MicroRNA signature of the human developing pancreas

Background MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. Results The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase altgorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. Conclusions We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas.


Background
MicroRNAs are small non-coding RNAs [1][2][3] that act mostly as translational repressors through binding to partially complementary sequences of target messenger RNAs. Recent studies indicate that in some cases micro-RNAs might act also as positive regulators of translation and transcription [4,5]. To date, more than 700 human microRNAs are annotated in the Wellcome Trust Sanger Institute microRNA database [6]. MicroRNAs play a fundamental role in regulation of gene expression, consequently affecting key biological events such as embryogenesis, stem cell proliferation/differentiation and organ development [7]. The method of conditional knockout of the microRNA-processing enzyme Dicer has been used to study the action of microRNAs in specific tissues and organs. This approach has confirmed that the expression of microRNAs is essential for morphogenesis of several systems, including the lungs, limbs, muscle, skin and neuronal development and differentiation [8][9][10][11][12] including the genesis of pancreatic islet cells [13]. Dicer-null animals displayed gross defects in all pancreatic lineages; the most dramatic reduction was seen in the endocrine cells, especially the insulinproducing beta cells. Another approach utilized to assess the role of microRNAs in organs/tissues is the loss of function, i.e., silencing or knock out of a single or cluster microRNAs. For instance, genetic deletion of the miR-1/miR-133 cluster, showed its crucial role in cardiac skeletal muscle development [14]. Several micro-RNAs have been described in the context of pancreatic development, pancreatic exocrine/endocrine microRNA expression or islet biology/physiology. For example, knockdown of miR-375, an islet microRNA that negatively controls insulin secretion [15], has a deleterious effect on the developing pancreas of zebra fish [16]. Mice lacking miR-375 (375KO) are hyperglycemic and exhibit an increase in total pancreatic alpha-cell numbers, whereas the pancreatic beta-cell mass is decreased [17]. Recent studies indicate that during human pancreatic development miR-7, miR-9, miR-375 and miR-376 are specific islet microRNAs expressed at high levels [18,19]. MiR-124a is known to target FOXA2 the transcription factor crucial for early pancreatic formation [20] by affecting genes such as PDX1, KCNJ11 (Kir6.2) and SUR1, essential for the normal development of the pancreas, glucose metabolism and insulin secretion [21], while miR-15a, -15b, -16 and -195 have important roles in regulating translation of NGN3, a transcription factor affecting the adoption of an endocrine fate during embryogenesis [22].
Research conducted over the last decade has outlined rather comprehensive "roadmap" of the major molecular events that shape mouse islet development [23,24]. Development of the human endocrine pancreas has not been studied as well as that of the mouse. The previous work has described expression of endocrine hormones as well as transcription factors, and presented morphological examination of islet formation during human fetal pancreatic development [25][26][27][28][29].
In this study we investigated the global expression profile of microRNAs in the human fetal pancreas from age 10 to 22 weeks of gestational age (wga) and found 212 microRNAs expressed throughout this entire period. Understanding the complex processes behind pancreatic development will require the identification of RNA targets that are controlled by the extensive microRNA network expressed throughout the process. To identify potential microRNA gene targets, we calculated the correlations between the expression profile of these 212 microRNAs and their corresponding mRNA targets, predicted by bioinformatic tools, that were found in the genome-wide RNA microarray study by Sarkar et al. [28]. These studies will advance our understanding of pancreatic development, potentially unveiling prospective therapeutic targets to treat diabetes.

Results and discussion
MicroRNA Expression profile during pancreatic development Using TaqMan Low Density Arrays (TLDA) from Applied Biosystems we have performed microRNA arrays with 10 samples acquired at different stages of human fetal pancreatic development. Specifically; 10 wga (two samples), 11 wga (one sample), 13 wga(one sample), 14 wga (three samples), 15 wga (one sample), 21 wga (one sample), 22 wga (one sample). The TLDA platform has been selected because it requires smaller amounts of RNA~100 ng per experiment, which is about~20 times less than what is required for other microarray hybridization platforms, furthermore it delivers quantitative output. We identified 212 microRNAs expressed throughout all studied gestational ages. Interestingly several microRNAs reported previously in studies of mouse pancreatic development/regeneration and islet function were also found expressed during human pancreatic development (Table 1).
For the correlation analysis we arranged the samples in 3 gestational periods: 10-11wga (three samples); 13-15 wga (five samples) and 21-22 wga (two samples) and classified the microRNAs in three groups according to their expression profile (Material and Methods). Groups I and II contain 4 and 35 microRNAs respectively. Their statistically significant (p < 0.05) expression change was either up-regulated (group I) or down-regulated (group II) at least from one gestational stage to the other. Group III comprises of 173 microRNAs with expression relatively unchanged (p > 0.05) ( Table 2 & Additional file 1, Table  S1). Table 2 was prepared using relative quantification (RQ) values normalized by nucleolar RNA (RNU48) as endogenous control (Additional file 1, Table S1). RQ is a measure of the abundance of microRNA transcripts at each developmental stage. The microRNAs in Table 2 are arranged according to their descending RQ starting at the stage 10 wga. MiR-7, miR-124, miR-9 and miR-375, are microRNAs previously reported as related to pancreatic islets [18][19][20]. We found that only statistically significant increase of expression was for miR-7. miR-375 showed an increasing trend, however slightly below the threshold value (p = 0.08), which might be the result of a low sample number. Additional file 2, Table S2 shows the average RQs for microRNAs in group I and II in each gestational period.
A hierarchical cluster representation of microRNAs clearly shows the pattern of expression of these three groups ( Figure. 1).

Inverse correlation between microRNA and mRNA during human pancreatic development
Although mechanistically, microRNAs inhibit mRNA translation, the expression of a given microRNA has also been associated with the downregulation of its target mRNA levels [30,31]. Therefore, to identify potential microRNA targets, we performed a statistical analysis looking for inverse correlations between the microRNAs in groups I and II (a total of 39 microRNAs) and the algorithm-predicted target genes that were found in a human pancreatic development genome-wide mRNA microarray gene expression study [28]. Predictive algorithms are a powerful tool to identify potential RNA targets [32]. The target RNAs were selected by two of the most commonly used computational microRNA predictive target programs, miRBase [33] and TargetScan [30,34]. There is a limitation to this kind of analysis because it utilizes only the sequence correlation of 3'UTR mRNA domain with microRNA seed sequences. This excludes naturally occurring microRNA targets with recognition motifs within the gene coding sequence. This kind of microRNA/mRNA interaction has been recently reported as well [35]. Computational predictions often generate long lists of target genes and these are not always concordant [32]. Therefore, the correlations were calculated with target genes predicted by each program individually, and also by the intersection of both algorithms, as this might increase the odds of identifying the bona fide RNA targets [36].
High confidence correlation (R 2 ≥0.8) analysis between microRNAs and gene targets identified 2832 and 9448 correlations using miRBase and TargetScan respectively (Additional file 3, Table S3 and Additional file 4, Table  S4). A total of 1018 genes were identified as potential targets by both predictive algorithms (Additional file 5, Table S5); microRNAs from group I correlated with decreased expression of 142 potential targets while micoRNAs from group II, correlated with the increasing expression of 876 potential targets (Table 3). For all microRNA groups the number of targets predicted by TargetScan was higher than by miRBase, which is in agreement with previous observations [37,38].

Genes involved in pancreatic development correlated with microRNAs
We searched for microRNAs that negatively correlated with genes from signaling pathways and transcription factors that are known to be involved in murine pancreatic development and/or regulate beta-cell development [39] (Table 4). Out of the 28 candidate genes, 11 were identified as potential targets for microRNAs. Most of the correlations corresponded to microRNAs from group II. Ten genes up-regulated throughout the development correlated with 19 decreasing microRNAs from group II. Decreasing gene SOX4, was identified as a potential target for miR-489 from group I. The identification of 19 microRNAs with decreasing expression along the pancreatic development, thus contributing to the upregulation of their potential targets, suggests a collaborative network of microRNAs and mRNAs during this period ( Figure. 2). Most microRNAs target more than one gene and conversely a given mRNA could be the target of several microRNAs. For example, NEU-ROD1, a key transcription factor involved in the Expressed in pancreatic adult and fetal endocrine cells [18,19,30].

miR-218
Expressed in mouse early fetal pancreas, controls the liver and pancreatic development regulator Onecut-2 in liver embryonic cells [34].

miR-146a
Increased expression in islets from db/db obese mice, contributes to fatty acids-induced beta-cell dysfunction [36]. Proinflammatory cytokines induce its expression in human islet and MIN6 cells [37].

miR-96
Increases mRNA and protein levels of granulophilin, a negative regulator of insulin exocytosis [33].
These microRNAs have been previously described as expressed in adult or developing pancreatic tissue and/or having a functional role in islets/insulinoma cells.
conversion of pancreatic progenitor cells into endocrine cells [40] was identified as a potential target of five microRNAs: miR-17-5p, miR-18a, miR-92, miR-103 and miR-494. The decrease in the expression of these five microRNAs would lead to the up-regulation of NEU-ROD1. It is apparent that microRNAs can target genes/ transcription factors that have a role at different stages of embryogenesis, for example miR-342 targets genes such as GATA-4, involved in the formation of definitive endoderm [41] and transcription factors FOXA2 and MAFB both involved in beta-cell differentiation and maturation [39,42].

Conclusions
In this study we determined the microRNA expression signature during human pancreatic development, identifying a total of 212 microRNAs expressed at least once in one of the four stages evaluated (10 to 22 wga). To further our understanding of gene expression regulation in the human developing pancreas, we compared this microRNA signature with the global expression of genes during the similar period of the developing pancreas. There are potential limitations to this kind of study that should be considered: 1) the study was designed under the assumption that microRNAs are associated with MicroRNAs from group I and II increase and decrease their expressions respectively throughout the entire studied period. Group III includes microRNAs with relatively constant expression throughout the period studied.
downregulation of their targets. However, some micro-RNAs were reported as positive regulators of transcription or translation [4,5], 2) the correlation between microRNAs and gene expression was performed with mRNAs only, but there are some microRNAs that regulate only protein translation; 3) all potential targets were identified by algorithms analyzing the 3'UTR of the mRNA, while some microRNAs can also interact with targets containing the recognition sequence in the coding region; 4) the expression of microRNAs and mRNAs was evaluated on different set of sample tissues, using different platforms by different laboratories; 5) in this study we have analyzed the pancreas as a whole, instead of looking at the cellular subsets. Therefore, it is not possible to determine if the observed changes corresponded to a specific cell population (e.g endocrine progenitors, mesenchymal tissue etc). Despite of these caveats, the correlation between the expression of hundreds of microRNAs with the analysis of thousands of genes expressed during human pancreatic development suggests the intertwined collaborative network of microRNAs and mRNAs. It also provides broad and useful information to explore potential two-way level of gene expression control regulation in which microRNAs target multiple mRNAs, and, target mRNAs are regulated in parallel by multiple microRNAs. Ultimately, the speculation regarding the roles of all these presumptive targets will be justified only if they are experimentally validated. Figure 1 Expression of microRNAs during human pancreatic development. The Heat-map figures were obtained using Cluster and Treeview software [44] with the data presented in Additional file 1, Table S1. This table shows correlation among expression profiles of microRNAs comprised into three groups from all studied fetal pancreatic samples; Group I, Group II and Group III. The color of each cell represents the expression of microRNA normalized to small nucleolar RNU48 RNA. Colorgram depicts intensity of expression from high (bright red) to low (black). Please note that even though in some cases the group III colorgram suggests a change of expression throughout the gestational stages (e.g miR-376, miR-378, etc), these were found to be not statistically significant.

Human fetal pancreas procurement
Human fetal pancreases from 10 to 22 weeks of gestational age (wga) were collected from fetal tissue immediately after elective termination of pregnancy. The healthy women admitted to local clinics were properly informed and gave their consent to use fetal tissues for research studies. The study is in compliance with US legislation and the guidelines of the University of Miami. Gestational age was determined on the basis of the last menstrual period, with ultrasonographic measurements of the Crown-Rump Length, and the biparietal diameter.

MicroRNA isolation
MicroRNA isolation was performed using mirVana microRNA Isolation kit from Ambion/Applied Biosystems (Foster City CA), following protocol for the total RNA isolation procedure provided by the manufacturer.

Quantitative microRNA profiling: criteria for inclusion of microRNAs in this study
We performed this study with 10 human fetal pancreases of following gestational stages: two samples (10 wga); one sample (11 wga); one sample (13 wga); three samples (14 wga); one sample (15 wga); one sample (21 wga) and one For the correlation analysis we arranged the samples in 3 gestational periods: 10-11 wga; 13-15 wga and 21-22 wga. Only microRNAs that amplified in 2 out 3 samples for 10-11 wga period, 4 out of 5 samples for 13-15 wga and 2 out of 2 samples in 21-22 wga were considered as positively expressed. Statistical analysis between the groups: 10-11 wga vs 13-15 wga; 10-11 wga vs 21-22 wga and 13-15 wga vs 21-22 wga was performed with two-tailed Student's t test (Additional file 1, Table S1). Values were considered significant when p < 0.05. The microRNAs were classified according to their expression profile into three groups. Group I contained microRNAs with the expression increased at least from one gestational stage to the other (p < 0.05). Group II contained microRNAs with the expression decreased at least from one gestational stage to the other (p < 0.05). In group III we included the microRNAs with the expression unchanged throughout the studied gestational stages (p > 0.05). Global gene expression profiling and histochemical analysis of the developing human fetal pancreas [28]. 22,000 genes were measured by 54,675 probe-sets on Human Genome HG U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA, USA). The mRNA hybridization data from genes with multiple probes were averaged prior to the correlation analysis. For the correlation analysis the Sarkar et al gene expression data were grouped in three periods: 9, 10 and 11 wga (9-11); 15 wga; and 20, 23 wga. The gene expression of these three periods was compared to the microRNA expression corresponding to the three following periods: 10-11 wga; 13-15 wga and 21-22 wga. The Pearson correlation of the expression profile between microRNA and a target gene was calculated in the R package. The correlation coefficient (r) was calculated to determine the regulatory role of the micro-RNA on its target genes. The correlation with r close to -1 depicts a strong negative correlation between micro-RNA and its target gene, indicating the suppression function of microRNA on its target gene. P-value is used to test H 0 : r = 0. Benjamini-Hochberg FDR was used to correct the original p-value for multiple test correction. The correlations with adjusted p-value < 0.05 are significant correlations, and among these correlations the ones with (R 2 ≥0.8) are significantly negative correlations where microRNAs suppress the expression of target genes in terms of expression in the three gestational ages.