miR-155 文献3

Elevated miR-155 Promotes Inflammation in Cystic Fibrosis by Driving Hyperexpression of Interleukin-8*□S Received for publication,October 28, 2010, and in revised form, January 24, 2011 Published, JBC Papers in Press, January 31, 2011, DOI 10.1074/jbc.M110.198390 Sharmistha Bhattacharyya‡, Nagaraja S. Balakathiresan‡, Clifton Dalgard§, Usha Gutti‡, David Armistead¶, Cathy Jozwik§, Meera Srivastava§, Harvey B. Pollard§, and Roopa Biswas‡1 From the ‡Department of Health Systems, Risk, and ContingencyManagement, Graduate School of Nursing, and the §Department of Anatomy, Physiology and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and ¶Applied Biosystems, Foster City, California 94404 Cystic Fibrosis (CF) is characterized by a massive proinflam- matory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link betweenmutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that spe- cificmicroRNAs (miRNAs)mightmediate this linkage. To iden- tify the potential link, we screened an miRNA library for differ- ential expression in �F508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR- 155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced lev- els of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells sup- pressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifi- cally lowered by either antagomir-155 or elevated expression of SHIP1.We therefore suggest that elevatedmiR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF. Cystic fibrosis (CF),2 the most common autosomal recessive disease in the United States and Europe, is caused bymutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1–4). CFTR mutations, of which the most com- mon is�F508-CFTR, cause amassive proinflammatory pheno- type in the lung, which manifests in the airway by high levels of IL-8 and other proinflammatory cytokines and chemokines (5–7). IL-8 is the most potent known chemotactic agent for neutrophils (8) and is constitutively secreted from CF lung epi- thelial cells (9). The enhanced secretion of IL-8 seems to be an intrinsic property of the CF epithelium because fetal CF lung epithelium also spontaneously secretes high IL-8 levels into the airway as early as the 15th week of gestation (10, 11). However, the mechanism by which a mutation in CFTR causes up-regu- lated levels of IL-8 expression remains poorly understood. The expression of proinflammatory genes, such as IL-8, is known to be regulated by post-transcriptionalmechanisms. For example, the stability of mRNAs encoding many inflammatory genes, including IL-8, is regulated by interactions between AU- rich elements (AREs) in the 3�-untranslated region (3�-UTR) and specific ARE-binding proteins. In the case of CF lung epi- thelial cells in culture, which constitutively secrete high levels of IL-8, we have recently reported that high levels of IL-8 mRNA are sustained by mutation-dependent reduction in the ARE-binding protein tristetraprolin (TTP) (12). We also found a similarly low level of TTP in primary CF lung cells, which had been obtained acutely by brush biopsy of CF patients. Finally, we found experimentally that elevation inTTPdirectly reduced the stability of IL-8 mRNA, and, concomitantly, reduced the level of secreted IL-8. Thus hypersecretion of IL-8 from the CF lung epithelium seems to involve a loss of post-transcriptional regulation byTTP.However, it also became clear that low levels ofTTPmight not be the onlymechanismunderpinningCF lung inflammation. For example, as mentioned above, the CF airway is characterized not only by high levels of IL-8 but also by high levels of TNF�, IL-6, and many other potent proinflammatory analytes (5–7). Furthermore, many of the CF-specific proin- flammatory processes have been associated with up-regulation of both TNF�/NF�B signaling (13, 14) and TGF�-1 signaling (15). Thus, the CFTR mutation appears to target a proinflam- matory regulatory mechanism with simultaneous deleterious effects on many proinflammatory genes. MicroRNAs (miRNAs) may provide such a pleiotropic mechanism. miRNAs mediate mRNA instability by action in the 3�-UTRs of target genes (16–19). There are nearly 1000 unique miRNAs in the human genome, each of which individ- ually targets �200 different mRNAs (20–25). Furthermore, it has been reported that, in response to certain kinds of stress, * This study was supported by USU-Intramural Funds (to R. B.), National Insti- tutes of Health (RO1-DK053051, to H. B. P.) and Cystic Fibrosis Foundation (to R. B. and H. B. P.). □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1 To whom correspondence and reprint requests should be addressed: Grad- uate School of Nursing, Rm. B4024, Uniformed Services University of the Health Sciences, Bethesda, MD 20814. E-mail: rbiswas@usuhs.mil. 2 The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis trans- membrane conductance regulator; miRNA, microRNA; TTP, tristetraprolin; ARE, AU-rich element; IPA, ingenuity pathway analysis; qPCR, quantitative PCR; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PIP2, phosphatidylino- sitol 4,5-bisphosphate. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 13, pp. 11604–11615, April 1, 2011 Printed in the U.S.A. 11604 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 13•APRIL 1, 2011 miRNAs can switch from a normally translational repressor mode to that of a translational activator (26, 27). Taken together, it appears that a relatively fewmiRNAs can regulate as much as 20–30% of the human genome. Recently, specific miRNAs have been reported to be associated with diabetes (28, 29), cancer (30–33), heart disease (34, 35), cell cycle (36), and development (17). Importantly, functional suppression of miRNAs can be achieved, both in vitro and in vivo, by antago- mirs, which are chemically engineered oligonucleotides that are antisense to miRNAs (37). However, the possibility of an association between specific miRNAs and the pathophysiology of cystic fibrosis has not yet been described. Thus, if aberrant elevation of a CF-specific miRNA could be identified, then it might lead to a candidate therapeutic agent. We report here the results of a hypothesis-based discovery study formiRNAs associatedwith the proinflammatory pheno- type for cystic fibrosis. We specifically hypothesized that uniquely expressedmiRNAsmight aid in identifying themech- anism by which mutant CFTR induces the characteristic pro- inflammatory phenotype in theCF lung. To test this hypothesis, we have performed a comprehensive and systematic analysis of all miRNAs in CF IB3-1 lung epithelial cells that are differen- tially affected by the presence of natural abundance �F508- CFTR. Among 22 miRNAs that are aberrantly expressed in CF cells, we have identified miR-155 as the most abundantly ele- vated species, both in vitro and in vivo. Furthermore, we found that a reduction in miR-155, mediated by either antisense or antagomir constructs, resulted in suppression of IL-8 mRNA and concomitantly reduced expression of IL-8 protein. The mechanism of miR-155 action in CF cells is to inhibit transla- tion of SHIP1, an inositol phosphate phosphatase. Loss of SHIP1 results in elevated signaling of the PI3K/Akt pathway, with downstream effects on the IL-8 system. We suggest that miR-155may play an important role in the regulation of inflam- mation in CF lung epithelial cells. EXPERIMENTAL PROCEDURES Reagents—LHC-8 media, trypsin-EDTA (0.05%), and Lipo- fectamine transfection reagent were purchased from Invitro- gen. Bronchial epithelial growth medium and the normal human bronchial epithelial cells were purchased from Lonza. The miRVana kit and RiboPure kit for isolation of total RNA from CF cells were purchased from Ambion Inc. (Austin, TX). Taqman low density V1 arrays, miRNA primer pools, and pMIR-Report luciferase vector were purchased from Applied Biosystems (Foster City, CA). Wortmannin was purchased from EMD Chemicals (Gibbstown, NJ), and CFTRinh-172 was obtained from Sigma. Cell Culture—IB3-1 CF lung epithelial cells and the control CFTR-repaired IB3-1/S9 cells were maintained in LHC-8 serum-free medium in humidified 5% CO2 as described previ- ously (13). RNA Isolation—Total RNA was isolated from the IB3-1 and IB3-1/S9 cells using the miRVana isolation kit (Ambion). The primary bronchial epithelial cells were obtained from lung brush biopsies of CF patients as described earlier (12), and the bloodwas collected fromCF patients and controls under a Uni- formed Services University of the Health Sciences Institutional ReviewBoard-approved protocol. The cells were stored in RNA later or TRIzol, and total RNA was isolated using the miRVana kit. Real-time Quantification of miRNAs by Stem-Loop RT-PCR— Multiplex Reverse Transcription was performed with the TaqMan microRNA reverse transcription kit (Applied Biosys- tems). Following reverse transcription, each reverse transcrip- tion reaction was diluted andmixedwith TaqMan gene expres- sion Master Mix (2�). 100 �l of the reverse transcription reaction-specific PCR mix was loaded into the corresponding fill ports of the TaqMan low density human microRNA panel version 1.0 (Early Access). The CF brush biopsy samples were similarly analyzed using the Taqman version 2.0 low density arrays. Individual microRNA assays were performed using spe- cific TaqMan MicroRNA assay kit (ABI). miRNA Expression Arrays—miRNA expression arrays were probed essentially as described (Ambion). Five micrograms of total RNA from IB3-1 or IB3-1/S9 cells were end-labeled with 30 �Ci of [�-33P]dATP (3000 Ci/mmol) by T4 polynucleotide kinase and purified using the QIAgen nucleotide removal kit. Membranes were first prehybridized in MicroHyb hybridiza- tion buffer (ResGen) at 37 °C for at least 30 min, followed by an overnight hybridization in the same solution containing RNA probe. Following hybridization membranes were washed twice with 2� SSC, 0.5% SDS at 37 °C. The second wash was per- formed in 1� SSC, 0.5% SDS at 37 °C. Membranes were exposed to a phosphor storage screen and scanned using a PhosphorImager, and hybridization signals were quantified using ImageQuant software (Amersham Biosciences). Statistical Data Analyses—Real-time PCR data were ana- lyzed using the R and the Bioconductor package as well as with STATMINER (a statistical analyses package from ABI). Data were filtered forCt values of�35, and the datawere normalized to the endogenous control gene RNU44. If an assay measure- ment was not detected in both experimental subgroups, the assay was not included in the pairwise statistical analysis. Fur- thermore, if an assay measurement was not detected in more than 50% of samples in an experimental subgroup, it was deemed undetected for that subgroup. For all detectable assays, an unpaired Student’s t test was performed on the �Ct values. Adjusted p values (false discovery rates) were calculated using the Benjamini Hochberg procedure. -Fold changes were calcu- lated using the comparativeCtmethod. Hierarchical clustering based upon Euclidean distances was performed on differen- tially expressed samples with p values of�0.05. Illumina mRNA Expression Processing and Analysis—Bea- darray data were obtained from whole-genome expression HumanRef-8 version 2.0 as well as human HT-12 BeadChips using the iScan system and BeadScan software (Illumina, San Diego, CA). Non-background, non-normalized array data were generated using BeadStudio 3.2.7 software. Preprocessing of array data bymodel-based or offset background correction and robust spline normalization was performed using MATLAB or the Lumi 1.8.3 package from Bioconductor 2.3 on the R 2.8 programming language platform. The processed Illumina array data are MIAME-compliant and have been submitted to the NCBI Gene Expression Omnibus (GEO) data base. Processed array data were analyzed using ingenuity pathway analysis miR-155 Regulates IL-8 Expression APRIL 1, 2011•VOLUME 286•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 11605 (IPA). MicroRNA and mRNA relationship analysis was gener- ated using TargetScan release 5.1 (Whitehead Institute) for miRNA biological target prediction and IPA. RESULTS CF Lung Epithelial Cells Express Mutation-specific miRNAs— As shown in Table 1, we used two independent methods to identify CF-specific microRNAs in CF lung epithelial cells. Using the new technology, quantitative Taqman� qPCR miRNAarray platform, we find that of 365miRNAs tested, only 22 significantly distinguish between the natural abundance IB3-1 CF cell and the wild type CFTR-repaired daughter cell, IB3-1/S9 (see Table 1, left). For this analysis, we analyzed three independent cultures of both IB3-1 and IB3-1/S9 cells and identified any miRNAs for which the -fold difference was at least�50%, and the p value for the difference was�0.05. Of the 22 differentially expressed miRNAs, 18 were elevated in the CF cells, and four were reduced. The data in Table 1, ordered by -fold change, indicate that the miRNAs with the highest differ- ential expression (�4-fold) are miR-155 and let-7c. We also validated the miR-155 and miR-let7c data with independent Taqman� miRNA assays (see Fig. 1B). Of the remaining 16 elevatedmiRNAs, all were changed by 1.3–3.4-fold. Of the four down-regulated miRNAs, only miR-615 has a �2-fold reduc- tion, whereas miR-660, miR-194, and miR-192 exhibit reduc- tions only in the 1.2–1.6-fold range. However, these differently expressed miRNAs all appear to contribute to a composite CF microRNA signature. For exam- ple, Fig. 1A shows that when all 22 microRNAs are compared using a hierarchical cluster algorithm, the dendrogram clearly distinguishes between three independent experiments with CF IB3-1 and three independent experiments with CFTR-repaired IB3-1/S9 daughter cells. Thus, despite quantitative differences in expression, all of the significantly aberrantmiRNAs, from the most aberrantmiR-155 to the least, appear tomake a concerted contribution to the CF phenotype in the CF IB3-1 lung epithe- lial cell system. To further validate the Taqman� data, we also tested the CF lung epithelial cell samples using a conventional, semiquantita- tive Ambion Bioarray� platform (see Table 1, right). The data with this different type of system consistently replicated 12 of the 18 miRNAs predicted by the Taqman� platform. The com- mon directions of change are indicated by upward or down- ward arrows. Horizontal double-headed arrows indicate satu- rated expression. The validatedmiRNAs includedmiR-155 and many of the other miRs with predicted high -fold differences. Several of the low -fold changes are also confirmed. However, consistent saturation problems were encountered with two of the high -fold different miRs, let7c and miR-21, and were not pursued further. Inasmuch as we had independently validated the let7c by PCR (Fig. 1B), we did not pursue this technical saturation problem further. Of the microRNAs with a�2-fold difference from control, 6 were not detectable at all on the microarray platform (markedND). Based on low levels in either platform, we also chose not to pursue this technical sensitivity problem any further. We therefore conclude that both the quan- titative Taqman� and the semiquantitative Ambion Bioarray� platforms agree with similar identifications for most of the miRNAs with high -fold differences between CF IB3-1 and wild type CFTR-repaired IB3-1/S9 cells, includingmiR-155. Ex Vivo Clinical CF Cells Also Exhibit Elevated miR-155 Levels—To test the extent to which microRNA data from the IB3-1 cell system might parallel microRNA expression in cells from CF patients, we used the Taqman� qPCR miRNA array platform tomeasuremicroRNA expression in bronchial brush- ings and neutrophils obtained from CF patients homozygous for the �F508 mutation. As a control for the CF lung epithelial cells obtained by brush biopsy, we measured miR-155 in pri- mary non-CF normal human bronchial epithelial cells. As a control for CF neutrophils, we used neutrophils from normal individuals. Of the 22 differentially expressed microRNAs found to be differentially expressed in the CF IB3-1 cell system, only massively elevated miR-155 expression was noted. We therefore validated this finding using independent Taqman� miRNAassays. As shown in Fig. 1C, we found thatmiR-155was significantly elevated in CF bronchial brushings (versus normal human bronchial epithelial cells, 10.8-fold elevated) and in CF neutrophils (CD66�CF versus control neutrophils, 2.4-fold ele- vated). We conclude that these differences therefore appear to closely parallel the differences in the CF lung epithelial IB3-1 cell system (IB3-1 versus IB3-1/S9, 8.6-fold elevated). Addition- ally, the inhibition ofWTCFTR functionwithCFTR-172 inhib- itor in CFBE41o-wtCFTR cells, a human bronchial epithelial cell line, has been shown to produce a similar effect on inflam- matory signaling as is observed in CF cells (38). As depicted in Fig. 1D, treatment of the IB3-1/S9 CFTR-repaired control cells TABLE 1 miRNA expression levels in IB3-1 CF cells relative to control cells (p< 0.05) The miRNAs that exhibit altered expression, analyzed by Taqman qPCR miRNA arrays, are listed with respective -fold changes in expression in the IB3-1 CF cells compared with IB3-1/S9 control cells; the 18 miRNAs up-regulated (1) and the four that are down-regulated (2) in CF cells are indicated. Additionally, themiRNA expression profile obtained by miRNA microarrays (Bioarray, Ambion) is also depicted. The miRNAs that indicate similar expression profile by these two inde- pendent methods are indicated in boldface type. NP, not present; ND, not detecta- ble;7, saturated expression. miR-155 Regulates IL-8 Expression 11606 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 13•APRIL 1, 2011 with CFTR-172 inhibitor induced an 8-fold up-regulation of miR-155. These data indicate that elevation inmiR-155 expres- sion is closely tied to failure of CFTR channel activity, either by chemical inhibition or by mutation. The data thus consistently suggest that further specific interest in miR-155 is warranted. Aberrantly Expressed miRNAs Have Predicted Effects on mRNA Expression in CF Cells—To further understand the mechanism by which miR-155 and the other CF-specific microRNAs might affect the CF phenotype, we used the ILLUMINA� bead chip system to analyze levels of �24,000 transcripts, in both CF IB3-1 andwild type CFTR-repaired IB3- 1/S9 cells. Table 2 is ordered by starting with top-ranked miR- 155 and then proceeding in the order of declining values of -fold elevation, followed by -fold reduction in CF cells. For each of the miRNAs, the changes are listed for the top six specific mRNAs, chosen from among the predicted target mRNAs for the respectivemiRNA. In each box,mRNAs that are targeted by more than one of the CF-specific microRNAs are shown in bol