproducible than possible otherwise. Using well-developed differential centrifugation based methods for micro-vesicle and exosome isolation , we fractionated the media isolated from the cultures 2 hours after SD, into 4 fractions (Figure 4a). The microvesicle, exosome, “hard spin” pellet (the latter is the pellet of a final 220K x g spin for 1 hr), and supernatant fractions were then assayed for the presence of the miRNA species examined in the previous figures. The results of these measurements (3 biological replicates – repeated experiments) are presented in figure 4(b-d) and were surprising in several respects. First, counter to the expectation that miRNA is confined to previouslydescribed cell-derived vesicles; all of the miRNAs are found at significant concentrations in the final supernatant fraction in serum depletion conditions. The second surprise is that many, but not all, of the miRNAs are also found both in the vesicles and the supernatant. The patterns of distribution showed a strong diversity of profiles, falling into 3 to 5 distinct patterns (Figure 4b-h).The most extreme differences are shown in figure 4 panels b - e, where we illustrate the distributions of several miRNA species. Note that there are several miRNAs that are found in almost equal proportions in all four fractions, while several that are almost5*miRNA export from mammalian cells8/29/09completely absent from all but the supernatant fraction. This result strongly counters the idea that the presence of miRNA in the final supernatant is due to disruption of cell derived vesicles during fractionation procedures. The data also suggest that many of the vesicles remain intact (as evidenced by the high levels of some miRNA seen in this fraction). Therefore, the absence of some of the miRNAs, present in the supernatant, in the vesicle fraction cannot be explained by ruptured vesicles. Thus, while themeasurements from all these fractions demonstrate some experimental noise, it is clear that the dramatic quantitative differences represent real qualitative differences between miRNA distributions. These observations of miRNA distribution demonstrate that the miRNA export system in the cells must somehow target specific miRNA to specific extracellular structures, but does not exclusively channel specific miRNAs to a given fraction. There are, however, some miRNAs that are found in the supernatant almost to the exclusion of all other fractions (miR-219 in figure 4b, for example). Anothersignificant observation from this data is that even though we showed that the two cell lines studied here export a similar spectrum of miRNAs (figure 1b) the distributions of miRNAs from the two cell lines demonstrate clear differences, as shown in figure 4 b-h. For example, miR-145 is present at very low levels in HepG2 derived microvesicles (Figure 4d), while when this same miRNA is exported from A549 it is present at significant levels in both microvesicles and exosomes (Figure 4c). On the other hand,there are some miRNAs, notably those that are almost uniformly distributed among the fractions, with the same distribution no matter which cell-line is the source. Theseobservations certainly indicate the degree of complexity of this export system that will bear further investigation.To further investigate the miRNA exporting process, we treated the HepG2 cells with different concentration of rotenone, a respiratory chain inhibitor to reduce the cellular ATP level, and brefeldin A (BFA), an inhibitor of protein secretion through the interference with the Golgi apparatus, for 30 minutes prior to SD. As expected, the cellular ATP level showed a significant decrease in rotenone treated cells while the LDH levels in media showed no significant changes in either treatment. We then examined levels of selected miRNAs with Q PCR in both cells and culture media. The level of6*miRNA export from mammalian cells8/29/09miRNAs inside the cells showed no significant changes in either treatment; however, the levels of most of the extracellular miRNAs examined displayed a dose dependent decrease with rotenone treatment only (see data in supplementary material). This finding suggests that: 1) most of the miRNA exporting process is probably energy dependent – an active transport process, and 2) during the time frame examined, the exporting process is not affected by BFA – a process independent of the Golgi apparatus. It is interesting to note that the extracellular levels of three miRNAs we measured, mir-671-3P, mir-943 and mir-302c are not affected by the decrease of cellular ATP level, suggesting that the export pathway is more complex and involves multiple branches.To examine the protein content of the exported complexes, we collected and concentrated the primary human fibroblast medium 2 hours after SD, digested the protein contents with trypsin and examined the peptides by mass spectrometry. We then identified all the proteins that were represented by more than one peptide – an unexpectedly large total of 197 proteins. Of these, 12 were known RNA-binding proteins (Table 1). Besides several ribosomal proteins, it is notable that there was a substantial level of a known nucleolar, RNA binding protein, nucleophosmin (NPM1), in the medium. Nucleophosmin isimplicated in the nuclear export of the ribosome . There was also a significant amount of nucleolin, a known NPM1 interacting protein, in the medium. Since NPM1 is known to be located primarily in the nucleolus and involved ribosomal RNA processing, it is surprising to observe it in significant levels outside the cell. Using an antibody tonucleophosmin, we identified the NPM1 protein in culture media from other cell lines as well as HepG2 at 2 hours after SD. We also examined NPM1 protein levels in different fractions of culture medium. The vast majority of the protein is present only in the supernatant fraction (as defined in figure 4a), and is undetectable by Western blot in either vesicle fraction. Thus, the distribution of NPM1 protein in different fractions of HepG2 medium, illustrated in figure 5a, parallels the distribution of miRNAs – by far the largest concentration is found in the supernatant (figure 5b). The presence of a RNAbinding protein in this fraction, where most of the miRNAs are found, suggests miRNAs may be bound by this protein. We carried out a number of experiments, including gel shifts and immunoprecipitations (data not shown), that confirm the binding of7*miRNA export from mammalian cells8/29/09nucleophosmin to miRNAs. To see whether NPM1 might be responsible for the stability of the external miRNA, we incubated synthetic miR-122 with purified NPM1 protein and challenged the mixture with RNaseA. We found that NPM1 alone can fully protect this miRNA from digestion (figure 5c.). It has been suggested that NPM-1 may be involved in shuttling RNAs and ribosomal proteins to the cytosol , and, in a recent report, that it can be found outside the cell . This shuttling mechanism may be relevant to apossible role in miRNA export. In knockdown experiments, using siRNA against NPM1 message, we reduced the of this protein in the cells and saw corresponding reductions in miRNA export (data not shown.) Our findings collectively are highly suggestive that the miRNA-associated protein nucleophosmin is involved both in some part of the miRNA exporting process and in protecting the external miRNAs outside the cell.We have demonstrated that miRNAs are actively exported from cells after serum deprivation. They appear to be exported in a pulse lasting about one hour after serum deprivation. This export system has not been reported before, to our knowledge, and strongly suggests both the existence of a complex response system and the possible involvement of miRNA in cell-cell communication. Our results also suggest thatprevious reports of extracellular vesicle-associated miRNAs represent only a part of the extracellular miRNA distribution, at least in our cell culture conditions. A significant fraction of extracellular miRNA is, in fact, not within the known cell-derived vesicles. It is yet unclear how they are packaged outside these vesicles. This leads us to speculate that there may be at least two pathways for the packaging and export of miRNAs – exosome/microvesicle mediated and “other particle” mediated processes. We were surprised to find a number of reportedly intracellular proteins in the culture media shortly after serum depletion. At least one of these associated RNA binding proteins, NPM1, may play a role in the packaging, and perhaps the export, of extracellular miRNAs. Its role in miRNA packaging/exporting is currently being investigated.The hypothesis of miRNA-dependent cell-cell communication based on the export of miRNA seems to be the most satisfying explanation for the cellular export phenomena of miRNA. There has been circumstantial evidence in favor of this interpretation, but we8*miRNA export from mammalian cells8/29/09provide here direct evidence that: demonstrates the export system in action, characterizes some of its properties, and supports the miRNA mediated cell-cell communication idea. Our biological observations indicate an important role of miRNAs, and enable future investigations of this potential communication system, unknown until now.There are important pieces of the puzzle still missing. We have no direct evidence yet that miRNA complexes are taken up by other cells, although vesicles containing miRNA are reported to be taken up by cells. We expect that miRNA outside of vesicles,complexed with proteins, could be targeted to specific cell surface receptors, and are currently investigating this important hypothesis. Our results raise significant questions about miRNA mediated cell-to-cell communication, and enable the characterization of such a system. The elucidation of a novel, and perhaps extensive, biological information transduction system between cells will certainly be of the utmost importance in understanding many biological processes in mammalian systems including development, stress response and tissue renewal.Methods Cell culture The HepG2, A549, T98 and BSEA2B cells were obtained from The American Type Culture Collection (ATCC, Manassas, VA) and grown in recommended medium containing 10% fetal bovine serum (FBS), 100U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37 °C under 5% CO2. We determined that this strain of HepG2 does not express CYP2E1. The primary human pulmonary fibrobast cells were obtained from Sciencell Research Laboratories (Sciencell, Carlsbad, CA) and grown in Fibroblast medium (Sciencell, Carlsbad, CA) on collagen I coated cell culture flasks (Becton Dickinson, Franklin Lakes, NJ). For serum depletion experiments, the cells were inoculated and grown for 24 hours with 10% FBS containing media prior to switching to serum-free media. Serum free medium contained the basic medium as provided by Invitrogen (Carlsbad, CA) with 100U/ml penicillin and 100µg/ml streptomycin. For rotenone and brefeldin A treatments, the cell were inoculated and9*miRNA export from mammalian cells8/29/09grown for 24 hours with 10% FBS prior to exposing the cells with different concentration of drugs for 30 minutes. Lactate dehydrogenase (LDH) levels were determined with LDH-Cytotoxicity assay kit from Abcam (Abcam Inc., Cambridge, MA) and ATP levels were determined with ENLITEN ATP assay system (Promega, Madison, WI). RNA extraction Total RNA including miRNA was isolated using miRNeasy kit (Qiagen, Germantown, MD) with minor modifications that have been previously described . In summary, 700ul of QIAzol reagent was added to 300ul of culture medium samples, followed by adding 140 ul of chloroform. The samples were mixed vigorously for 15 seconds and centrifuged at 12,000x g for 15 minutes at 4°C to separate the aqueous and organic layers. After transferring the aqueous phase to a new collection tube, 1.5 volumes of ethanol were added and the samples were then applied to RNeasy mini column. Total RNA was eluted from the membrane with 30 ul of RNase-free water. Quantitative RT-PCR The cDNA was generated using the miScript Reverse Transcription kit (Qiagen, Germantown, MD). In brief, miRNAs were polyadenylated by using poly(A) polymerase and cDNA was generated with reverse transcriptase using a tag containing oligo-dT primers. The tag on oligo-dT served as universal primer in QPCR step. Human miScript Assay 384 set v10.1 (Qiagen, Germantown, MD) was used for real-time PCR analysis. To reduce pipetting error, the Matrix Hydra eDrop (Thermo Scientific, Hudson, NH) was used to mix the cDNA sample and qPCR master reagent. The data was analyzed by SDS Enterprise Database 2.3(Applied Biosystems, Foster City, CA). Microarrays miRNA microarrays were performed using the manufacturer’s (Agilent, Santa Clara, CA) protocol as previously described [ 3] . 100ng of total RNA was dephosphorylated with calf intestinal alkaline phosphate, and denatured with heat in the presence of dimethyl10*miRNA export from mammalian cells8/29/09sulfoxide (DMSO). T4 RNA ligase added the Cyanine 3-cytidine biphosphate (pCp) to the dephosphorylated single stranded RNA. MicroBioSpin 6 columns (Bio-Rad, Hercules, CA) were used to remove any unincorporated cyanine dye from the samples. The purified labeled miRNA probes were hybridized to human miRNA V2 oligo microarrays in a rotating hybridization oven at 10 rpm for 20h at 55 C. After hybridization, the arrays were washed in Agilent GE Wash Buffer 1 and 2 with Triton X-102. Then the array slides were dried immediately by a nitrogen stream and scanned at 5-um resolution by using a PerkinElmer ScanArray Express array scanner.Fractionation of culture mediumThe cells were grown as stated above. The serum free media were collected and centrifuged at 1000 x g for 10 minutes to remove cell debris. This supernatant (25 ml) was transferred to a new tube and spun at 16K x g for 60 minutes, the pellet microvesicles, were washed and resuspended in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphate dibasic, 2 mM Potassium Phosphate monobasic at pH of 7.4). The supernant of the 16K x g spun was transferred to a new tube and further centrifuged at 120K x g for 60 minutes to pellet the exosome particles. The exosome depleted supernatant was then spun at 220K x g for 60 minutes. The final supernatant was concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore, Billerico, MA) to a final volume of 0.5ml. The pellets, microvesicles, exosomes, and 220K x g pellet were resuspended in 0.5ml PBS, so that the total volume of medium contributing to each was identical.Mass Spectrometry Sample Preparation and Data AnalysisCells were grown as above and the serum free media was concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore, Billerico, MA). The concentrated media was enzymatically digested with trypsin and desalted with C18 Ultramicrospin columns (The11*miRNA export from mammalian cells8/29/09Nest Group, Southborough, MA). After drying in a Savant speedvac (Thermo Scientific, Waltham, MA) the sample was re-suspended and run on Q-TOF Ultima API Mass Spectrometer (Waters, Bedford, MA). The results were analyzed using SEQUEST (v.27) against a human International Protein Index (IPI) database (v.3.38).Western blotsConcentrated medium and pellet samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). Human NPM1 was detected on the western blot blocked with 5% nonfat dry milk using an anti-NPM1 monoclonal antibody (Sigma, St. Louis, MO). Near-infrared (NIR) IRDye 680 labeled secondary antibodies (Li-Cor, Lincoln, NE) were used to visualize the NPM1 antibody. The membrane was scanned using the Odyssey infrared imaging system (Li-Cor, Lincoln, NE).Acknowledgments: This work was supported by, NSF (FIBR grant EF0527023), the Battelle Memorial Institute, the DoD, and the University of Luxembourg. The authors gratefully acknowledge the excellent work of the microarray facility at ISB, technical help from David Huang, and stimulating discussions with Clay Marsh, Ohio State University Medical School, Richard Gelinas, ISB & Battelle Memorial Institute, and Leroy Hood, ISB. We also thank Leroy Hood, Richard Gelinas and John Aitchison for helpful comments on the manuscript.12*miRNA export from mammalian cells8/29/09References:1.2. 3. 4. 5. 6. 7.8. 9.Valadi, H., et al., Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol,. 9(6): 654-9. (2007) Hunter, M.P., et al., Detection of microRNA expression in human peripheral blood microvesicles. PLoS One, 3(11): e3694 (2008) Wang, K., et al., Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A,. 106(11): 4402-7 (2009) Mitchell, P.S., et al., Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A, 105(30): 10513-8 (2008) Nana-Sinkam, S.P., et al., Integrating the MicroRNome into the study of lung disease. Am J Respir Crit Care Med,. 179(1): 4-10 (2009) Zhuge, J., and Cederbaum, A.I., Serum deprivation-induced HepG2 cell death is potentiated by CYP2E1, Free Radic. Biol. Med., 40(1), 63-74 (2006) Maggi, L. et al., Nucleophosmin serves as a rate-limiting nuclear export chaperone for the mammalian ribosome, Mol. Cell Biol., 28(23), 7050-7065 (2009) Leask, A., Hijacking ZIP Codes: posttranscriptional regulation of CCN2 by nucleophosmin, J. Cell Commun. Signal., 3, 85-86 (2009) Nawa, Y., et al., Nucleophosmin may act as an alarmin:implications for severe sepsis, J. Leukoc. Biol., 86, p. 1-9 (2009)13*miRNA export from mammalian cells8/29/09Figure legends Figure 1. Differential miRNA spectra between cultured cells and the medium. a. Intra and extra-cellular miRNA spectra for 6 different cell lines in culture as measured by miRNA arrays. The cell lines are as indicated (T98 is derived from human glioblastoma multiforma, BEAS2B is derived from lung bronchial epithelium, and HPF are primary pulmonary fibroblast cells isolated from human lung tissue). Heat maps represent the levels of miRNA for the cell lines as indicated (red highest, green lowest). The brackets indicating “in” or “out” mark groups of miRNAs that show striking differences between the intra (“in”) and extra (“out”) cellular concentrations specifically for HepG2 cells. b. Q-PCR measurements of selected miRNAs shown in 1a that illustrate different profiles for HepG2 (red) and A549 (blue). The histogram shows the external versus internal levels of selected miRNAs, in a log2 scale. The extra-cellular levels are indicated above, the intra-cellular below. c. Extra-cellular levels of miRNAs expressed from A549 and HepG2 cells . The upper frame represents the relative levels (expressed in terms of log2), while the lower frame represents the standard deviations of these measurements in the upper frame (3 biological replicates) also in terms of log2. Figure 2. Time course of levels of miRNA exported into the medium. Samples were taken at various indicated time points taken serum deprivation. The serum free medium contains no proteins or miRNAs. Most of the miRNA measured shown very similar behavior. All vertical axes are in log2. a. The levels for 16 miRNAs measured by quantitative PCR from A549 (left frame and HepG2 (right frame). The points are the means of 3 biological replicates each. b. The time course measurements for three selected miRNAs from these same cell lines (same frame positions) that show the standard deviations for the replicates.Figure 3. Decay of miRNAs in the medium. The cells in culture were serum deprived for 2 hours, then the medium was removed and incubated under the same conditions for varying periods of time. miRNA measurements were made (3 biological replicates) by specific quantitative PCR on each sample. a. The time course measurements of 4 selected14*miRNA export from mammalian cells8/29/09miRNAs showing diverse profiles, and their standard deviations. b. Fitting the decay curves by least squares gives an effective decay constant, plotted here for the 4 miRNAs in c. c. Model kinetics for the export response of the majority of the miRNAs considered here, showing the rate of export as a function of time (in red) and the accumulated level as a function of time (in blue). d. the intracellular response in HepG2 cells of three selected miRNAs. g. the extracellular levels from the same experiment ( three biological replicates) of the same three miRNAs. e. a “score card” showing which miRNA showed definitive evidence of decreasing in the cell immediately after SD (marked with a cross indicating that the average of values at 0.5 and 1 hr after SD show a decrease) together with a profile of intra and extracellular levels averaged over the set of all 24 miRNA measured (3 biological replicates of each.). f. An expanded view of the averaged profile shown in e. The 0 hr point shown is that of the miRNA prior to the serum deprivation, even though the serum free medium begins at 0 hr with no miRNAs present.Figure 4. miRNA levels in fractionated serum-free culture medium for the two cell lines. The medium was fractionated (2 hours after serum deprivation) by differential centrifugation into four fractions using standard protocols for isolating microvesicles (size range: 100nm to 1000nm), exosomes (size range: 30nm to 100nm) , one higher g spin pellet (220K for 1 hour), and the remaining supernatant. Pellets were resuspended in 0.5 ml of PBS and 200 ul of the solution were used for miRNA isolation. a.Experimental design for fractionation of medium. Panels b through e show 2 selected sets of 3 miRNAs profiles from the two cell lines compared. The bars show the standard deviations of 3 biological replicates.Figure 5. Protein exported upon serum deprivation. a. NPM1 protein is observed in Western analysis of the concentrated medium from HepG2 cells. This protein is exported into the medium, but is not found in any fraction except the supernatant (as defined in legend to figure 4 ) as shown by Westerns on the fractions. b. The relative concentrations of all miRNAs measured in this work were averaged for each fraction to obtain a relative measure of miRNA levels in each fraction. These averages for HepG215*miRNA export from mammalian cells8/29/09cells are are shown in the histogram using a scale of log to base 2. c. NPM1 protein protects microRNA from RNase degradation. Synthetic mir-122 (100pmole) was mixed with different proteins, NPM1 (blue bars, 3 pmole), TGF-β (purple bars, 4 pmole) or BSA (yellow bars, 1.5 nmole) for 30 minutes followed by adding RNase A (7 nmole) for another 30 minutes and incubation at 37oC. The miRNA levels were determined by QPCR. Control experiments: omitting RNAse A, protein, or microRNA were also included, as indicated on top of the graph. The scale is log2 of relative mir-122 levels. Table I. We show here all of the known RNA binding proteins for which we observed2 or more peptide fragments in the medium (2hrs after SD) .16*miRNA export from mammalian cells8/26/09Figure 1 aExtracellularS2 B HP F 2 ep G A5 49Intracellular2 A5 49 BE AS ep G 2B HP FT9 8BE AHT9 8HOUTOUTINOUTINFigure 1 bRelative ratio of miRNA concentration (log 2)20.00 15.00 10.00 5.00 0.00 5.00 10.00 15.00 20.00mExtracellularIntracellular1b 89 Ri * 29 -6 iR m m 3p 629 iR 52 -6 iR m m 37 -1 iR 5p 115 iR m m 90 -1 iR 53 -5 iR m 0b -1 iR m m 5p d20 -5 iR m 82 -3 iRA549HepG21*miRNA export from mammalian cells8/26/09Figure 1 cFigure 1c.A549 HepG-2A549, Std. DevHepG-2, Std. Dev.Figure 2a.,',,2ZEh ^EKZZEh ^EKZ*miRNA export from mammalian cells8/26/09Figure 2b.,',,Figure 3a.1224487212244872hrs3*miRNA export from mammalian cells8/26/09b.4*miRNA export from mammalian cells8/26/09Figure 3c.,Figure 3d.,','Z^^5*miRNA export from mammalian cells8/26/09Figure 3e.DHepG-2mir-125 mir-133b mir-134 mir-135a mir-141* mir-16 mir-17 mir-17-3p mir-199b mir-219 mir-22 mir-23a mir-23b mir-26b mir-29a mir-29c mir-30d mir-323 mir-342 mir-452 mir-515 mir-671 mir-671-3pZE,'+ + + + + + + ++ + + + + + + + + +Figure 3f.DZE,'6*miRNA export from mammalian cells8/26/09Figure 3g.mediumLog2(relative level)cellst (hours)Figure 4a.DW>W ^^D