MicroRNA regulation as one type of the epigenetic modifications.

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MicroRNA regulation as one type of the epigenetic modifications.

MicroRNAs’ role as new regulators of gene expression was not identified until a decade ago. These small 19 to 22 nts RNA molecules took their place in the complex cell biology as a post-transcriptional regulator.

MicroRNA genes account for approximately 1% of the genome in different species, and each of them has many different conserved or non-conserved mRNA targets. MicroRNAs are transcribed by RNA polymerase II as primary transcripts featured by hairpin structures (pri-microRNAs) and processed by RNAse III Drosha into 70 to 100 nts long pre-microRNAs. These precursors are exported by Exportin 5 to the cytoplasm, where an following step handled by the RNAse III Dicer forms a dsRNA of approximately 22 nts, called miRNA/miRNA*. The mature single stranded microRNA is then incorporated in the microRNA-containing RNA-induced silencing complex (miRISC), while the other strand is likely to be degraded.

So what is the mechanism of microRNA derived the post-transcriptional regulation.

First, miRNAs can direct endonucleolytic cleavage of mRNAs. Endonucleolytic cleavage of mRNA is generally favored by fully complementary base-pairing between the miRNAs and the target mRNA (Mallory et al. 2004;Yekta et al. 2004;Guo et al. 2005). However perfect base pairing between the miRNAs and the mRNA is not always enough to induce cleavage, indicating that there can be extra requirements for a RISC complex to catalyze cleavage (Chen 2004). One additional requirement for cleavage activity is that a specific Argonaute protein be contained within RISC. For example, in mammalian cells, Ago2 was identified as the only one of the four mammalian Ago proteins (Ago1, Ago2, Ago3 and Ago4) that is able to direct cleavage (Liu et al. 2004;Meister et al. 2004). Ago2 contains an RNaseH-like domain and has all of the crucial active residues to carry out slicer activity. Moreover, it is confirmed that mutations in the RNaseH domain of Ago2 disrupt small RNA-mediated cleavage (Liu et al. 2004;Song et al. 2004). These results define the minimal RISC composition needed for miRNA-directed cleavage in mammals.

Moreover, the miRNs are able to induce translational repression, on which there are multiple potential mechanisms to explain.

Firstly, miRNA required mRNA targeting is based on the number and type of base pair matches in the 3´ UTR and binding site (Bagga et al., 2005; Aleman et al., 2007). Once the miRNA matches with its targets, inhibition of translation via miRNA is a result from deadenylation of the poly-A tail and then decapping of the mRNA sequence (Giraldez et al., 2006; Wu et al., 2006). As the consequence, the mRNA sequence becomes unstable and easily responsive to degradation, resulting in downregulation of mRNA abundance and subsequently decrease in translation.

Another mechanism of posttranscriptional regulation by miRNA began to come up since research revealed that mRNA’s level did not always decrease with gene translation. (Wang et al., 2006; Thermann and Hentze, 2007). Similar start with the miRNA binding to the RISC and targeting the 3´ UTR of the mRNA, however, instead of subsequent inducing the degradation of mRNA , activation the blocking of initiation proteins from binding to the 5´ cap of the mRNA is the next step (Chendrimada et al., 2007; Kiriakidou et al., 2007). AGO2 contains a similar binding region to mRNA as the eukaryotic translation initiation factor 4E (eIF4E) (Kiriakidou et al., 2007). It is possible that AGO2 competes with eIF4E for binding to the 5´ cap of the mRNA sequence, the existence of this binding loop subsequently blocks translation initiation. It has also been reported that argonaute protein binds to the proteins of the 60S ribosomal subunit including eIF6 (Chendrimada et al., 2007). As a result, the mRNA sequence cannot be translated while the mRNA is intact and has no change in abundance.

A third proposed mechanism of miRNA’s regulation is the miRNA:mRNA complex will be translocated to cytoplasmic foci in the cell, referred as processing bodies (P-bodies), after the miRISC complex binds the mRNA target (Chan and Slack, 2006). This is supported by finding that P-bodies is consist of components including argonaute protein, miRNA, and target mRNA (Liu et al., 2005). P-bodies contain no ribosomal proteins for translation while manage many enzymes and factors for mRNA turnover and translation repression(Liu et al., 2005). For example, trinucleotide repeat containing 6A (GW182) which is an RNA-binding protein and a dipeptidyl carboxypeptidase (Dcp1/Dcp2) decapping complex which is able to remove of the 5´ cap and degradation of the mRNA sequence, are co-localized in P-bodies and bind to argonaute proteins (Rehwinkel et al., 2005; Behm-Ansmant et al., 2006; Ikeda et al., 2006). However, it is reported that P-bodies may just act as a storage unit for mRNA, because interference of the P-bodies does not disrupt the miRNA induced RNAi pathway or the degree of translational repression (Chu and Rana, 2006; Eulalio et al., 2007). Thus, P-bodies might have an role in miRNA regulation of translation and serve as a unit of temporary storage before mRNA degradation (Eulalio et al., 2007).

What epigenetics has been found associated with diabetes.

2 Type 1 diabetes (T1D) is an autoimmune disease causing by T cell mediated destructive effect on insulin-secreting beta cells [69]. TID concordance rate in monozygotic twins varies from 13–67.7% [7]. One possible reason may be the variation in risk of human leukocyte antigen (HLA) genotypes associated with T1D. HLA is a risk gene for T1D, and the heterozygous DR3/DR4 genotype contributes to the highest risk of T1D susceptibility, but the major of individuals carrying the DR3/DR4 genotype do not develop diabetes [70]. This spectrum of genetic risk for T1D leaves room for epigenetics, which regulate the gene expression without changing the gene. There are three major mechanism of epigenetic regulation on T1D.

DNA methylation in T1D.

Rakyan and his group [49] performed a comparative study on T1D-discordant monozygotic twin pairs, learning the difference of epigenome-wide association in CD14+ monocytes. They identified 132 different CpG sites that were significantly linked with diabetic condition. Some of the genes were discovered hypomethylated or hypermethylated, such as GAD2 and HLA-DQB1 which are identified to be associated with risk of T1DM. Moreover, T1D-associated methylation in variable positions emerges in the islet autoantibody positive individuals years before clinical confirmed diagnosis, indicating that the methylation arises early in the progression of the T1D. In addition, a 3-CpG-hypomethylation condition that seemed to be exist only in T1D patients was identified [50]. These three CpG pattern are adjacent to the transcription initiation site in the insulin promoter gene, that they could be a predictive biomarker for T1DM. Furthermore, 19 perspective CpG sites were associated with the onset time of nephropathy, the major complication of T1D. Another hypermethylated CpG site was found in the UNC13B gene, which is known to be linked to the risk of diabetic nephropathy [51]. Another research team demonstrated that there is detectable hypomethylated insulin DNA in the blood of newly-diagnosed T1D patients and this finding might provide a new means for detection the beta-cell death [52].

Histone modification in T1D.

Researchers used ChIP-chip to compare genome-wide histone H3K9me2 patterns in peripheral lymphocytes and monocytes between non-diabetes controls and T1D patients[54]. In lymphocytes, they found a significant increase of H3K9me2 in some T1D high risk genes, e.g. CTLA4 gene. This is supported by the mechanism of curcumin treatment, which is used to provide protection against T1D nephropathy, is to increase acetylation of histone H3 [55]. Moreover, HDAC expression was repressed in blood mononuclear cells in T1D patients [2-85]. And it is reported the upregulation of the NF-κB-p65 gene due to the histone methylation in the NF-κB-p65 gene promoter region can be caused by hyperglycemia, which is the major syndrome of T1D.

MicroRNA regulation in T1DM.

Numberous microRNAs are involved in the pathogenesis of T1D. miR-326 are reported to be associated with β-cell death [57]. MiR-326 expression levels in peripheral lymphocytes was analyzed from T1D patients who possessed autoantibodies to glutamic acid decarboxylase and insulinoma antigen-2. miR-326 was observed that have a high level expression among T1D patients with autoantibodies, compared to T1D patients with no autoantibodies. Upregulation of miR-21 was found to decrease the levels of PDCD4 in beta cells, which is a tumor suppressor that induced the cell death via activation of the pathway contain BAX [2-89]. That means miR-21 might make the beta cell more resistant to cell death. miR-342 and miR-191 were repressed in regulatory T cells from T1D patients, while miR-510 was upregulated [2-90].

DNA methylation in T2D.

Recent gene candidate studies identified several epigenetic regulations in pancreatic islets of T2D patients. The expression of the PPARGC1A gene, a transcriptional co-activator that modulates genes which involved in energy metabolism, is downregulated in pancreatic islets of patients with T2D and this altered expression is related to the increased levels of DNA methylation in the promoter region [125]. DNA methylation is also enhanced at the INS promoter and at the distal PDX1 promoter and enhancer in islets of T2D patients compared with non-diabetic controls, and the mRNA levels is inversely correlated [126, 127]. Interestingly, DNA methylation at the INS promoter and distal PDX1 promoter changes accordingly with levels of glycosylated hemoglobin (HbA1c—a marker of poor glycemic control) in patients with T2D, meanwhile the methylation at the INS1 and PDX1 promoters also increases in rat INS1 cells in vitro exposure to high glucose condition for 72 h [126, 127].

A comprehensive study that addresses the role of epigenetic alterations in the pathogenesis of T2D in humans has recently uncovered 254 genes with different DNA methylation condition compared to controls [59]. Strikingly, the vast majority of these genes show decreased DNA methylation in T2D islets, have low or intermediary CpG density in promoters, and some of them have significant but also opposite different mRNA expression compared with control islets. What’s more significant, none of these DNA methylation alterations identified in islets were discovered in blood cells of diabetic patients or in control islets with exposure to high glucose medium in vitro, suggesting that they may play a role in the pathogenesis of the disease via just acting specifically in islets.

Recent some research results suggest that the occurrence of epigenetic alterations in the pancreas might be partly result from underlying DNA sequence variants. A genome-wide DNA methylation survey identified several other differentially methylated sites in the vicinity of SNPs associated with the disease in previous GWAS studies, such as THADA, JAZF1, SLC30A8, TCF7L2, KCNQ1, and FTO [128]. The top-ranking association was a CpG site located in the first intron of the FTO gene that showed a small (3.35 %) but significant hypomethylation in cases relative to controls [129]. Importantly, although the T2D-associated SNP in the FTO region influences the levels of DNA methylation, the hypomethylation in the T2D group is independent of the sequence polymorphism and persists in individuals that carry the risk alleles. The identification of diabetes-associated DNA methylation alterations in a tissue (e.g., blood) that is not directly involved in insulin secretion or action further suggests their occurrence in early stages of development. In summary, together these studies provide evidence for both sequence-influenced and sequence-independent DNA methylation variations at loci that predispose to T2D.

miRNA regulation IN T2D

miRNAs are essential for normal pancreas development and are implicated in diabetes.47 There being deleterious effects of knocking down miRNAs on β-cell function, hyperglycemia and lipotoxicity have profound effects on miRNA expression, demonstrating that miRNAs are dynamically regulated in response to environmental conditions in the β-cells.

The miR375 is enriched in pancreatic islets, not detected in other tissue types,47 and its deletion in mice leads to hyperglycemia and a reduction in the number of β-cells,51 and impaired islet development in zebra fish,52 suggesting that its presence is critical for normal programming of β-cells and maintenance of glucose homeostasis. Knockdown of miR375 in MIN6 cells led to enhanced glucose-stimulated insulin secretion (GSIS), while overexpression led to a 40% reduction in insulin secretion.47,53 The mechanism of how it reduces insulin secretion is partly understood, as it was shown to target the myotrophin (Mtpn) mRNA, which encodes a protein that is involved in actin depolymerization and vesicle fusion and plays a role in the late stage of insulin granule exocytosis.53 Li et al. The miR124a is co-expressed with miR375, and also targets myotropin56 suggestive of coordinate regulation. The miR124a also targets the transcription factor FOXA2,57,58 which plays a role in β-cell differentiation.

In Goto-Kakizaki rats, a spontaneous model of T2D, 30 miRNAs were identified as differentially expressed in islets as compared with control Wistar rats, 24 of which were upregulated in the diabetic islets.59 Lovis et al.16 performed miRNA profiling on MIN6B1 β-cells grown in the presence or absence of excess palmitate for 3 d and observed that out of 132 miRNAs that were expressed in these cells, several were upregulated in response to palmitate treatment, including miR34a and miR146, both in MIN6B1 cells and freshly isolated rat islets, as well as nontreated diabetic (db/db) mouse islets.16 Overexpression of miR34a and miR146 amplified palmitate-induced apoptosis, while knockdown blunted the effects of lipotoxicity.16 miR146a expression is regulated by NF-κB, a transcription factor that plays a role in cytokine-mediated signaling.60 Effects of inflammation-induced β-cell dysfunction on miRNA expression were confirmed with a T1D mouse model, whereby miRNA profiling in 4- and 8-week-old (spontaneously diabetic at this age) NOD mice islets revealed an upregulation in miR21, miR34a, miR146a/b, and miR29a/b/c in β-cells from islets of 8-weekold mice.61 Overexpression of miR29a/b/c in MIN6 cells was associated with decreased pro-insulin expression and GSIS and increased apoptosis.61 miR29a and miR29b target monocarboxylate transporter 1, which leads to reduced insulin release.62 Onecut2, a target of miR29, was decreased by miR29 overexpression, leading to increased expression of granuphilin, a negative regulator of exocytosis that interacts with proteins involved in vesicular transport,63 providing another link between miR29 and the pathway of insulin granule exocytosis. The miR9 also targets Onecut-2 and affects insulin secretion through a similar pathway.64 Recently, Ramachandran et al., showed that miR9 also targets the 3'UTR of Sirtuin 1, a NAD-dependent protein acetylase.65

Thus, groups of miRNAs have been identified that play a role in pancreas development (cell lineage specification and differentiation) as well as mature β-cell function (insulin expression, secretion and signaling).66 The studies described herein are relevant to the human condition; miR9, miR29a, miR30d, miR34a, miR124a, miR146a and miR375 were elevated in the serum of T2D patients,67 and as discussed above, appear to be involved in β-cell function and the pathogenesis of T2D. With inflammation, hyperglycemia and lipotoxicity as key factors in the pathogenesis of T2D and predisposing factors to β-cell dysfunction, miRNAs may provide a link between altered fat metabolism (e.g., increased ceramide signaling), chronic hyperglycemia, inflammation (increased pro-inflammatory cytokine-induced signaling) and β-cell apoptosis.54