Live and heat-treated probiotics differently modulate IL10 mRNA stabilization and microRNA expression
2015; Elsevier BV; Volume: 137; Issue: 4 Linguagem: Inglês
10.1016/j.jaci.2015.08.033
ISSN1097-6825
AutoresAudrey Demont, Fériel Hacini‐Rachinel, Rémi Doucet-Ladevèze, Catherine Ngom‐Bru, Annick Mercenier, Guénolée Prioult, Carine Blanchard,
Tópico(s)Gut microbiota and health
ResumoFor the last century, microorganisms, such as probiotics, have been widely used in food or as food supplement for varied health benefits. In inflammatory diseases and healthy animals, studies have shown a probiotic-induced IL-10 overproduction1de Moreno de L.A. del C.S. Zurita-Turk M. Santos R.C. van de Guchte M. Azevedo V. et al.Importance of IL-10 modulation by probiotic microorganisms in gastrointestinal inflammatory diseases.ISRN Gastroenterol. 2011; 2011: 892971PubMed Google Scholar or an increase in regulatory FoxP3+ cell number.2Smelt M.J. de Haan B.J. Bron P.A. van S.I. Meijerink M. Wells J.M. et al.L. plantarum, L. salivarius, and L. lactis attenuate Th2 responses and increase Treg frequencies in healthy mice in a strain dependent manner.PLoS One. 2012; 7: e47244Crossref PubMed Scopus (60) Google Scholar Interestingly, in vivo and in vitro studies have convincingly demonstrated differential activities of live and heat-treated organisms.3Taverniti V. Guglielmetti S. The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept).Genes Nutr. 2011; 6: 261-274Crossref PubMed Scopus (351) Google Scholar Yet, little is known about the molecular mechanism involved in this modulation of IL10 mRNA expression. Several articles have tried to correlate this cytokine expression to Toll-like receptors, pathogen-associated molecular patterns, and nuclear factor kappa b activation, but the results were not always convincing and other modes of activation were frequently suggested. Although probiotics are usually not in direct contact with blood cells, the use of PBMCs constitutes an appropriate ex vivo human system to study the intracellular cytokine expression pathways. Microorganisms are naturally present in many raw foods, such as milk-derived products, and optimal heat treatments are used to prevent them from replicating. Yet, biologically active bacterial components may still persist, be degraded, or be produced, thus modifying the impact of microorganisms on the host immunity. In this study, we hypothesized that heat-treated bacteria may alter the stabilization of IL10 mRNA via the differential expression of microRNAs (miRNA). IL-10 is produced and secreted by PBMCs upon stimulation with different live and heat-treated probiotics. Interestingly, heat-treated Lactobacillus paracasei Nestlé Culture Collection (NCC) 2461 highly and significantly induced IL-10 secretion in the supernatant (4556 ± 1926 pg/mL; P < .05) compared with the live strain (968 ± 505 pg/mL) or the unstimulated control (6.8 ± 1.4 pg/mL) (Fig 1, A). Cellular localization using flow cytometry identified the CD14+ cells as the major IL-10 producers in this in vitro system (see Fig E1 in this article's Online Repository at www.jacionline.org). These results indicated that live and heat-treated L paracasei NCC 2461 induced differential levels of IL-10 in PBMCs. The IL10 mRNA levels were quantified using real-time PCR. A 9.3-fold increase in IL10 mRNA was observed in PBMCs treated with heat-treated L paracasei NCC 2461, whereas stimulation with live L paracasei NCC 2461 induced only a 4.5-fold increase in IL10 mRNA expression compared with unstimulated PBMCs. Interestingly, the disconnect between the 2- to 3-log induction in IL-10 protein level (4556 ± 1926 pg/mL with the heat-treated probiotic strain compared with 6.8 ± 1.4 pg/mL in unstimulated PBMCs; Fig 1, A) and the only 9.3-fold induction of IL10 mRNA expression (Fig 1, B) is striking and suggests that the IL10 mRNA might have an excellent translation efficiency under stimulation with heat-treated L paracasei NCC 2461. Numerous mechanisms explaining a differential mRNA/protein ratio exist; here, we hypothesized that it was possibly due to a stabilization of the mRNA. Indeed, treatment of L paracasei–stimulated PBMCs with actinomycin D revealed that IL10 mRNA (but not IFNG; see Fig E2 in this article's Online Repository at www.jacionline.org) was specifically and significantly stabilized by the heat-treated L paracasei NCC 2461 (half-life of 9.33 ± 2.51 hours; P < .05) compared with its live counterpart (half-life of 1.74 ± 1.76 hours) or the unstimulated control (half-life of 0.84 ± 0.66 hours) (Fig 1, C). This mRNA stabilization was observed in all 5 PBMC donors tested (Fig 1, D). All together, these results suggest that heat-treated L paracasei NCC 2461 stabilizes IL10 mRNA. There are several AU-rich elements in the 3′ untranslated region (UTR) of IL10 mRNA that have been shown to regulate IL10 gene expression level.4Powell M.J. Thompson S.A. Tone Y. Waldmann H. Tone M. Posttranscriptional regulation of IL-10 gene expression through sequences in the 3′-untranslated region.J Immunol. 2000; 165: 292-296Crossref PubMed Scopus (143) Google Scholar PBMCs, transfected with a plasmid containing the IL10-3′UTR downstream of the luciferase (Luc) gene (Luc/IL10-3′UTR) and stimulated by heat-treated L paracasei NCC 2461 produced a significantly higher level of luciferase activity (3.6 ± 0.6 vs 0.6 ± 0.11; P < .05) than did transfected unstimulated cells (Fig 2, A), suggesting a role for IL10-3′UTR in heat-treated L paracasei NCC 2461–induced IL10 mRNA stabilization. We then investigated whether microRNA could be involved in this mechanism. We identified that live and heat-treated L paracasei NCC 2461 differently influenced microRNA expression (Fig 2, B). These results suggest that the physiological stage of the studied probiotic strain (live or heat-treated) may differently affect the host immunity, underlining the need to properly control probiotics cultures. Numerous microRNAs were specifically dysregulated in PBMCs stimulated by heat-treated L paracasei (Table E1), but surprisingly only few microRNAs specific for IL10 were downregulated when compared with live L paracasei NCC 2461–treated or unstimulated cells (Fig E2). MiR-27a, previously shown to regulate IL10 mRNA level,5Xie N. Cui H. Banerjee S. Tan Z. Salomao R. Fu M. et al.MicroRNA-27a regulates imflamatory response of macrophages by targeting interleukin 10.J Immunol. 2014; 193: 327-334Crossref PubMed Scopus (96) Google Scholar, 6Yeh C.H. Chen T.P. Wang Y.C. Lin Y.M. Fang S.W. MicroRNA-27a regulates cardiomyocytic apoptosis during cardioplegia-induced cardiac arrest by targeting interleukin 10-related pathways.Shock. 2012; 38: 607-614Crossref PubMed Scopus (22) Google Scholar was significantly (P < .005) decreased by the heat-treated probiotic (Fig 2, C). Transfection of the miR-27a in cells was able to decrease IL10-3′UTR-dependent luciferase activity in PBMCs stimulated with the heat-treated probiotic (Fig 2, D). These results suggest that miR-27a may play a role in heat-treated probiotic-induced IL-10 expression, but other pathways are certainly also involved. We are currently considering the possibility that specific probiotic sequences may regulate the host immune function by a direct modulation of mRNA stabilization or indirectly through regulation of microRNA function. Probiotic strains may contain free RNA sequences that could be released on bacterial heat treatment because of partial membrane disruption. Multiple copies of sequences with more than 70% similarity with miR-27a have been identified in several probiotic strains (Fig E4). This concept of cross-kingdom regulation by microRNAs was recently described for rice, which could regulate mammalian host cell gene expression via the plant MIR168a.7Zhang L. Hou D. Chen X. Li D. Zhu L. Zhang Y. et al.Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA.Cell Res. 2012; 22: 107-126Crossref PubMed Scopus (734) Google Scholar In conclusion, we identified that heat-treated L paracasei NCC 2461 modulated IL-10 expression level by specifically stabilizing IL10 mRNA. In our study, the bacterial heat treatment led to a differential regulation of microRNAs. This illustrates that depending on probiotics' physiological state, they may differently stabilize specific host cell mRNAs, thus potentially leading to varying effects. Importantly, we demonstrated that beyond classical molecular pattern recognition by dedicated receptors, epigenetic mechanisms may also play a role in host-microbe interaction. Finally, in this study, we illustrate how miRNA regulation by probiotics may lead to enhanced IL-10 production and partly explain the anti-inflammatory effects observed in clinical trials with specific probiotic strains. We thank Sophie Nutten for critical review of the manuscript. The bacterial strain Lactobacillus paracasei NCC 2461 (CNCM I-2116) from the Nestlé Culture Collection was grown in a 15-L bioreactor (new MBR, Zürich, Switzerland) using industrial media (fermentation composition media provided by Nestlé PTC, Konolfingen, Switzerland). Biomass was harvested when entering the stationary phase. Bacterial cells were then spun down at 5000g for 20 minutes and resuspended in PBS until heat treatment. Glycerol 15% (v/v) was added to live samples, and the exact number of colony-forming units (CFUs) was determined by plating serial dilutions on agar plates. Bacteria were heat-treated at 140°C for 15 seconds by a plate heat exchanger (Alfa Laval RF 372, Alfa Laval Mid Europe AG, Dietlikon, Switzerland). Heat treatment resulted in no residual CFU in the preparation. All samples were kept frozen at −80°C until use. Human PBMCs were isolated from filters obtained from the transfusion center of the Centre Hospitalier Universitaire Vaudois (Lausanne, Switzerland). Ethical approval was obtained to perform this study. The cells trapped in the filters were flushed back and diluted 1:2 with HBSS (Sigma, Lachen, Switzerland). After a Histopaque gradient centrifugation (Sigma), mononuclear cells were collected at the interface and washed twice with 40 mL HBSS according to the manufacturer's protocol. Cells were then resuspended in Iscove's modified Dulbecco's medium (Sigma) supplemented with 10% heat-inactivated FCS (Bioconcept, Paris, France), 1% l-glutamine (Sigma), 1% penicillin/streptomycin (Sigma), and 0.1% gentamycin (Sigma). PBMCs (1.5 × 106 cells/well) were then incubated with live and heat-treated bacterial strains (1.5 × 107 equivalent CFU/well) in 24-well plates. The effects of live and heat-treated bacteria were tested in triplicate on PBMCs (ratio 1:8) isolated from 5 individual donors. An inhibitor of transcription (actinomycin D, 10 μg/mL) was added to PBMCs to evaluate the RNA stability of PBMCs. PBMCs were stimulated with probiotics for 24-hour incubation and subsequently cultured with actinomycin D for 1, 6, and 20 hours. To assess whether intermediate protein synthesis was involved, before adding live or heat-treated bacterial strains PBMCs were incubated with or without 20 μg/mL cycloheximide (inhibitor of protein synthesis) for 30 minutes to inhibit the protein synthesis. Then strains were added (7 × 106 equivalent CFU/well) and incubated for 18 hours at 37°C in 5% CO2. The levels of cytokines (IFN-γ; IL-10) were determined in the cell culture supernatants after 2- to 28-hour incubation using electrochemiluminescence-based multiplex assay (Meso Scale Discovery, Gaithersburg, Md) following the manufacturer's instructions. Results are expressed as means (pg/mL) ± SEM of 4 donors per bacterial preparation. RNA was extracted with TRIzol reagent following the manufacturer's instruction (Invitrogen, 15596-018, Life Technologies Europe B.V., Zug, Switzerland). Briefly, supernatants were removed and kept at −20°C until cytokine measurement and cells were lysed directly by adding TRIzol. PBMCs and TRIzol were scraped with cell-scrapers. The homogenized samples were incubated for 5 minutes at room temperature. One volume of chloroform was added to 5 volumes of TRIzol reagent for 2 minutes at room temperature and centrifuged at 12,000g for 15 minutes at 4°C. Two volumes of the upper aqueous phase were transferred and mixed with 1 volume of isopropanol. Samples were incubated for 10 minutes at room temperature and centrifuged at 12,000g for 15 minutes at 4°C. The supernatant was removed, and the pellet was washed with 75% ethanol and centrifuged at 8000g for 5 minutes at 4°C. Ethanol was removed, and the pellet was dried for 1 hour. The pellet was dissolved in RNase-free water. The concentration was measured at an OD of 260 nm (NanoDrop), and the purity was determined by measuring the ratio A260/A280. Reverse transcription was performed on 750 ng of total RNA using qScript cDNA Synthesis Kit (Quanta BioSciences, #95047-025; VWR International, Dietikon, Switzerland) following the manufacturer's instruction. Reverse transcription was performed on a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Rotkreuz, Switzerland) using the following cycle program: 5 minutes at 22°C, 30 minutes at 42°C, and 5 minutes at 85°C to finish at 4°C. Quantitative PCR was performed with the synthesized cDNA using TaqMan PCR. Each reaction mixture contained TaqMan Universal Master Mix 1×, primers IL10 and IFNG (Hs00174086_m1, Hs99999041_m1, Applied Biosystems), 50 ng cDNA, and water. After 1 cycle of 50°C for 2 minutes and 95°C for 10 minutes to activate the polymerase, real-time PCR was carried out for 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute by using ABI-Prism 7900HT (Applied Biosystems). The mRNA were quantified and normalized with a house-keeping gene (HPRT). The mRNA expression was determined for each gene (IL10 and IFNG) on the basis of average Ct value obtained. The results were calculated by using the formula 2−Δct. Three replicates of all samples were included, and experiments were performed on 5 donors. To control for possible DNA contamination in the RNA samples, "non-RT" controls were included in all experiments and no amplification above background levels was observed. Water controls were also included for each gene in each run, and no amplification above background levels was observed. After 18 hours of culture, BD GolgiStop (BD Biosciences, Allschwil, Switzerland) was added for 4 hours to PBMCs. Cells were harvested, washed once in ice-cold PBS, and suspended in a small amount of PBS. Cells were then incubated with 10% human serum for 15 minutes at 4°C to block Fc receptors. Specific fluorochrome-conjugated antibodies (CD3-phycoerythrin, CD14-fluorescein isothiocyanate, CD4-Allophycocyanin) were incubated for 30 minutes at 4°C. Cells were washed, fixed and permeabilized with Cytofix/Cytoperm solution (BD Bioscience) for 20 minutes at 4°C, and pelleted cells were stained for intracellular cytokines using phycoerythrin-conjugated antibodies against human IL-10 or IFN-γ for 30 minutes at 4°C in the dark. Phycoerythrin-conjugated isotype controls were used in parallel. Finally, cells were washed and resuspended in PBS-BSA for flow cytometry analysis using FACS Fortessa (BD Biosciences) and FlowJO software version 7.6.5 (Tree Star, Ashland, Ore). The RNA extracted from PBMCs (Trizol, Invitrogen) stimulated with live or heat-treated bacteria and unstimulated PBMCs were pooled from 3 donors. Reverse transcription (RT) was performed on 500 ng total RNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, # 4366596). The total RNA was mixed with 100 mM deoxyribonucleotide triphosphates, 50 U/μL Multiscribe RT enzyme, 10× RT buffer, 20 U/μL RNase inhibitor, and 3 μL Megaplex Primer Pool A or B (Applied Biosystems, #4444750) in a final volume of 15 μL. Reverse transcription was performed using GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) with the following cycle program: 30 minutes at 16°C, 30 minutes at 42°C, and 5 minutes at 5°C to finish at 4°C. TaqMan Array Human MicroRNA Set Cards v2.0 from Applied Biosystems (#4400238) was loaded and run according to the manufacturer's instruction on a quantitative ABI-Prism 7900HT (Applied Biosystems). The results were normalized to 3 endogenous controls. In exploratory experiment, no false-discovery rate correction was applied to conserve all potential microRNA targeting IL10. In the experiment aiming at confirming miR-27a expression, Dunn's multiple comparisons test correction was used. The list of the dysregulated microRNA is provided in Table E1. The results were calculated by using the formula 2−Δct. Fold increase result expression was normalized to expression levels in the unstimulated group. Results were transferred and analyzed in the GeneSpring Software (Agilent Technologies, Santa Clara, Calif) and data were analyzed through the use of QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN, Redwood City, Calif; www.qiagen.com/ingenuity). PBMCs were left to adhere and then transiently transfected with lipofectamin iRNAiMAX (Invitrogen, Life Technologies) and 0.01 nM miR-27a mimic or inhibitor or the respective negative controls for 6 hours. The supernatant were collected for cytokine quantification, and cells were lyzed for RNA quantification. When cells were transfected with plasmids, cells were left to adhere and then transiently cotransfected with lipofectamin 2000 (Life Technologies) and 1 μg PGL3-promoter vector containing the Firefly luciferase gene followed by the 3′URT of the IL10 mRNA, and 50 ng of phRL-TK vector (Renilla Luciferase). Cells were then washed and stimulated with the probiotics for 18 hours (overnight). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wis). For the transfection of microRNA, mirVana miRNA control and mimic were obtained from Ambion (Life Technologies) and transfected in a 24-well plate with 2 μL Lipofectamine RNAiMax Reagent (Invitrogen) per well in OPTI-MEM. The effects of live and heat-treated bacteria were studied on PBMCs (ratio 1:8). Transfection experiments are representative of at least 2 experiments tested in quadruplicates. Transfection efficacy was approximately 20% based on flow cytometry analyses of cells transfected with Cy3 Dye-Labeled Anti-miR Negative Control #1 from Ambion (Life Technologies).Fig E2IFN-γ expression in PBMCs stimulated with live or heat-treated L paracasei NCC 2461. A, IFN-γ protein secreted in the supernatant. B, IFNG mRNA expression in PBMCs stimulated with live or heat-treated L paracasei NCC 2461. C, IFNG mRNA half-life in PBMCs. Experiments shown are means of triplicates and representative of 5 independent PBMC donors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Comparative expression of the microRNA targeting IL10 mRNA. Fold changes in microRNA expression in PBMCs stimulated with L paracasei (live or heat-treated) compared with unstimulated cells were plotted using Ingenuity Pathway Analysis. *Only 1 of the mircoRNAs is shown while several are in the list. Here, miR-27a is downregulated, miR-27b is upregulated. Only miR-27b upregulation is shown here. See Table E1 for exact fold changes of individual microRNAs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Number of sequences with at least 70% similarity to miR-27a and control sequence in the genome of 6 probiotic strains. A control sequence present in the pre–miR-27a sequence and the miR-27a sequence were compared with the chromosomes of 6 probiotic strains. The 2 sequences of 20-21 nucleotides were compared after reverse translation U->T against the genome of 6 strains using FUZZNUC (EMBOSS package) allowing for 4 to 6 mismatches (80% to 70% similarity).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1MicroRNA expression in probiotic-stimulated PBMCsFold changeIDNotesSymbolEntrez gene nameHeat-treated NCC 2461 vs medium 53.047hsa-miR-144miR-144-3p(miRNAs w/seed ACAGUAU) 50.723hsa-miR-9miR-9-5p(and other miRNAs w/seed CUUUGGU) 49.290hsa-miR-152DmiR-148a-3p(and other miRNAs w/seed CAGUGCA) 34.995hsa-miR-100DmiR-100-5p(and other miRNAs w/seed ACCCGUA) 34.944hsa-miR-99aDmiR-100-5p(and other miRNAs w/seed ACCCGUA) 26.359hsa-miR-523miR-523-3p(miRNAs w/seed AACGCGC) 22.424hsa-miR-642miR-642a-5p(miRNAs w/seed UCCCUCU) 19.960hsa-miR-652miR-652-3p(miRNAs w/seed AUGGCGC) 16.956hsa-miR-18aDmiR-18a-5p(and other miRNAs w/seed AAGGUGC) 14.922hsa-miR-125a-3pmiR-125a-3p(miRNAs w/seed CAGGUGA) 14.447hsa-miR-146b-3pmiR-1231-3p(and other miRNAs w/seed GCCCUGU) 12.654hsa-miR-22miR-22-3p(miRNAs w/seed AGCUGCC) 10.560hsa-miR-222DmiR-221-3p(and other miRNAs w/seed GCUACAU) 10.288hsa-miR-330miR-330-3p(and other miRNAs w/seed CAAAGCA) 9.971hsa-miR-155miR-155-5p(miRNAs w/seed UAAUGCU) 9.444hsa-miR-411miR-411-5p(and other miRNAs w/seed AGUAGAC) 9.171hsa-miR-486-3pmiR-486-3p(and other miRNAs w/seed GGGGCAG) 8.313hsa-miR-133amiR-133a-3p(and other miRNAs w/seed UUGGUCC) 8.257hsa-miR-942miR-7028-3p(and other miRNAs w/seed CUUCUCU) 7.741hsa-let-7cDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 5.993hsa-miR-361miR-361-5p(miRNAs w/seed UAUCAGA) 5.895hsa-miR-15aDmiR-16-5p(and other miRNAs w/seed AGCAGCA) 5.099hsa-miR-21miR-21-5p(and other miRNAs w/seed AGCUUAU) 4.475hsa-miR-526bmiR-526b-5p(miRNAs w/seed UCUUGAG) 3.997hsa-miR-1miR-1-3p(and other miRNAs w/seed GGAAUGU) 3.964hsa-miR-200bDmiR-200b-3p(and other miRNAs w/seed AAUACUG) 3.947hsa-miR-32DmiR-92a-3p(and other miRNAs w/seed AUUGCAC) 3.447hsa-miR-27bDmiR-27a-3p(and other miRNAs w/seed UCACAGU) 3.419hsa-miR-130aDmiR-130a-3p(and other miRNAs w/seed AGUGCAA) 3.394hsa-miR-636miR-636(miRNAs w/seed GUGCUUG) 3.220hsa-miR-362miR-362-5p(and other miRNAs w/seed AUCCUUG) 3.042mmu-miR-187miR-187-3p(miRNAs w/seed CGUGUCU) 2.926hsa-miR-148bDmiR-148a-3p(and other miRNAs w/seed CAGUGCA) 2.896hsa-let-7fDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 2.847hsa-miR-199a-3pmiR-199a-3p(and other miRNAs w/seed CAGUAGU) 2.740hsa-miR-1290miR-1290(miRNAs w/seed GGAUUUU) 2.671hsa-miR-29aDmiR-29b-3p(and other miRNAs w/seed AGCACCA) 2.534hsa-miR-15bDmiR-16-5p(and other miRNAs w/seed AGCAGCA) 2.526hsa-miR-28DmiR-708-5p(and other miRNAs w/seed AGGAGCU) 2.503hsa-miR-454DmiR-130a-3p(and other miRNAs w/seed AGUGCAA) 2.491hsa-miR-1305miR-1305(miRNAs w/seed UUUCAAC) 2.455hsa-miR-221DmiR-221-3p(and other miRNAs w/seed GCUACAU) 2.430hsa-miR-132DmiR-132-3p(and other miRNAs w/seed AACAGUC) 2.364hsa-miR-1271miR-96-5p(and other miRNAs w/seed UUGGCAC) 2.359hsa-miR-664miR-664-3p(and other miRNAs w/seed AUUCAUU) 2.313hsa-miR-210miR-210-3p(miRNAs w/seed UGUGCGU) 2.198hsa-miR-146amiR-146a-5p(and other miRNAs w/seed GAGAACU) 2.194hsa-miR-363DmiR-92a-3p(and other miRNAs w/seed AUUGCAC) 2.083hsa-miR-190bmiR-190a-5p(and other miRNAs w/seed GAUAUGU) 2.060hsa-miR-519b-3pmiR-519a-3p(and other miRNAs w/seed AAGUGCA) 2.060hsa-miR-212DmiR-132-3p(and other miRNAs w/seed AACAGUC) −2.007hsa-miR-197miR-197-3p(and other miRNAs w/seed UCACCAC) −2.057hsa-miR-18aDmiR-18a-5p(and other miRNAs w/seed AAGGUGC) −2.059hsa-miR-30a-5pmiR-30c-5p(and other miRNAs w/seed GUAAACA) −2.070hsa-miR-328miR-328-3p(and other miRNAs w/seed UGGCCCU) −2.109hsa-miR-193bmiR-193a-3p(and other miRNAs w/seed ACUGGCC) −2.370hsa-miR-1275miR-1275(and other miRNAs w/seed UGGGGGA) −2.402hsa-miR-148aDmiR-148a-3p(and other miRNAs w/seed CAGUGCA) −2.508hsa-miR-31miR-31-5p(and other miRNAs w/seed GGCAAGA) −2.586hsa-miR-95miR-95-3p(miRNAs w/seed UCAACGG) −2.712hsa-miR-429DmiR-200b-3p(and other miRNAs w/seed AAUACUG) −2.752hsa-miR-432miR-432(and other miRNAs w/seed CUUGGAG) −2.993hsa-miR-34aDmiR-34a-5p(and other miRNAs w/seed GGCAGUG) −3.004hsa-miR-638miR-638(miRNAs w/seed GGGAUCG) −3.650hsa-miR-509-5pmiR-509-5p(and other miRNAs w/seed ACUGCAG) −3.671hsa-miR-545miR-545-3p(miRNAs w/seed CAGCAAA) −3.984hsa-miR-26bmiR-26a-5p(and other miRNAs w/seed UCAAGUA) −6.601hsa-miR-27aDmiR-27a-3p(and other miRNAs w/seed UCACAGU) −7.205hsa-miR-577miR-577(miRNAs w/seed AGAUAAA) −8.346hsa-miR-1249miR-1249-3p(and other miRNAs w/seed CGCCCUU) −8.353hsa-miR-505miR-505-3p(miRNAs w/seed GUCAACA) −8.359mmu-miR-496miR-503-3p(and other miRNAs w/seed GAGUAUU) −8.765mmu-miR-134miR-3118(and other miRNAs w/seed GUGACUG) −11.042hsa-miR-1244miR-1244(miRNAs w/seed AGUAGUU) −11.467hsa-miR-571miR-571(miRNAs w/seed GAGUUGG) −12.837hsa-miR-127miR-127-3p(miRNAs w/seed CGGAUCC) −12.892hsa-miR-539miR-539-5p(miRNAs w/seed GAGAAAU) −14.478hsa-miR-769-5pmiR-769-5p(miRNAs w/seed GAGACCU) −15.122rno-miR-29cDmiR-29b-3p(and other miRNAs w/seed AGCACCA) −19.458hsa-miR-449bDmiR-34a-5p(and other miRNAs w/seed GGCAGUG) −22.429hsa-miR-598miR-598-3p(miRNAs w/seed ACGUCAU) −25.956hsa-miR-107miR-103-3p(and other miRNAs w/seed GCAGCAU) −26.273hsa-miR-449DmiR-34a-5p(and other miRNAs w/seed GGCAGUG) −28.571hsa-miR-125bmiR-125b-5p(and other miRNAs w/seed CCCUGAG) −30.393hsa-miR-15aDmiR-16-5p(and other miRNAs w/seed AGCAGCA) −31.668hsa-miR-130bDmiR-130a-3p(and other miRNAs w/seed AGUGCAA) −32.432hsa-miR-433miR-433-3p(miRNAs w/seed UCAUGAU) −33.203hsa-miR-485-3pmiR-485-3p(and other miRNAs w/seed UCAUACA) −53.414hsa-miR-30a-3pmiR-30a-3p(and other miRNAs w/seed UUUCAGU) −57.982hsa-miR-450amiR-450a-5p(and other miRNAs w/seed UUUGCGA) −60.486hsa-miR-708DmiR-708-5p(and other miRNAs w/seed AGGAGCU) −70.142hsa-miR-99bDmiR-100-5p(and other miRNAs w/seed ACCCGUA) −75.581hsa-miR-324-5pmiR-324-5p(miRNAs w/seed GCAUCCC) −95.606hsa-miR-138miR-138-5p(miRNAs w/seed GCUGGUG)Heat-treated NCC2461 vs live NCC2461 150.942hsa-miR-30e-3pDmiR-30a-3p(and other miRNAs w/seed UUUCAGU) 118.263mmu-miR-491miR-491-5p(and other miRNAs w/seed GUGGGGA) 50.723hsa-miR-9miR-9-5p(and other miRNAs w/seed CUUUGGU) 45.557hsa-miR-27bDmiR-27a-3p(and other miRNAs w/seed UCACAGU) 40.176hsa-miR-139-5pmiR-139-5p(miRNAs w/seed CUACAGU) 39.009hsa-miR-101miR-101-3p(and other miRNAs w/seed ACAGUAC) 37.788hsa-miR-526bmiR-526b-5p(miRNAs w/seed UCUUGAG) 34.995hsa-miR-100DmiR-100-5p(and other miRNAs w/seed ACCCGUA) 33.864hsa-miR-362miR-362-5p(and other miRNAs w/seed AUCCUUG) 33.566hsa-miR-103miR-103-3p(and other miRNAs w/seed GCAGCAU) 30.687hsa-miR-339-5pmiR-339-5p(and other miRNAs w/seed CCCUGUC) 29.653hsa-miR-31DmiR-31-5p(and other miRNAs w/seed GGCAAGA) 27.195hsa-miR-532miR-532-5p(and other miRNAs w/seed AUGCCUU) 26.359hsa-miR-523miR-523-3p(miRNAs w/seed AACGCGC) 22.424hsa-miR-642miR-642a-5p(miRNAs w/seed UCCCUCU) 19.960hsa-miR-652miR-652-3p(miRNAs w/seed AUGGCGC) 19.887hsa-miR-210miR-210-3p(miRNAs w/seed UGUGCGU) 19.633hsa-miR-423-5pmiR-423-5p(and other miRNAs w/seed GAGGGGC) 17.681hsa-miR-502-3pmiR-501-3p(and other miRNAs w/seed AUGCACC) 16.478hsa-miR-148aDmiR-148a-3p(and other miRNAs w/seed CAGUGCA) 15.813hsa-miR-671-3pmiR-671-3p(and other miRNAs w/seed CCGGUUC) 14.970hsa-miR-212DmiR-132-3p(and other miRNAs w/seed AACAGUC) 14.922hsa-miR-125a-3pmiR-125a-3p(miRNAs w/seed CAGGUGA) 14.724hsa-miR-190bmiR-190a-5p(and other miRNAs w/seed GAUAUGU) 14.456hsa-miR-411miR-411-5p(and other miRNAs w/seed AGUAGAC) 14.447hsa-miR-146b-3pmiR-1231-3p(and other miRNAs w/seed GCCCUGU) 14.371hsa-miR-410miR-344d-3p(and other miRNAs w/seed AUAUAAC) 12.970mmu-miR-495miR-495-3p(and other miRNAs w/seed AACAAAC) 12.394hsa-miR-223DmiR-223-3p(miRNAs w/seed GUCAGUU) 10.291hsa-miR-15bDmiR-16-5p(and other miRNAs w/seed AGCAGCA) 10.049hsa-miR-1275miR-1275(and other miRNAs w/seed UGGGGGA) 9.942hsa-miR-99bDmiR-100-5p(and other miRNAs w/seed ACCCGUA) 9.034hsa-miR-574-3pmiR-574-3p(miRNAs w/seed ACGCUCA) 8.954hsa-miR-185miR-185-5p(and other miRNAs w/seed GGAGAGA) 8.909hsa-miR-106bDmiR-17-5p(and other miRNAs w/seed AAAGUGC) 8.782hsa-miR-99aDmiR-100-5p(and other miRNAs w/seed ACCCGUA) 8.725hsa-miR-942miR-7028-3p(and other miRNAs w/seed CUUCUCU) 8.424hsa-miR-339-3pmiR-339-3p(miRNAs w/seed GAGCGCC) 7.484hsa-miR-29bDmiR-29b-3p(and other miRNAs w/seed AGCACCA) 7.458hsa-let-7fDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 7.105hsa-miR-125a-5pmiR-125b-5p(and other miRNAs w/seed CCCUGAG) 6.934hsa-miR-486-3pmiR-486-3p(and other miRNAs w/seed GGGGCAG) 6.715hsa-miR-425-5pmiR-425-5p(and other miRNAs w/seed AUGACAC) 6.712hsa-miR-660miR-660-5p(and other miRNAs w/seed ACCCAUU) 6.704hsa-miR-132DmiR-132-3p(and other miRNAs w/seed AACAGUC) 6.622hsa-miR-192miR-192-5p(and other miRNAs w/seed UGACCUA) 6.616hsa-let-7bDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 6.434hsa-miR-28miR-708-5p(and other miRNAs w/seed AGGAGCU) 6.260hsa-miR-296miR-296-5p(miRNAs w/seed GGGCCCC) 6.070hsa-let-7aDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 6.034hsa-miR-142-5pmiR-142-5p(and other miRNAs w/seed AUAAAGU) 5.993hsa-miR-361miR-361-5p(miRNAs w/seed UAUCAGA) 5.960hsa-miR-532-3pmiR-532-3p(miRNAs w/seed CUCCCAC) 5.895hsa-miR-15aDmiR-16-5p(and other miRNAs w/seed AGCAGCA) 5.324hsa-miR-518fDmiR-518a-3p(and other miRNAs w/seed AAAGCGC) 5.099hsa-miR-21DmiR-21-5p(and other miRNAs w/seed AGCUUAU) 4.931hsa-miR-625miR-625-5p(and other miRNAs w/seed GGGGGAA) 4.518hsa-miR-454DmiR-130a-3p(and other miRNAs w/seed AGUGCAA) 4.446hsa-miR-1260miR-1260a(and other miRNAs w/seed UCCCACC) 4.146hsa-let-7eDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 4.119hsa-miR-155miR-155-5p(miRNAs w/seed UAAUGCU) 4.042hsa-miR-223DmiR-223-3p(miRNAs w/seed GUCAGUU) 3.997hsa-miR-1miR-1-3p(and other miRNAs w/seed GGAAUGU) 3.985mmu-miR-374-5pDmiR-374b-5p(and other miRNAs w/seed UAUAAUA) 3.964hsa-miR-200bDmiR-200b-3p(and other miRNAs w/seed AAUACUG) 3.947hsa-miR-32DmiR-92a-3p(and other miRNAs w/seed AUUGCAC) 3.728hsa-miR-16DmiR-16-5p(and other miRNAs w/seed AGCAGCA) 3.592hsa-miR-19aDmiR-19b-3p(and other miRNAs w/seed GUGCAAA) 3.583hsa-miR-454DmiR-130a-3p(and other miRNAs w/seed AGUGCAA) 3.394hsa-miR-636miR-636(miRNAs w/seed GUGCUUG) 3.385hsa-miR-140-3pmiR-140-3p(and other miRNAs w/seed ACCACAG) 3.362hsa-miR-146aDmiR-146a-5p(and other miRNAs w/seed GAGAACU) 3.291hsa-miR-30bDmiR-30c-5p(and other miRNAs w/seed GUAAACA) 3.127hsa-miR-422amiR-378a-3p(and other miRNAs w/seed CUGGACU) 3.103hsa-miR-20bDmiR-17-5p(and other miRNAs w/seed AAAGUGC) 3.099hsa-miR-197miR-197-3p(and other miRNAs w/seed UCACCAC) 3.049rno-miR-7miR-7a-5p(and other miRNAs w/seed GGAAGAC) 3.042mmu-miR-187miR-187-3p(miRNAs w/seed CGUGUCU) 3.020hsa-miR-374DmiR-374b-5p(and other miRNAs w/seed UAUAAUA) 2.970hsa-miR-484miR-344a-5p(and other miRNAs w/seed CAGGCUC) 2.913hsa-miR-376amiR-376a-3p(and other miRNAs w/seed UCAUAGA) 2.897hsa-miR-24miR-24-3p(and other miRNAs w/seed GGCUCAG) 2.842hsa-miR-30cDmiR-30c-5p(and other miRNAs w/seed GUAAACA) 2.839hsa-miR-31DmiR-31-5p(and other miRNAs w/seed GGCAAGA) 2.832hsa-miR-590-3PmiR-590-3p(miRNAs w/seed AAUUUUA) 2.830hsa-miR-301DmiR-130a-3p(and other miRNAs w/seed AGUGCAA) 2.766hsa-miR-191miR-191-5p(and other miRNAs w/seed AACGGAA) 2.747hsa-miR-1254miR-1254(and other miRNAs w/seed GCCUGGA) 2.739hsa-miR-133amiR-133a-3p(and other miRNAs w/seed UUGGUCC) 2.636hsa-miR-20aDmiR-17-5p(and other miRNAs w/seed AAAGUGC) 2.627hsa-miR-331miR-331-3p(miRNAs w/seed CCCCUGG) 2.600hsa-miR-19bDmiR-19b-3p(and other miRNAs w/seed GUGCAAA) 2.599hsa-miR-26bDmiR-26a-5p(and other miRNAs w/seed UCAAGUA) 2.585hsa-miR-92aDmiR-92a-3p(and other miRNAs w/seed AUUGCAC) 2.554hsa-miR-29aDmiR-29b-3p(and other miRNAs w/seed AGCACCA) 2.547hsa-miR-126miR-126a-3p(and other miRNAs w/seed CGUACCG) 2.539hsa-miR-342-3pmiR-342-3p(miRNAs w/seed CUCACAC) 2.533hsa-miR-144miR-144-3p(miRNAs w/seed ACAGUAU) 2.531hsa-miR-199a-3pmiR-199a-3p(and other miRNAs w/seed CAGUAGU) 2.485mmu-miR-93DmiR-17-5p(and other miRNAs w/seed AAAGUGC) 2.479hsa-miR-376cmiR-376c-3p(and other miRNAs w/seed ACAUAGA) 2.386hsa-miR-25DmiR-92a-3p(and other miRNAs w/seed AUUGCAC) 2.381hsa-miR-26aDmiR-26a-5p(and other miRNAs w/seed UCAAGUA) 2.368hsa-let-7cDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 2.359hsa-miR-186miR-186-5p(miRNAs w/seed AAAGAAU) 2.359hsa-miR-664miR-664-3p(and other miRNAs w/seed AUUCAUU) 2.306hsa-miR-21DmiR-21-5p(and other miRNAs w/seed AGCUUAU) 2.276hsa-miR-146bDmiR-146a-5p(and other miRNAs w/seed GAGAACU) 2.257hsa-miR-200cDmiR-200b-3p(and other miRNAs w/seed AAUACUG) 2.234hsa-miR-106aDmiR-17-5p(and other miRNAs w/seed AAAGUGC) 2.177hsa-miR-222DmiR-221-3p(and other miRNAs w/seed GCUACAU) 2.149hsa-miR-221DmiR-221-3p(and other miRNAs w/seed GCUACAU) 2.130hsa-let-7dDlet-7a-5p(and other miRNAs w/seed GAGGUAG) 2.089hsa-miR-335miR-335-5p(and other miRNAs w/seed CAAGAGC) 2.084hsa-miR-29cDmiR-29b-3p(and other miRNAs w/seed AGCACCA) 2.080hsa-miR-150miR-150-5p(and other miRNAs w/seed CUCCCAA) 2.060hsa-miR-519b-3pmiR-519a-3p(and other miRNAs w/seed AAGUGCA) 2.022hsa-miR-1305miR-1305(miRNAs w/seed UUUCAAC) 2.008hsa-miR-590-5pDmiR-21-5p(and other miRNAs w/seed AGCUUAU) −2.275hsa-miR-18amiR-18a-5p(and other miRNAs w/seed AAGGUGC) −2.868hsa-miR-130aDmiR-130a-3p(and other miRNAs w/seed AGUGCAA) −3.145hsa-miR-539miR-539-5p(miRNAs w/seed GAGAAAU) −4.294hsa-miR-29aDmiR-29b-3p(and other miRNAs w/seed AGCACCA) −4.442hsa-miR-501miR-501-5p(miRNAs w/seed AUCCUUU) −4.580hsa-miR-148bDmiR-148a-3p(and other miRNAs w/seed CAGUGCA) −4.725hsa-miR-342-5pmiR-342-5p(and other miRNAs w/seed GGGGUGC) −5.500hsa-miR-1227miR-1227-3p(miRNAs w/seed GUGCCAC) −6.308hsa-miR-222DmiR-221-3p(and other miRNAs w/seed GCUACAU) −6.411hsa-miR-483-5pmiR-483-5p(miRNAs w/seed AGACGGG) −6.427hsa-miR-432miR-432(and other miRNAs w/seed CUUGGAG) −6.929hsa-miR-26bDmiR-26a-5p(and other miRNAs w/seed UCAAGUA) −6.992hsa-miR-363DmiR-92a-3p(and other miRNAs w/seed AUUGCAC) −7.362hsa-miR-518bDmiR-518a-3p(and other miRNAs w/seed AAAGCGC) −9.878hsa-miR-27aDmiR-27a-3p(and other miRNAs w/seed UCACAGU) −10.100hsa-miR-505miR-505-3p(miRNAs w/seed GUCAACA) −12.811hsa-miR-30dDmiR-30c-5p(and other miRNAs w/seed GUAAACA) −18.215mmu-miR-134miR-3118(and other miRNAs w/seed GUGACUG) −18.842hsa-miR-502miR-502-5p(and other miRNAs w/seed UCCUUGC) −20.700hsa-miR-627miR-627-5p(miRNAs w/seed UGAGUCU) −28.711hsa-miR-598miR-598-3p(miRNAs w/seed ACGUCAU) −31.629hsa-miR-130bDmiR-130a-3p(and other miRNAs w/seed AGUGCAA) −51.340hsa-miR-30a-3pDmiR-30a-3p(and other miRNAs w/seed UUUCAGU)© 2000-2015 QIAGEN. 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