miR-181 targets multiple Bcl-2 family members and influences apoptosis and mitochondrial function in astrocytes
Highlights
► miR-181a targets multiple Bcl-2 family members. ► Reducing miR-181a protects neural cells from ischemic stress. ► Reducing miR-181a protects mitochondrial function.
Introduction
Mitochondria are known to play a leading role in the decision between cell death and cell survival via control of signaling to induce apoptosis. A key family in the regulation of apoptosis via the mitochondrial pathway is the Bcl-2 family consisting of about 20 pro- and anti-apoptotic proteins divided into three groups. The anti-apoptotic multidomain members, such as the prototype B cell lymphoma-2 (Bcl-2), contain BH1-4 domains. The pro-apoptotic multidomain proteins often referred to as the Bax subfamily contain domains BH1-3. The BH3-only group is pro-apoptotic (Adams and Cory, 2007). These proteins control apoptosis by regulating opening of the mitochondrial membrane permeability pore. In addition to their role in regulating mitochondrial permeability, recent work indicates a role for these proteins in control of mitochondrial fission and fusion (Rolland and Conradt, 2010) and in cellular homeostasis with respect to metabolism, calcium signaling, endoplasmic reticulum function, and autophagy (Danial et al., 2010). Bcl-2 decreases after brain ischemia (Martinez et al., 2007) and overexpression of pro-survival Bcl-2 family proteins protects against cerebral ischemia in vivo (Kitagawa et al., 1998, Zhao et al., 2003) and in vitro (Xu et al., 1999). The neuroprotective mechanism involves maintaining mitochondrial function (for review see Ouyang and Giffard, 2004a). However, the regulation of the Bcl-2 family following cerebral ischemia is not fully understood.
Tissue and cell type specific expression of microRNAs (miRNAs) is now appreciated to provide an additional level of control of protein expression by silencing specific messengerRNAs (mRNAs) to which the miRNA binds with sequence specificity, usually to a sequence within the mRNA 3′UTR. This results either in mRNA silencing or degradation. Because the sequence recognition site or seed in the miRNA is generally only 6 bp long, each of the several hundred miRNAs has many targets, and each mRNA can be targeted by multiple miRNAs. miR-181 has been studied particularly in the setting of immune cell differentiation and leukemia (Chen et al., 2004, Li et al., 2007, Zimmerman et al., 2010). These prior studies have identified several targets for miR-181 including phosphatases SHP-2, PTPN22, DUSP5, and DUSP6, and Bcl-2 family member Mcl-1. In addition Kazenwadel and colleagues identified a key role of miR-181 in controlling Prox 1 levels and thereby determining endothelial cell fate as either lymphatic or blood phenotype (Kazenwadel et al., 2010).
These earlier studies found that the miR-181 family, especially miR-181a and miR-181b, are enriched in brain (Chen et al., 2004, Miska et al., 2004) and their aberrant expression has been associated with brain diseases. hsa-miR-181a and hsa-miR-181b are reduced in human gliomas and glioma cell lines, and expression is negatively correlated with tumor grade (Shi et al., 2008). miR-181a sensitizes human malignant glioma cells to radiation (Chen et al., 2010). While miR-181 is enriched in brain, we do not yet know the cell type specific expression patterns. Our prior results show that in brain cortex, Bcl-2 expression decreases with age, being highest from embryonic day 14 to postnatal day 0, then declining (Xu et al., 2004). Bcl-2 expression was much higher in cultured astrocytes than in cultured neurons, so this study was performed in astrocytes.
Despite their abundance and functional importance, very few studies to date have evaluated the role of miRNAs in ischemic brain damage and in particular in the regulation of cell death and mitochondrial function. Profiling studies of miRNAs following brain ischemia showed changes in miR-181 (Jeyaseelan et al., 2008, Yuan et al., 2010). Due to the high expression of miR-181 in brain, its broad conservation through evolution and the observation that its expression changes after cerebral ischemia we decided to investigate the role of miR-181 in brain cell response to ischemic stress.
At least three RNA species, long primary miRNA (pri-miRNA), ~ 70-nucleotide (nt) precursor miRNA (pre-miRNA), and ~ 22-nt mature miRNA, are made from miRNA genes through transcription and sequential endonucleolytic maturation steps (Kim, 2005). The miR-181 family consists of four members (miR-181a, miR-181b, miR-181c, and miR-181d) that map to two distinct genomic loci in clusters (Fig. 1a) (mirbase.org). In this study, we first identified putative apoptosis related targets of the broadly conserved miR-181 using computational prediction algorithms to identify potential target sequences in the mRNA 3′UTRs of three Bcl-2 family members. Using the dual luciferase reporter assay we validated the targets. We then examined the relationship between miR-181 expression and Bcl-2 family protein expression in primary astrocytes. Lastly we verified that mitochondrial function was improved by decreasing miR-181 in stressed astrocytes, as we previously found by directly increasing expression of Bcl-2 (Ouyang and Giffard, 2004a).
Section snippets
pri-miRNA, miRNA, 3′UTRs, luciferase reporter assay
Since pri-miR-181a and b or pri-miR-181c and d are only about 100 nt apart (Fig. 1a), we made pri-miR-181ab and pri-miR-181cd constructs. Wild-type mature miR-181a-d and their seed mutant sequences are shown in Fig. 1b. DNA fragments containing the pri-miR-181ab or pri-miR-181cd hairpin (or their seed mutants) and ~250 nt flanking sequence on each side were cloned downstream of the PGK promoter in MWX-PGK-IRES-GFP (Fig. 1c); MWX-PGK-IRES-GFP was a kind gift from Dr. Chang-Zheng Chen at Stanford
miR-181 targets the 3′UTRs of three Bcl-2 family members
Using computational miRNA target prediction algorithms, as detailed at TargetScan (http://targetscan.org, Release 5.1) and Microcosm Targets (http://www.ebi.ac.uk/enright-srv/microcosm), we identified three members of the Bcl-2 family with mRNA 3′UTRs that were potential targets: Bcl-2 and Mcl-1 in the pro-survival subgroup and Bim/Bcl-2-like 11 (Bcl-2-L11) a BH3-only pro-apoptotic protein. Interestingly, the seed sequences of all these 3′UTRs targeted by miR-181 are highly evolutionarily
Discussion
This is the first investigation of the regulation of Bcl-2 family members in astrocytes by miRNA. Apoptosis is controlled at many levels, often reflecting the specific cell type and tissue involved. Recent work has begun to define ways in which apoptosis may also be controlled by changes in miRNA, though much of it has been in cancer, and some in development. To date research on miRNAs in brain ischemia has focused on profiling expression changes, with several studies employing different models
Acknowledgements
This work was supported in part by NIH grants NS053898 and GM49831 to RGG. The authors would like to thank Chang-Zheng Chen for the plasmid and William Magruder for help in preparing the manuscript.
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These authors contributed equally to this work.