Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 β: identification of miR-153 target genes with functions related to IA-2β in pancreas and brain
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We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2β. We also identified miR-153 target genes that correlated with IA-2β localisation and function.
A bioinformatics approach was used to identify miR-153’s genomic organisation. To analyse the co-regulation of miR-153 and IA-2β, quantitative PCR analysis of miR-153 and Ia-2β (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2β single knockout and Ia-2/Ia-2β double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out.
Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2β, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2β in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2β knockout and Ia-2/Ia-2β double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2β expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels.
This study suggests the involvement of miR-153, IA-2β and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.
KeywordsDiabetes Glucose stimulation IA-2β MicroRNA miR-153 Neurodegeneration
Ia-2 single knockout
Ia-2β single knockout
Dense core vesicle
Ia-2/Ia-2β double knockout
Ingenuity Pathway Analysis
National Institutes of Health
Primary RNA transcript
Protein tyrosine phosphatase
Synaptosomal-associated protein 25
Transcription start site
Vesicle-associated membrane protein 2
Islet-associated protein (IA) 2 and IA-2β are major autoantigens in type 1 diabetes . Based on sequence, IA-2 and IA-2β, also known as ICA512 and phogrin, respectively, are members of the transmembrane protein tyrosine phosphatase (PTP) family, but are enzymatically inactive with standard PTP substrates because of two critical amino acid substitutions in the PTP domain . However, recent studies have shown that IA-2β has low phosphatidylinositol phosphatase activity . Both proteins consist of an intracellular, transmembrane and luminal domain and are produced in neuroendocrine cells throughout the body (e.g. pancreatic islets, brain) [4, 5, 6, 7, 8]. Knockout mice of the individual Ia-2 (also known as Ptprn) and Ia-2β (also known as Ptprn2) genes and Ia-2/Ia-2β double knockout (DKO) mice do not develop diabetes, but show abnormal glucose tolerance, impaired insulin secretion and reduced insulin content in beta cells [9, 10, 11, 12]. Moreover, DKO mice display defects in neurotransmitter release (dopamine, noradrenaline [norepinephrine] and serotonin), resulting in behavioural and learning disturbances, seizures and reduced lifespan . These findings demonstrate that IA-2 and IA-2β not only affect the secretion of insulin, but also that of neurotransmitters, raising the possibility that defects in the IA-2–IA-2β pathway might not only lead to glucose intolerance, but also to neurological disorders.
MicroRNAs (miRNAs) function as a rheostat of the genome and proteome by controlling gene expression at the post-transcriptional level . These short (~21 to 23 nucleotides [nt]), conserved, non-coding RNA molecules are transcribed from the genome as miRNA precursor molecules, which are processed to single stranded miRNA by a series of ribonucleases. The mature miRNA is integrated into the RNA-induced silencing complex (RISC) and binds partial complementary mRNA sequences that mostly reside in the 3′ untranslated region (UTR) of mRNA. Binding of the miRNA to its target mRNA results in either translational repression or mRNA destabilisation . In mammals, about one thousand conserved miRNAs have been identified and can be localised as either intragenic (intronic or exonic) or intergenic . Intragenic miRNAs can be transcriptionally regulated by their own regulatory elements and/or co-regulated with their host gene. A single miRNA can influence the translation of several mRNA sequences at once, potentially affecting parallel biological pathways. Dysregulation of miRNA expression contributes to various diseases, including cancer , and cardiovascular  and neurodegenerative diseases .
Examination of the genomic sequence of IA-2 (also known as PTPRN) and IA-2β (also known as PTPRN2) revealed that two miRNAs, miR-153-1 and miR-153-2, are embedded in these genes. Because of the importance of IA-2 and IA-2β in type 1 diabetes, we investigated whether miR-153 expression is co-regulated with IA-2 and IA-2β; we also determined whether it might be involved in regulating target genes that play a role in similar cellular functions to those affected by IA-2 and IA-2β, such as secretion of neurotransmitters in the brain and insulin release by beta cells in pancreas. This study provides the first evidence of a miR-153–IA-2β–target gene regulatory pathway and sets the stage for further investigations to obtain a greater mechanistic insight into this pathway.
Animal care and use
Targeted disruption of the individual Ia-2 and Ia-2β genes has been described previously [10, 11, 12, 20]. Because female Ia-2 −/− /Ia-2β −/− mice are infertile, male Ia-2 −/− /Ia-2β −/− mice were bred to female Ia-2 +/− /Ia-2β −/− mice to generate Ia-2 −/− /Ia-2β −/− (DKO) mice. Ia-2 +/+ /Ia-2β +/+ wild-type mice served as controls. Animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committees of the USA at the National Institutes of Health (NIH).
Cell culture reagents were purchased from Invitrogen (Ghent, Belgium). Antibodies were derived from the following sources: mouse anti-β-actin (A5441; Sigma-Aldrich [Diegem, Belgium]), mouse anti-alpha-synuclein (SNCA) (610787; BD-Biosciences [Erembodegem, Belgium]), mouse anti-parkin (PARK2) (MAB5512; Millipore [Overijsse, Belgium]), rabbit anti-synaptosomal-associated protein 25 (SNAP25) (AB1762; Millipore) and mouse anti-bassoon (monoclonal antibody SAP7F407; Enzo Life Sciences, [Antwerpen, Belgium]). Total RNA from mouse pancreas, heart and brain was purchased from Biochain (Kampenhout, Belgium). Total RNA and miRNA from wild-type and DKO mouse islets were extracted using a kit (miRNeasy Mini kit; Qiagen [Antwerpen, Belgium]) according to the manufacturer’s protocols.
MIN6B mouse insulinoma cell line (ATCC, Molsheim, France) was cultured at 37°C in 95% air and 5% CO2 in DMEM supplemented with 15% vol./vol. heat-inactivated fetal bovine serum, 2 mmol/l l-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The human neuroblastoma SH-SY5Y cell line (ATCC) was cultured in DMEM/F12 medium supplemented with 10% vol./vol. fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin; the culture process was at 37°C in 95% air and 5% CO2.
Glucose-stimulated gene expression assay in fresh mouse islets
Islets from 3- to 4-month-old sex-matched mice were isolated as previously described , with slight modifications according to the manufacturer’s protocol for Collagenase P (Roche, Indianapolis, IN, USA), and stimulated with various concentrations of glucose, followed by RNA extraction and quantitative PCR analysis. See electronic supplementary material (ESM) Methods for further details.
Glucose-stimulated gene expression assay in MIN6B cells
MIN6B cells were stimulated with various concentrations of glucose, followed by RNA extraction and quantitative PCR analysis. See ESM Methods for further details.
Quantitative RT-PCR (mRNA)
Total RNA was extracted using a kit (mirVana Paris kit; Applied Biosystems [Ghent, Belgium]) and following the manufacturer’s instructions. RT-PCR was performed with a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s instructions; for quantitative PCR we used the Taqman gene expression assay (Applied Biosystems) for Ia-2, Ia-2β, primary RNA transcript (pri) pri-miR-153 (Mm03306386_pri), Snca, Syt1, Syt4, Bsn, Pclo, Vamp2, Snap25, Gapdh, Tbp and β-actin according to the manufacturer’s instructions and using a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland).
Quantitative RT-PCR (miRNA)
Total RNA was extracted using a kit (mirVana Paris kit; Applied Biosystems) and following the manufacturer’s instructions. Each RT-PCR reaction was performed in triplicate. The Taqman miRNA reverse transcription kit (Applied Biosystems) and the Taqman Universal PCR master mix (Applied Biosystems) were used according to the manufacturers’ instructions. The quantitative PCR procedures were carried out following the instructions provided with the Taqman miRNA assays (Applied Biosystems). The following miRNA assay was used: mmu-miR-153 (Applied Biosystems). Relative miR-153 expression was calculated by using the comparative cycle threshold method and normalised to the following reference genes: RNU19 (also known as Snora74a), Sno135 (also known as Snord65) and miR-16.
Bioinformatics identification of potential miR-153 targets involved in neurotransmitter and insulin release
Analysis of co-production of IA-2β and predicted miR-153 targets (predicted by Targetscan www.targetscan.org release 6.2, accessed June 2012; 629 top predicted conserved targets) in brain and pancreas was performed by searching the literature. Overlap of IA-2β and miR-153 target gene expression was further validated using the following online gene expression databases: BIOGPS (http://biogps.gnf.org, accessed Sept 2012), the type 1 diabetes database (T1Dbase)(www.t1dbase.org, accessed Sept 2012) and the Allan brain atlas (www.brain-map.org/, accessed Sept 2012) (data not shown). Targets that met these criteria were used for further analysis to determine possible protein × protein interactions and gene–function relationships between the predicted targets using Ingenuity Pathways Analysis (IPA) 9.0 (Ingenuity Systems, www.ingenuity.com, accessed Sept 2010).
Protein extraction and western blot analysis
Cultured cells were rinsed with cold PBS and lysed in the cell disruption buffer from the mirVANA Paris kit (Applied Biosystems). Note that with this extraction procedure, co-isolation of proteins and high-quality RNA (including the small RNA species) from the same sample is possible, allowing direct comparative measurements (of miRNAs, mRNA and protein) within a given tissue or cells. This protocol was used to extract RNA and proteins from SH-SY5Y cells, and from adult wild-type, Ia-2 single knockout (AKO), Ia-2β single knockout (BKO) and DKO mouse brains. Proteins were analysed by western blotting according to standard procedures. See ESM Methods for further details.
Transfections, DNA cloning and luciferase assay
MIN6B and SH-SY5Y cells were transfected using a transfection reagent (HiPerFect; Qiagen) or Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions and further processed for quantitative PCR analysis or luciferase reporter assays. See ESM Methods for further details.
Localisation and co-regulation of miR-153 and IA-2/IA-2β genes
Next, we analysed mirR-153 expression in mouse MIN6B cells and isolated mouse pancreatic islets after glucose stimulation (Fig. 2d,e). We detected a significant increase in the expression of miR-153, Ia-2 and Ia-2β after glucose stimulation of MIN6B cells at 25 mmol/l glucose compared with 5 mmol/l glucose (Fig. 2d). Finally, increasing glucose levels from 3.3 to 16.7 mmol/l dramatically increased miR-153, Ia-2 and Ia-2β expression in cultured isolated mouse islets (Fig. 2e). These studies show that miR-153, Ia-2 and Ia-2β expression is increased after glucose stimulation in MIN6B cells and isolated mouse pancreatic islets.
These results also show that whereas miR-153 expression was almost completely lost in DKO islets (Fig. 2c), miR-153 levels in DKO brain were still detectable at about 50% of the levels found in wild-type mouse brain (Fig. 2b). This might indicate that in addition to miR-153 being generated from a transcript and transcription start site (TSS) shared with the host gene Ia-2β, an alternative tissue-specific transcript and TSS for miR-153 might exist independently of its host gene Ia-2β. Moreover, the targeted Ia-2β allele was generated by deleting its promoter region . Thus, loss of the Ia-2β promoter rules out the possibility that any remaining miR-153 expression is generated from shared regulatory elements in the promoter region of its host gene, Ia-2β. We performed an in silico analysis of putative miR-153-2 TSSs in humans, using a miRStart database search . A putative miR153-2 TSS was identified at 38,909 bp from the start of the miR-153 precursor (ESM Fig. 2). Further experimental evidence, outside the scope of the current study, would be required to determine whether this is a conserved and tissue-specific miR-153 TSS.
Bioinformatics identification of potential miR-153 targets involved in neurotransmitter and insulin release
Gene–function relationships between miR-153 target genes
Relevant biological function
Fusion of synaptic vesicles
3.40 × 10–12
BSN, SNAP25, SYT1, VAMP2
Secretion of neurotransmitter
4.21 × 10–12
PTPRN2, SNAP25, SNCA, SYT1, SYT4, VAMP2
Transport of synaptic vesicles
5.63 × 10–12
PCLO, SNCA, SYT1, SYT4, VAMP2
2.48 × 10–11
PCLO, SNAP25, SNCA, SYT1, SYT4, VAMP2
Exocytosis by cells
2.43 × 10–10
PCLO, SNAP25, SNCA, SYT1, VAMP2
Release of neurotransmitter
5.64 × 10–10
PCLO, SNAP25, SNCA, SYT1, SYT4, VAMP2
Excitatory postsynaptic potential
4.20 × 10–8
SNAP25, SNCA, SYT1, VAMP2
Presence of fusion pores
1.13 × 10–7
Quantity of synaptic vesicles
1.82 × 10–7
PCLO, PTPRN2, SNCA
Miniature excitatory postsynaptic currents
3.00 × 10−7
SNAP25, SYT1, VAMP2
Synthesis of neurotransmitter
6.70 × 10−7
SNAP25, SNCA, SYT1
Exocytosis of vesicles
7.10 × 10−7
SNAP25, SYT1, VAMP2
Flux of noradrenaline
2.38 × 10−6
6.55 × 10−6
BSN, SNAP25, SNCA, SYT1
Exocytosis of synaptic vesicles
3.96 × 10−5
Secretion of dopamine
4.58 × 10−5
Secretion of l-glutamic acid
5.95 × 10−5
Exocytosis of granules
7.09 × 10−5
1.64 × 10−4
SNAP25, SNCA, SYT1
Fusion of cellular membrane
1.92 × 10−4
Analysis of regulation of predicted miR-153 target genes in vivo and in vitro
These data indicate that changes in miR-153 abundance in various settings can result in significant changes of predicted target gene expression.
This study describes the localisation of miR-153-1 and miR-153-2 in introns of the human IA-2 and IA-2β genomic loci, respectively. We show that only miR-153-2 is evolutionarily conserved and partly co-regulated with IA-2β expression, as loss of Ia-2β expression in BKO mice results in a strong reduction of miR-153 expression. We identified several potential miR-153 targets that correlate with IA-2β expression and function. We found significant changes in mRNA expression of several predicted target genes after glucose stimulation of MIN6B cells, as well as in isolated mouse pancreatic islets, where endogenous miR-153 levels are increased, and in BKO mouse brain and isolated islets, which have reduced miR-153 levels. Moreover, significant reductions of bassoon, SNAP25 and SNCA abundance in SH-SY5Y cells was found upon overproduction of miR-153 (Fig. 4f). It should be noted that in vivo miRNAs mainly function as rheostats or act via neutral repression, rather than being a binary off-switch to dampen protein output [15, 40].
The first point that arises from our study is that although in BKO mice mRNA expression of Ia-2β is completely lost , reduced levels of miR-153 can still be detected in BKO and DKO brain, whereas miR-153 expression in DKO islets is almost completely lost (Fig. 2b, c). Interestingly, the effect of loss of miR-153 on predicted target gene expression appears to be more pronounced in isolated mouse knockout islets than in mouse knockout brains (Fig. 4a,b), correlating with the stronger reduction of miR-153 levels in knockout islets than in knockout brain. Moreover, the targeted Ia-2β allele was generated by deleting its promoter region . Thus, loss of the Ia-2β promoter rules out the possibility that any remaining miR-153 expression that was detected in BKO and DKO mice was generated from shared regulatory elements in the promoter region of its host gene, Ia-2β, thus predicting the presence of an independent and tissue-specific miR-153 promoter region. Using miRStart software , we were able to detect putative transcription regulatory elements for miR-153-2 (ESM Fig. 2). Another study has also predicted a promoter region for miR-153-2 independently of the Ia-2β TSS . However, a recent study failed to find evidence that human miR-153-2 has a classical TSS, but instead contains a CpG island and transcription factor binding sites that could initiate transcription . Interestingly, demethylation of methylated cytosines in two identified miR-153-2 CpG islands increases miR-153 expression . Based on these studies and our findings, it is clear that miR-153-2 transcription unit can be regulated independently of Ia-2β expression in a tissue-specific manner, but more detailed studies will be required to elucidate the exact transcriptional regulation of miR-153-2 expression.
Based on our present findings, earlier studies [9, 10, 11, 12, 13] that described the use of material from BKO or DKO mice should be judged with some caution, as it is possible that (some of) the observed results could have been caused by loss of miR-153 expression and potential upregulated expression of their targets. This issue probably needs to be considered in general for other studies that have generated targeted deletion in genes that contain intragenic miRNAs.
The present study points to the existence of a potential miR-153–IA-2β–target gene pathway, which is involved in vesicle release in brain and pancreas. Indeed, it has already been shown that IA-2β localises to synaptic vesicles and that loss of IA-2β results in decreased dopamine release in PC12 cells [13, 43]. Furthermore, DKO mice display defects in neurotransmitter release (dopamine, noradrenaline and 5-hydroxytryptamine (5-HT)) . Various targets of miR-153 have the same localisation and function in the same pathways as IA-2β. For example, both SNCA and IA-2β localise to DCVs of beta cells, where loss of SNCA increases insulin release and loss of IA-2β decreases insulin release [9, 39]. In addition, SNCA is a presynaptic protein that regulates synaptic vesicle dynamics and trafficking, and physically interacts with at least three other predicted miR-153 target genes, i.e. Vamp2, Pclo and Snap25, to stimulate soluble n-ethyl maleimide sensitive factor (NSF) attachment protein receptor (SNARE) complex assembly . Interestingly, mild overabundance of SNCA in mice resulted in a marked reduction of neurotransmitter release , similar to the phenotype observed in BKO mice . Moreover, it has been shown that miR-153 expression is reduced in synaptosomes in comparison with the expression in total brain [32, 46, 47]. Finally, miR-153-1 has been associated with one of the type 1 diabetes mellitus loci (IDDM13), indicating that miR-153 may be a susceptibility candidate for human type 1 diabetes . Taken together, the presence of miR-153-2 in the Ia-2β gene locus could be part of a feedback loop including miR-153 targets, and possibly regulating release of neurotransmitters at synapses and insulin secretion by beta cells. This possibility underscores the importance of the IA-2β–miR-153–target gene pathway in brain and pancreas, and sets the stage for further investigations to obtain a greater mechanistic insight into this pathway.
We thank G. Carmona (National Institute of Dental and Craniofacial research/NIH, Bethesda, MD, USA) for technical help.
This work was supported, in part, by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD, USA, a Marie Curie Intra-European Fellowship (EIF proposal number 041333) awarded to W. Mandemakers, and a Methusalem grant (Flemish community and University of Leuven, Belgium) and European Research Commission (ERC) grant (EU Commission) to B. De Strooper.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript
WM, LA, HX, LAC, SSH, VAM, SM, TC, ALN and BDS conceived and designed the experiments. WM, LA, HX, LAC, SSH and AS performed the experiments. All authors analysed the data. WM, LA, ALN and BDS were responsible for drafting the paper. All authors revised and approved the final version of paper.
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