Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family
2011; Springer Nature; Volume: 30; Issue: 10 Linguagem: Inglês
10.1038/emboj.2011.94
ISSN1460-2075
AutoresLeon N. Schulte, Ana Eulálio, Hans‐Joachim Mollenkopf, Richard Reinhardt, Jörg Vogel,
Tópico(s)interferon and immune responses
ResumoArticle5 April 2011free access Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family Leon N Schulte Leon N Schulte Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Ana Eulalio Ana Eulalio Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Hans-Joachim Mollenkopf Hans-Joachim Mollenkopf Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Richard Reinhardt Richard Reinhardt Genome Centre Cologne at MPI for Plant Breeding Research, Cologne, Germany Search for more papers by this author Jörg Vogel Corresponding Author Jörg Vogel Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Leon N Schulte Leon N Schulte Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Ana Eulalio Ana Eulalio Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Hans-Joachim Mollenkopf Hans-Joachim Mollenkopf Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Richard Reinhardt Richard Reinhardt Genome Centre Cologne at MPI for Plant Breeding Research, Cologne, Germany Search for more papers by this author Jörg Vogel Corresponding Author Jörg Vogel Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany Max Planck Institute for Infection Biology, Berlin, Germany Search for more papers by this author Author Information Leon N Schulte1,2, Ana Eulalio2, Hans-Joachim Mollenkopf2, Richard Reinhardt3 and Jörg Vogel 1,2 1Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany 2Max Planck Institute for Infection Biology, Berlin, Germany 3Genome Centre Cologne at MPI for Plant Breeding Research, Cologne, Germany *Corresponding author. Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Strasse 2/D15, Würzburg D-97080, Germany. Tel.: +49 931 3182 576; Fax: +49 931 3182 578; E-mail: [email protected] The EMBO Journal (2011)30:1977-1989https://doi.org/10.1038/emboj.2011.94 There is a Have you seen? (May 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info MicroRNAs have well-established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type-dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly upregulate NF-κB associated microRNAs; strikingly, these regulations which are induced by bacterial lipopolysaccharide (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti-bacterial defence. However, a suppression of the global immune regulator miR-155 in endotoxin-tolerant macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let-7 family as the common denominator of Salmonella-regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let-7 relieves cytokine IL-6 and IL-10 mRNAs from negative post-transcriptional control. Our results establish a paradigm of microRNA-mediated feed-forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens. Introduction MicroRNAs are a class of genome-encoded small RNAs, of ∼22 nt in length, that govern post-transcriptional repression of target mRNAs in a wide range of biological processes including development, cellular differentiation, apoptosis, fat metabolism and growth control. They also have important roles in human disorders including cancer, neurodegenerative and cardiovascular diseases (Filipowicz et al, 2008; Stefani and Slack, 2008; Bartel, 2009). Regarding the host response to pathogens, microRNAs have well-established roles in viral infections (Ding and Voinnet, 2007; Umbach and Cullen, 2009). Several host microRNAs such as miR-29a and miR-32 exert direct anti-viral activity by downregulating viral mRNAs (Lecellier et al, 2005; Pedersen et al, 2007; Nathans et al, 2009). Conversely, some DNA viruses encode microRNAs and utilize the host machinery to regulate either host or viral mRNAs for their own benefit (Pfeffer et al, 2004; Gottwein et al, 2007; Umbach and Cullen, 2009). A role of microRNAs in bacterial infections was first discovered in plants where Arabidopsis miR-393 contributed to resistance against the extracellular pathogen Pseudomonas syringae, presumably by repressing auxin signalling (Navarro et al, 2006). In addition, P. syringae was shown to secret effector proteins (Navarro et al, 2008) that similarly to virus-encoded suppressor proteins (Burgyan, 2008; de Vries and Berkhout, 2008) antagonize the small RNA-directed basal immunity of the host. Before the work in plants, mammalian microRNAs were implicated in bacterial infections because of associations with immunity and inflammation (Baltimore et al, 2008; Lindsay, 2008). That is, recognition of pathogen-associated molecular patterns (PAMPs) by Toll-like receptors (TLRs) was found to differentially regulate several microRNAs, for example, TLR4-mediated sensing of bacterial lipopolysaccharide (LPS) and downstream NF-κB activity induced miR-146a/b and miR-155 (Taganov et al, 2006; O'Connell et al, 2007; Tili et al, 2007; Androulidaki et al, 2009; Ceppi et al, 2009; Liu et al, 2009). Moreover, mice deficient in miR-155 display an altered immune response, and fail to be immunized by an attenuated bacterial pathogen (Salmonella) (Rodriguez et al, 2007). Subsequently, miR-155 was also shown to be induced by Helicobacter pylori (Xiao et al, 2009; Fassi Fehri et al, 2010), a pathogen of the human stomach. Whereas P. syringae and H. pylori remain extracellular during infection, many bacterial pathogens actively invade host cells or become intracellular after ingestion by phagocytic immune cells such as macrophages. During their multistage infection, intracellular pathogens extensively manipulate the signalling and gene expression cascades of the host for survival and replication (Diacovich and Gorvel, 2010). Whether and how host microRNAs are regulated by invasive and intracellular bacteria remained unknown. Salmonella enterica serovar Typhimurium (henceforth, Salmonella) is an intensely investigated intracellular bacterial pathogen that causes gastroenteritis in humans and lethal typhoid fever in mice (Cossart and Sansonetti, 2004; Mastroeni et al, 2009). Both host-cell invasion and intracellular replication by Salmonella can be recapitulated in defined cell lines in vitro (Valdez et al, 2009). Pathogenesis is mediated by secreted effector proteins that Salmonella injects into eukaryotic cells via the two type 3 secretion systems (T3SS) that are encoded by the major pathogenicity islands, SPI-1 (invasion) or SPI-2 (intracellular survival). These effectors act in concert to subvert the host cell cytoskeleton, signal transduction pathways, membrane trafficking and pro-inflammatory responses, by directly interacting with host proteins (Galan, 2009; McGhie et al, 2009). This paper reports microRNA responses of phagocytic (RAW 264.7) and non-phagocytic (HeLa) cells during the course of infection with Salmonella. Using defined bacterial mutants impaired in invasion, intracellular survival or both, we show that bacterial LPS through the innate immune response induces long-lasting microRNA programs that prevail during the course of infection. While these programmes vary considerably depending on cell type and status, downregulation of the let-7 microRNA family appears to be a generic and consistent response, even in endotoxin-tolerant macrophages with mute TLR4-based innate immunity. We provide evidence suggesting that let-7 family members post-transcriptionally repress production of the major cytokine, interleukin-10 (IL-10), via multiple pairing sites in the 3′ UTR of IL-10 mRNA. Collectively, our data suggest that differential expression of microRNAs caused by bacterial infection results in cell type-dependent feed-forward activation of cytokine expression. Results Salmonella infection regulates host microRNAs We identified differentially regulated microRNAs by comparative deep sequencing of a total of 14 cDNA libraries prepared from the small RNA population (19–34 nt) of host cells before or after Salmonella infection, or in mock-treated cells. Experiments were performed in two of the most commonly used cell lines for in vitro study of Salmonella infections, that is, murine RAW 264.7 cells due to their macrophage-like characteristics and human HeLa cells as a common epithelial infection model. In addition to infecting both cell types with wild-type bacteria (Figure 1), we determined microRNA changes in RAW 264.7 cells after challenge with Salmonella mutants deleted for the SPI-1 or SPI-2 major virulence regions (Supplementary Figure S1). On average, ∼50 000 cDNAs were sequenced per library, and typically >70% of all cDNAs in a library matched the known or predicted microRNAs (Supplementary Table S1) compiled by mirBASE version 14 (Griffiths-Jones et al, 2008). Figure 1.Salmonella infection regulates host microRNA expression in (A) RAW 264.7 and (B) HeLa cells. Small RNA libraries of uninfected cells at 0 h, cells infected with wild-type Salmonella at 24 h p.i. and mock-treated cells at 24 h p.i., were analysed by 454 sequencing, and microRNA expression changes were calculated by comparison of cDNA hits in the libraries (24 h infection versus 0 h and 24 h mock versus 0 h). The graphs show log2 fold changes in infected cells (y axis) versus log2 fold changes in mock-treated cells (x axis). Download figure Download PowerPoint Calculation of microRNA expression changes in RAW 264.7 cells as detected by 454 sequencing at 24 h p.i. (post infection) with wild-type Salmonella versus mock treatment (Figure 1A; Supplementary Table S2) readily detected the expected upregulation of miR-21 (3.4-fold), miR-146a/b (4.0-fold) and miR-155 (25.7-fold), all of which are known to accumulate following stimulation of macrophages with bacterial LPS (Taganov et al, 2006). Intriguingly, we observed significant downregulation of several let-7 family members, namely let-7a/c/d/f/g/i and miR-98. All of the above regulations were validated in independent northern blot (Figure 2A) and quantitative real-time PCR (qRT–PCR; Figure 2B) assays. Analysis of additional time points (4 and 8 h p.i.) by both cDNA sequencing and qRT–PCR showed that the upregulation or downregulation of miR-155 and the let-7 family, respectively, is a fast response and most dramatic within the first 4 h of infection (Supplementary Figure S2). Figure 2.Expression of let-7 microRNAs is downregulated upon Salmonella infection. (A) Northern blot analysis of let-7a, miR-155, miR-146a and miR-21 expression in RAW 264.7 cells after 24 h of mock treatment or infection with Salmonella wild type, or with mutants defective in invasion (ΔSPI-1), intracellular replication (ΔSPI-2) or both (ΔSPI-1/2). (B) qRT–PCR analysis of expression changes of let-7a, miR-155 and miR-146a in RAW 264.7 cells as above. Mean values±s.d. from three independent experiments are shown. (C) Invasion/infection assays performed in RAW 264.7 cells with Salmonella wild type and the three virulence mutants. Bacteria were enumerated at 4 and 24 h p.i. Mean values±s.d. from three independent experiments are shown. #Significant difference between wild-type infection and mock control (P-value 0.05). Download figure Download PowerPoint In HeLa cells, a significant upregulation of microRNAs by Salmonella was limited to miR-1308 (Figure 1B; Supplementary Table S3), a candidate microRNA of unknown function that derives from tRNA (tRF-5005; Lee et al, 2009). In contrast, miR-21, miR-146a/b or miR-155 remained unaffected, which is in accord with a lack of expression of the LPS sensing components TLR2 or the TLR4 co-factor MD-2 in this epithelial cell type (Wyllie et al, 2000; Taganov et al, 2006; O'Connell et al, 2007). Intriguingly, our sequencing data suggested that downregulation of let-7 microRNAs also occurred in HeLa cells (Figure 1B; Supplementary Table S3), and this was further validated by qRT–PCR and northern blot analyses (Supplementary Figure S3B and C). Taken together, these results suggest that Salmonella impacts on microRNA expression in a cell type-dependent manner and generically promotes downregulation of the let-7 family in both phagocytic and non-phagocytic cells. Extracellular stimulus, rather than invasion, drives the microRNA response to Salmonella Previous studies of mammalian microRNA alterations induced by bacterial pathogens were limited to extracellular stimulation of TLR4 by treatment with purified LPS or by non-invasive H. pylori (Baltimore et al, 2008; Zhang et al, 2008; Xiao et al, 2009). To evaluate the contributions of extracellular versus intracellular bacteria, we sequenced small RNA populations of RAW 264.7 cells infected with Salmonella mutants defective in invasion (ΔSPI-1), intracellular replication (ΔSPI-2) or both (ΔSPI-1/2). Invasion/infection assays confirmed the expected behaviour in RAW 264.7 cells (Figure 2C): ΔSPI-1 bacteria were poorly internalized yet then replicated at the same rate as the wild type (compare 4 and 24 h p.i.); the ΔSPI-2 strain showed normal uptake at 4 h p.i. followed by intracellular clearance as evident from reduced bacterial load at 24 h p.i.; only few intracellular bacteria were recovered for the ΔSPI-1/2 strain at either infection time point. These infection defects notwithstanding, the three mutants induced alterations to the levels of miR-21, miR-146, miR-155 and the let-7 family similar to those induced by wild-type Salmonella (Figure 2A and B). Importantly, downregulation of the let-7 microRNAs proceeds at the same speed with the wild-type or the ΔSPI-1/2 strain as determined by qRT–PCR at 4 h p.i. (Supplementary Figure S2E). To further distinguish the effects of intracellular and extracellular bacterial stimuli, RAW 264.7 cells were infected with Salmonella expressing green fluorescent protein (GFP), and the fraction of cells which had internalized bacteria were separated from the uninfected fraction by fluorescence-activated cell sorting (FACS). Using an MOI of 1, ∼10% of the host cells contained Salmonella at 24 h p.i. (Figure 3A). Comparison of let-7, miR-146 and miR-155 levels between the two fractions revealed no difference in the regulation of these microRNAs (Figure 3C). Figure 3.An extracellular Salmonella stimulus is sufficient to induce a microRNA response in the host cells. (A) Naive (N) and (B) endotoxin-tolerant (ET) RAW 264.7 cells were infected with Salmonella expressing GFP. At 24 h p.i., the fraction of cells with internalized bacteria (GFP+) were sorted from the uninfected cells (GFP−) by FACS. (C) qRT–PCR analysis of expression changes of let-7a, miR-155 and miR-146a in the infected (GFP+) and uninfected (GFP−) RAW 264.7 cells. Mean values±s.d. from three independent experiments are shown. #No significant difference between GFP+ and GFP− fraction (P-value>0.05). Download figure Download PowerPoint Salmonella may induce apoptosis and cytotoxicity in macrophages by the activity of its secreted SipB protein (Hersh et al, 1999). The congruent patterns of microRNA changes by Salmonella strains that do (wild type, ΔSPI-2) or do not encode SipB (ΔSPI-1 and ΔSPI-1/2) argued, however, against a significant contribution of apoptosis or cytotoxicity to the altered microRNA levels observed upon Salmonella infection. To assess this further, we quantified the apoptosis and cytotoxicity levels by Annexin V and propidium iodide (PI) staining, and by measurement of LDH release. At 4 and 24 h p.i., the invasive wild-type and ΔSPI-2 strains caused a mild increase in the fraction of apoptotic cells and in cytotoxicity, while no significant apoptosis or cytotoxicity was observed in infections with ΔSPI-1 and ΔSPI-1/2 bacteria (Supplementary Figure S4). Moreover, as shown below, non-pathogenic Escherichia coli K12 bacteria impact on miR-155 and let-7 microRNAs similar to Salmonella, which further argues that apoptosis or cytotoxicity effects have little if any role in the regulations reported here. Thus, the sensing of extracellular bacterial antigens sets the microRNA response to Salmonella, at least for the first 24 h of infection. Repression of let-7 in macrophages is triggered by LPS Whereas the induction of miR-21, miR-146 and miR-155 through TLR signalling had been well established, the signal to downregulate let-7 was unknown. Since extracellular bacteria were sufficient, we hypothesized that TLR sensing of main surface antigens of bacteria might as well mediate the repression of let-7. To test this hypothesis, RAW 264.7 cells were exposed to purified LPS or FliC flagellin—sensed by TLR4 or TLR5, respectively—for 4 or 24 h, and miRNA expression was analysed by qRT–PCR. In line with previous reports (O'Connell et al, 2007), LPS strongly induced miR-155 expression in RAW 264.7 cells (Figure 4A). Intriguingly, LPS also fully recapitulated the let-7 repression observed with Salmonella (Figure 4B). By contrast, the TLR5 ligand FliC impacted neither on miR-155 nor let-7 (Figure 4A and B). To ascertain that TLR4 signalling mediates let-7 repression, we treated bone marrow-derived macrophages from TLR4+/+ and TLR4−/− mice with heat-killed Salmonella; as predicted, the downregulation of let-7 was only observed in TLR4+/+ cells (Supplementary Figure S5). Figure 4.Regulation of miR-155 and let-7 can be recapitulated with purified Salmonella LPS and pathogenic/non-pathogenic Escherichia coli. Expression changes of (A) miR-155 and (B) let-7a upon stimulation of RAW 264.7 cells with the indicated concentrations of LPS and FliC or wild-type Salmonella as determined by qRT–PCR. Expression changes of (C) miR-155 and (D) let-7a upon challenge of RAW 264.7 cells with wild-type Salmonella, enteropathogenic E. coli (EPEC) or non-pathogenic E. coli K12 as determined by qRT–PCR. Mean values±s.d. from three independent experiments are shown. #No significant difference compared with Salmonella infection (P-value>0.05). ##Significant difference compared with Salmonella infection (P-value 0.05); ##no significant difference compared with naive wild-type infection (P-value>0.05); ###significant difference compared with naive wild-type infection (P-value <0.05). Download figure Download PowerPoint By contrast, the NF-κB-dependent microRNAs still responded to extracellular stimuli. That is, miR-21, miR-146 and let-7 levels were invariably altered in endotoxin-tolerant macrophages by wild-type Salmonella and any of the three mutants (northern blot and qRT–PCR results in Figure 5C and D; additional data with sorted macrophage populations for wild-type Salmonella in Figure 3B and C). The exception was miR-155, which showed a similar pattern as the NF-κB-dependent mRNAs, being poorly induced by the three Salmonella mutants in endotoxin-tolerant cells. The induction levels of miR-155 also indicated a requirement for intracellular sensing such that the invasive ΔSPI-2 mutant caused higher (∼5-fold) upregulation of miR-155 than the non-invasive ΔSPI-1 or ΔSPI-1/2 mutants (∼2-fold). Regardless of the deviant behaviour of miR-155, the macrophage desensitization experiment shows that there is a core set of microRNAs that respond to Salmonella in a very robust manner; this set includes the let-7 family. let-7 targets the major immune-modulatory cytokines IL-6 and IL-10 Macrophages are central players in the regulation of the immune response to systemic bacterial infection, part of which is achieved by the secretion of interleukins for intercellular communication. To obtain evidence of a physiological relevance of let-7 downregulation in this process, we focused on potential targeting of cytokine mRNAs by this microRNA family. Bioinformatic analysis had predicted the let-7 family members to repress several important immune-related genes (Krek et al, 2005; Lewis et al, 2005; Asirvatham et al, 2008) including IL-6 and IL-10, two major cytokines of the acute innate immune response. To test whether let-7 post-transcriptionally regulates the mouse IL-6 and IL-10 genes via predicted RNA–RNA complementarity (Figure 6A), the corresponding full-length 3′ UTRs were cloned downstream of the Renilla luciferase ORF in a reporter vector that also expresses firefly luciferase (our internal reference). The reporters were introduced into mouse embryonic fibroblast (MEF) cells, a highly transfectable murine fibroblast line. Co-transfection of synthetic oligo-ribonucleotides mimicking let-7a or let-7d caused 1.8-fold (IL-6) or 3.5-fold (IL-10) downregulation of the reporters, as compared with transfections with no or control microRNA (Figure 6B and C). Regulations were considered specific since neither microRNA regulated Renilla luciferase expression in the parental vector (Figure 6B and C). Figure 6.let-7 microRNA family targets the cytokines IL-6 and IL-10. (A) Schematic representation of let-7a complementarity to the 3′ UTRs of IL-6 or IL-10. Shaded regions denote the IL-6 or IL-10 sequences that were deleted or mutated in the reporter vector below. (B, C) MEF cells were transfected with a mixture containing the psicheck2 plasmid (vector) or the Renilla luciferase reporter plasmids carrying the 3′ UTRs of IL-6 or IL-10, or mutants thereof (indicated above the graphs), in combination with microRNA mimics. (D) Same experiment as in (C) but using microRNA inhibitors instead of mimics. Renilla luciferase activity is normalized to that of the Firefly luciferase and set to 100 in cells with no microRNA mimic or inhibitor. Mean values±s.d. from three independent experiments are shown. Download figure Download PowerPoint IL-6 and IL-10 mRNAs contain let-7 binding sites in their 3′ UTRs The 3′ UTR of the mouse IL-6 gene was predicted to contain one binding site (positions 318–324) complementary to the seed sequence (7 nt) of all let-7 family members; the interaction would be stabilized by an additional seven base-pair complementarity with the 3′ region of let-7 microRNAs (Figure 6A). Deletion of or point mutations in the anti-seed region in the IL-6 reporter abrogated repression by let-7a and let-7d (Figure 6B and data not shown), supporting the prediction that let-7 directly targets the IL-6 mRNA. In contrast to IL-6, deletion of the single let-7 binding site initially predicted in the IL-10 reporter (3′ UTR positions 144–150) only slightly reduced repression by let-7a or let-7d, to ∼2-fold as compared with 3.5-fold of the parental IL-10 reporter (Supplementary Figure S6A). Since some microRNAs were shown to act at multiple sites in the same mRNA, we used the RNAhybrid algorithm (Rehmsmeier et al, 2004) to predict additional let-7 binding sites. Using this approach, we identified two potential let-7 binding sites that are complementary to 6 nt of the seed sequence of the let-7 family members (positions 113–118 and 135–140) in the 3′ UTR of IL-10 (Figure 6A). Similar to the first binding site, individual deletions of these two additional sites reduced but did not abolish repression of the IL-10 reporter by let-7; however, simultaneous deletion or mutation of all three sites rendered it refractory to the action of let-7a as well as let-7d (Figure 6C; Supplementary Figure S6A), suggesting that the targeting of all three binding sites is essential for post-transcriptional repression of IL-10. The identified let-7 binding sites in the IL-6 and IL-10 mRNAs are conserved in sequence throughout mammalian genomes, suggesting an evolutionary conserved targeting mechanism (Supplementary Figure S6B and C). The post-transcriptional control of IL-10 was further confirmed with a let-7 anti-sense LNA (anti-let-7), which acts to repress the endogenous let-7 microRNAs. MEF cells showed an ∼2-fold increase in IL-10 reporter activity when co-transfected with the anti-let-7 as compared with a control inhibitor or anti-miR-155 (Figure 6D), supporting our model that a depletion of the endogenous let-7 pool alleviates IL-10 repression. Again, this effect was specific as the anti-let-7 did not impact on the mutant IL-10 reporter in which the three let-7 binding sites are mutated. Salmonella infection post-transcriptionally activates cytokine expression To address whether the observed regulations in the 3′ UTR reflected regulation of the native target mRNAs, we measured let-7-dependent changes in both 3′ UTR reporter activity and IL-6/IL-10 levels in RAW 264.7 macrophages, inducing cytokine production by infection with wild-type Salmonella (Supplementary Figure S7). Increasing concentrations of let-7a or let-7d RNA mimics (Supplementary Figure S7A) caused a concomitant, up to two-fold reduction of 3′ UTR reporter activity and the level of the respective secreted cytokine within a 2.5–20 nM range of co-transfected microRNA mimic (Supplementary Figure S7B–E); above this range, the regulation of both 3′ UTR reporter and secreted cytokine was equally saturated. Conversely, we antagonized let-7 in RAW 264.7 cells with anti-sense LNA oligonucleotides before infection with Salmonella, expecting to see an increase in both the activity of co-transfected 3′ UTR reporters, and the corresponding secreted cytokines. Again, the observed ∼1.4-fold increase in 3′ UTR reporter activity correlated well with a determined increase in secreted IL-6 and IL-10 (Supplementary Figure S7F–H). In other words, the let-7-mediated regulation of the 3′ UTR reporter is representative of regulation of the endogenous target mRNA. To determine the degree of
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