Repression of human activation induced cytidine deaminase by miR-93 and miR-155
© Borchert et al; licensee BioMed Central Ltd. 2011
Received: 6 May 2011
Accepted: 10 August 2011
Published: 10 August 2011
Activation Induced cytidine Deaminase (AID) targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis.
The mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID expression. MicroRNAs (miRs) have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells.
We have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA.
Together, our findings demonstrate that miR-93 and miR-155 constitutively suppress AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located within the AID 3'UTR supports the recent suggestion that many miR regulations have been overlooked due to the prevalence of truncated 3'UTR annotations.
KeywordsAICDA AID CSR hypermutation microRNA miR-93 miR-155 SHM UTR 3'UTR
In antigen activated B cells the Activation Induced cytidine Deaminase (AID) protein is required for initiating mutagenesis and recombination of the immunoglobulin (Ig) genes to promote immunity. AID is a cytidine deaminase that converts single-stranded genomic cytidine into uracil, and its activity is pronounced at the Ig variable (V) and switch (S) regions [1–5]. While spontaneously generated uracil in the genome is faithfully corrected as a part of normal DNA repair , AID-induced uracils at the Ig loci are mutagenically resolved by multiple DNA repair factors [4, 7]. Although AID targets the Ig loci in activated B cells, activity at other genomic sites has the potential to create oncogenic DNA damage. Indeed, transcribed genes across the genome have now been found to undergo AID deamination (reviewed by [8, 9]), and recently, deep sequencing of AID-ChIPed template revealed broad AID-associations with expressed loci . In addition, AID-dependent DNA breaks were recently identified within multiple types of repetitive elements  suggesting a broad ability for AID to target genomic regions harboring single-stranded character.
AID misexpression results in DNA damage that promotes cancer [11–16]. In lymphoid cells improper AID expression has been connected with mutations at c-MYC, PIM1, RhoH and PAX5 oncogenes, promoting diffuse large B cell lymphoma . Similarly, Burkitt's lymphoma is characterized by AID-induced mutation and a reciprocal translocation between the c-MYC proto-oncogene and the IgH loci [9, 18–20]. While AID expression was initially thought to be B cell-specific, recent evidence indicates AID may promote the development of various non-lymphoid oncologies. In gastric cancer, the upregulation of AID leads to point mutations and copy number alterations of CDKN2A and CDKN2B tumor suppressor genes . In the same manner, AID misexpression in human colonic cells increases the mutation rate of TP53 by ~10-fold . In addition, AID is expressed at various levels in ~1/3 of primary lung cancers  and in numerous breast cancer cell lines  (where AID misexpression may be due in part to observations that estrogen is capable of inducing AID expression > 20 fold ). Since errant AID activity can introduce significant genomic damage, the maintenance of genome stability outside of the activated B cell environment likely depends upon multiple molecular AID restraints.
Recent evidence suggests that one level of AID regulation comes from microRNAs (miRs). These short, ~20 nt, noncoding RNAs can regulate networks of genes and function by repressing translation or directing mRNA destruction through partial sequence complementarity to 3' untranslated regions (3'UTRs) of mRNAs [26, 27]. MiRs are key regulators of cellular differentiation, proliferation and apoptosis, and aberrant miR expression has been associated with a myriad of human diseases, including cancer (reviewed by ). A subset of miRs typically misexpressed in malignancy (oncomiRs) can function as oncogenes or tumor suppressors with impacts on cellular transformation and metastasis. One such oncomiR, miR-155, represses murine AID, and the disruption of the miR-155 recognition site or miR-155 itself results in increased AID-induced c-MYC translocation  and BCL6 mutagenesis . This may provide one explanation for why disruption of miR-155 is associated with Burkitt's lymphoma  and suggests the existence of parallel miR repressions in humans.
In order to better define the regulators of human AID in cells other than antigen-activated B cells, we examined the 3'UTR of human AID for miR complementarities. Consistent with the role of miR-155 in regulating AID in mice [29, 30], we find a moderate miR-155 site in human AID. Surprisingly, we also find a previously uncharacterized, yet exceptional, complementarity to miR-93 in the AID 3'UTR. We show both of these miRs interact with the AID 3'UTR in the cell to regulate its translation, and that loss of either miR results in increased AID protein levels. Based on the involvement of AID in generating oncogenic genome mutations, our results suggest that miR-93 and miR-155 act as dual genome sentries to prevent errant translation of AID mRNA.
Reagents and cell lines
Oligonucleotide sequences are detailed in Additional File 1, Table S1. Human embryonic kidney (HEK293) cell line was obtained from GenLantis (San Diego, CA), Burkitt's lymphoma (Ramos) and breast cancer (MCF-7) cells were both purchased from the American Type Culture Collection, (ATCC, Manassas, VA). HEK293 and MCF-7 cells were cultured in MEM (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Ramos cells were cultured in RPMI (Mediatech) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). All tissue culture media were supplemented with 25 mg/ml streptomycin and 25 I.U. penicillin (Mediatech). Cells were cultured in a humidified atmosphere with 5% CO2 at 37°C. For luciferase assays, HEK293 s were cultured in MEM (10% FBS and 1% PS) in 12-well plates. At 90% confluency, cells were transfected following the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) protocol. At 35 h, existing media was replaced with 1 ml fresh media. For miR expression assays, MCF-7 s were cultured in MEM (10% FBS and 1% PS) in 6-well plates. At 90% confluency, cells were transfected following the Lipofectamine 2000 (Invitrogen) protocol and harvested after 48 h. For miR sponge assays, MCF-7 s were cultured in MEM (10% FBS and 1% PS) in 6-well plates. At 70% confluency, cells were transfected following the Lipofectamine 2000 (Invitrogen) protocol. At 48 h, existing media was replaced with 2 ml fresh media and cell transfections were repeated as initially performed. Cells were then harvested after an additional 48 h culture.
At 36 h post transfection, cells were scraped from well bottoms and transferred to 1.5 ml Eppendorf tubes. Eppendorfs were centrifuged at 2000 RCF for 3 min, followed by supernatant aspiration and cell resuspension in 300 μl of PBS. Cells were lysed by freeze thaws and debris removed by centrifuging at 3000 RCF for 3 min. 50 μl of supernatant was transferred to a 96-well MicroLite plate (MTX Lab Systems, Vienna, VA) then firefly and Renilla luciferase activities measured using the Dual-glo Luciferase® Reporter System (Promega, Madison, WI) and a 96-well plate luminometer (Dynex, Worthing, West Sussex, UK). RLUs were calculated as the quotient of Renilla/firefly RLU and normalized to mock.
MCF-7 cells (at ~1 × 106 cells/ml) were pelleted by centrifugation, existing media removed, and cells resuspended in SDS lysis buffer containing protease inhibitors and transferred to 1.5 ml Eppendorf tubes. Proteins were electrophoresed through a 4-12% SDS-polyacrylamide gradient gel (Invitrogen) and transferred to immobilon-P PVDF membranes (Millipore, Temecula, CA). Membranes were blocked for 1 hour in 5% (w/v) nonfat milk in phosphate-buffered saline containing 0.05% Tween 20, washed, and incubated with primary antibody overnight at 4°C using the following dilution: anti-AID (Santa Cruz Biotechnology, Santa Cruz, CA, sc-25620) - 1:500 and anti-PCNA (Santa Cruz) - 1:10000. Membranes were washed and incubated with secondary Abs: HRP conjugated goat anti-mouse and goat anti-rabbit (Invitrogen) at 1:10000 dilution. Immunoreactive bands were visualized with ECL Plus (GE, Piscataway, NJ) and signals were detected by using the Storm 840 PhosphorImager and IMAGEQUANT software (GE).
Unless otherwise indicated, PCR amplifications were performed in 40 μl reactions at standard concentrations (1.5 mM MgCl2, 0.2 mM dNTP, 1× Biolase PCR buffer, 0.5 U Taq (Bioline USA, Inc., Randolph, MA), 0.5 uM each primer) and using standard cycling parameters (94°C - 3 min, (94°C - 30 s, 55°C - 30 s, 72°C - 60 s) × 30 cycles, 72°C - 3 min) then cloned into Topo PCR 2.1 (Invitrogen). RT-PCRs were performed at 65°C using MonsterScript Reverse Transcriptase (#MSTA5110, Epicentre, Madison, WI) and gene specific or random nonamer primers. Resultant amplicons were cloned into Topo PCR 2.1 and sequenced. Antisense reporter, Ctl 3'LR, was constructed by oligonucleotide primer extension (25 cycles with 10 s extensions) with primers containing 5' Xho-I and 3' Spe-I restriction enzyme sites immediately flanking sequences perfectly complementary to mature siLacZ. Antisense reporter, AID 3'LR, was constructed by standard PCR with primers containing 5' Xho-I and 3' Spe-I restriction enzyme sites. Following digestion, amplicons were ligated into the Renilla luciferase 3'UTR of psiCheck2 (Promega) vector linearized with Xho-I and Spe-I. The presence of an independently transcribed firefly luciferase in these reporters allowed normalization for transfection efficiency. Sponge expression constructs were generated by concatamerizing PCR using the primers indicated in Additional File 1, Table S1. Resulting amplicons were separated on a 1% agarose gel and a band excised from the appropriate lane at ~400 bp. Gel extractions were cloned into Topo PCR 2.1 and sequenced. Concatamers were next excised from Topo PCR 2.1 and cloned into the pEGFP expression vector (Clontech, Mountain View, CA) using BamHI and Not I restriction sites.
The human AID 3'UTR contains sequences complementary to miR-93 and miR-155
MiR-93 and miR-155 interact with the AID 3'UTR to inhibit its expression
MiR-93 and miR-155 can repress endogenous AID
Endogenous miR-93 and miR-155 restrain AID protein translation
Model for AID repression
Disruptions of miR-155 and miR-93 are associated with AID-induced oncogenesis
Highlighting the importance of tightly controlled AID expression, novel AID regulations continue to be described. In 2008, miR-155 was identified as an important repressor of AID translation in mice, and loss of this regulation is lymphomagenic [29, 30]. Our data now demonstrate directly that expression of endogenous miRs -93 and -155 represses AID translation in MCF-7 breast cancer cells. While a direct correlation between miR-93 activity and AID-induced oncogenesis remains to be described, miR-93 perturbations have been found to enhance cell survival, possess oncogenic activities, and augment tumor growth through regulating integrin-β8, the tumor suppressor gene FUS1, the Cdk inhibitor p21, and tumor protein 53-induced nuclear protein 1 (TP53INP1) [39–42]. Our results suggest that in addition to these known oncogene regulations, loss of miR-93 (or miR-155) may permit AID upregulation and mutator phenotypes in non-B cell oncologies (e.g. breast, colon, stomach and lung [21–24]). However, it is possible that additional miR AID regulations may still be described, and our research does not seek to eliminate other potential regulators.
MicroRNA target sites obscured by repetitive element insertions
Our identification of an extensive and uncharacterized 3'UTR for human AID reveals the presence of sequence elements that may confound miR regulatory relationships for other mRNAs. We find that the AID 3'UTR is ~1000 nt longer than previously annotated likely due to mispriming from an internal stretch of 20 adenosines. The full length AID 3'UTR contains a previously unidentified regulatory sequence, an unusually pronounced miR-93 complementarity, (Figure 1) conferring marked repression to targeted transcripts (Figure 2 and Additional File 2, Figure S1). Clearly, this finding agrees with recent suggestions that cryptic miR complementarities may actually be quite common with nearly 50% of human and mouse 3'UTRs likely extending well beyond their annotated termini [43, 44]. Since 3'UTRs contain miR binding sites, our analysis of AID is consistent with the notion that other miR posttranscriptional regulations may have been overlooked because of truncated 3'UTR annotations. This may be particularly pronounced in humans as 5-10% of all human 3'UTRs contain at least one Alu repeat, a common miR-associated retro-element characterized by a central 10-40 nt adenosine linker [32, 45–51].
In the present study, two human microRNAs, miR-155 and miR-93, were each shown to repress the translation of human AID through interactions with the AID 3'UTR in the MCF-7 breast carcinoma cell line which aberrantly expresses AID. Together, our data suggest that low-level errant AID expression and subsequent genome damage may be prevented through protective miR-93 and/or miR-155 regulation. In addition, our identification of a miR-93 target site for AID located downstream of an internal adenosine repeat highlights the possibility that other miR regulations may have been overlooked because of truncated 3'UTR annotations.
List of abbreviations
Activation Induced Deaminase
expressed sequence tag
human embryonic kidney
Michigan Cancer Foundation - 7 breast cancer line
microRNA target site
mRNA untranslated region
3'UTR luciferase reporter.
The authors would like to recognize support from the School of Biological Sciences and the College of Arts and Sciences at Illinois State University, Phi Sigma Honor Society fellowship to NWH, the National Cancer Institute, National Institutes of Health, 01R15CA137608 to EDL, and the American Cancer Society, Illinois Division, Inc. grant # 215165 to EDL.
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