Leptin Modulates the Expression of miRNAs-Targeting POMC mRNA by the JAK2-STAT3 and PI3K-Akt Pathways

The central control of energy balance involves a strongly regulated neuronal network within the hypothalamus and the brainstem. In these structures, pro-opiomelanocortin (POMC) neurons are known to decrease food intake and to increase energy expenditure. Thus, leptin, a peripheral signal that relays information regarding body fat content, modulates the activity of POMC neurons. MicroRNAs (miRNAs) are short non-coding RNAs of 22–26 nucleotides that post-transcriptionally interfere with target gene expression by binding to their mRNAs. It has been demonstrated that leptin is able to modulate the expression of miRNAs (miR-383, miR-384-3p, and miR-488) that potentially target POMC mRNA. However, no study has identified the transduction pathways involved in this effect of leptin on miRNA expression. In addition, miRNAs targeting POMC mRNAs are not clearly identified. In this work, using in vitro models, we have identified and confirmed that miR-383, miR-384-3p, and miR-488 physically binds to the 3′ untranslated (3′UTR) regions of POMC mRNA. Importantly, we show that leptin inhibits these miRNAs expression by different transduction pathways. Taken together, these results allowed us to highlight the miRNA involvement in the regulation of POMC expression downstream of the leptin signaling and satiety signal integration.


Introduction
The regulation of energy expenditure involved hormonal (leptin and insulin), metabolic (nutrients), and nervous signals [1,2]. The various signals mentioned before are integrated by the hypothalamus and the brainstem [1,2]. In this process, leptin has been shown to exert its satietogenic effect by stimulating the pro-opiomelanocortin (POMC) neurons located in the hypothalamic arcuate nucleus and nucleus of solitary tract in the brainstem [1][2][3][4]. Leptin is a polypeptide hormone (16 KDa) encoded by the Ob gene [5]. Leptin bind the long isoform of its receptor (LepRb) located on the membrane of POMC neurons [6,7]. After binding of leptin, three distinct cellular transduction pathways are stimulated by the Janus kinase (JAK2) protein [8,9]. Initially, JAK2 phosphorylates intracellular chain of the receptor on threonine amino-acid that are binding site for cytoplasmic proteins such as signal transducer and activators of transcription 3 (STAT3) [8,9]. STAT3 proteins form homodimers that are transported into the nucleus where they bind to specific DNA sequences, and activate the transcription of target genes such as that of POMC [8]. A large number of studies demonstrated that the JAK2 / STAT3 pathway is essential for the anti-obesity effects of leptin [1,8,9]. It has been also established that

Gene Ontology and KEGG Pathways Analysis
The gene ontology biological process (GO) terms and The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms enriched in predicted target genes were determined using mirPath v.3 from DIANA TOOLS bioinformatics resources [30].

Statistical Analysis
All data are expressed as mean ± SEM. Statistical analysis was performed by using GraphPad 6.0 software (San Diego, CA, USA). Significant differences between the control and treated groups were determined using ANOVA followed by a PLSD Fischer post-hoc test; p < 0.05 was considered statistically significant.

Effect of Leptin on POMC Expression in mHypoA-POMC/GFP Cell Line
qRT-PCR was performed in order to determine the effect of leptin on POMC mRNA levels in the mHypoA-POMC/GFP cell line. Treatment with graded concentrations of leptin induced a dose-dependent increase in POMC mRNA levels with a maximum effect observed at 1 µM leptin (+113%; p < 0.01) (Figure 1). Time-course experiments revealed that the effect of leptin on POMC mRNA levels was significant only for 3 h of treatment ( Figure 1).
qRT-PCR was performed in order to determine the effect of leptin on POMC mRNA levels in the mHypoA-POMC/GFP cell line. Treatment with graded concentrations of leptin induced a dosedependent increase in POMC mRNA levels with a maximum effect observed at 1 μM leptin (+113%; p < 0.01) ( Figure 1). Time-course experiments revealed that the effect of leptin on POMC mRNA levels was significant only for 3 h of treatment ( Figure 1).

miR-383, miR-384-3p, and miR-488 Target POMC 3′UTR
We have previously identified through in silico approach three miRNAs that could target the POMC and whose expression is under the control of leptin [13]. To confirm the effect of miR-383, miR-384-3p, and miR-488 on POMC, we transfected POMC 3′UTR with miR mimics. miR-488 was used as a positive control for the experiment, given that it has already been reported to target POMC [31,32]. The ratio of Renilla to firefly luciferase activity was significantly decreased for miR-383, miR-384-3p, and miR-488 (−47%, −31%, and −18%, respectively) ( Figure 2), validating our experimental conditions, and strongly suggesting that POMC may be considered as a target of miR-383, miR-384-3p, and miR-488.

miR-383, miR-384-3p, and miR-488 Target POMC 3 UTR
We have previously identified through in silico approach three miRNAs that could target the POMC and whose expression is under the control of leptin [13]. To confirm the effect of miR-383, miR-384-3p, and miR-488 on POMC, we transfected POMC 3 UTR with miR mimics. miR-488 was used as a positive control for the experiment, given that it has already been reported to target POMC [31,32]. The ratio of Renilla to firefly luciferase activity was significantly decreased for miR-383, miR-384-3p, and miR-488 (−47%, −31%, and −18%, respectively) ( Figure 2), validating our experimental conditions, and strongly suggesting that POMC may be considered as a target of miR-383, miR-384-3p, and miR-488.

Regulation of the Expression of the miRNAs of Interest by Leptin via the STAT3, AKT, and ERK Pathways
In order to determine if leptin regulates miRNAs expression in this model, mHypoA-POMC/GFP were treated with 5 µM leptin. This concentration was determined to be sufficient for the regulation of Pomc mRNA expression ( Figure 1). Leptin treatment in mHypoA-POMC/GFP culture for 2 h significantly decreased miR-383, miR-384-3p, and miR488 levels ( Figure 3A-C). Leptin is known to act primarily through the JAK2-STAT3, PI3K-Akt, and MEK 1/2/ERK pathways within POMC neurons [10]. Then to clarify the pathways involved in the regulation of miRNAs expression by leptin, cryptotanshinone (1 µM), MK 2206 (100 nM), and PD 98059 (10 µM) were used as specific blocker of JAK2-STAT3, PI3K-Akt, and MEK 1/2/ERK pathways, respectively. These concentrations were determined to be sufficient in regulating miRNAs expression without inducing significant cellular death. In the mHypoA-POMC/GFP cell line, pre-treatment with MK 2206 (100 nM) blocked the decrease of miR-383 in response to leptin ( Figure 3A). The inhibitory effect of leptin on the expression of miR-383 was not blocked either by cryptotanshinone (1 µM) or PD 98059 (10 µM) ( Figure 3A). For miR-384-3p and miR-488, inhibition of JAK2-STAT3 pathway by pre-treatment with cryptotanshinone (1 µM) for 24 h attenuated the decrease of miRNAs expression in response to leptin ( Figure 3B,C). Pre-treatment with MK 2206 (100 nM) or PD 98059 (10 µM) has no effect on the inhibitory action of leptin on the expression of miR-384-3p and miR-488 ( Figure 3B,C). Cryptotanshinone (1 µM), MK 2206 (100 nM), and PD 98059 (10 µM) had no effect alone on the expression of the miRNAs of interest ( Figure 3A-C). These results suggest that leptin may influence miR-383, miR-384-3p, and miR-488 expression though different mechanism of action.  was used as a positive control for experiments. Renilla activity was under the control of POMC 3′UTR, and firefly activity was used for normalization. The data are expressed as the mean ± SEM of at least three independent experiments. The values are presented as means ± SEM. *** p < 0.001.

Regulation of the Expression of the miRNAs of Interest by Leptin via the STAT3, AKT, and ERK Pathways
In order to determine if leptin regulates miRNAs expression in this model, mHypoA-POMC/GFP were treated with 5 μM leptin. This concentration was determined to be sufficient for the regulation of Pomc mRNA expression (Figure 1). Leptin treatment in mHypoA-POMC/GFP culture for 2 h significantly decreased miR-383, miR-384-3p, and miR488 levels ( Figure 3A-C). Leptin is known to act primarily through the JAK2-STAT3, PI3K-Akt, and MEK 1/2/ERK pathways within POMC neurons [10]. Then to clarify the pathways involved in the regulation of miRNAs expression by leptin, cryptotanshinone (1 μM), MK 2206 (100 nM), and PD 98059 (10 μM) were used as specific blocker of JAK2-STAT3, PI3K-Akt, and MEK 1/2/ERK pathways, respectively. These concentrations were determined to be sufficient in regulating miRNAs expression without inducing significant cellular death (data not shown). In the mHypoA-POMC/GFP cell line, pre-treatment with MK 2206 (100 nM) blocked the decrease of miR-383 in response to leptin ( Figure 3A). The inhibitory effect of leptin on the expression of miR-383 was not blocked either by cryptotanshinone (1 μM) or PD 98059 (10 μM) ( Figure 3A). For miR-384-3p and miR-488, inhibition of JAK2-STAT3 pathway by pretreatment with cryptotanshinone (1 μM) for 24 h attenuated the decrease of miRNAs expression in response to leptin ( Figure 3B,C). Pre-treatment with MK 2206 (100 nM) or PD 98059 (10 μM) has no effect on the inhibitory action of leptin on the expression of miR-384-3p and miR-488 ( Figure 3B,C). Cryptotanshinone (1 μM), MK 2206 (100 nM), and PD 98059 (10 μM) had no effect alone on the expression of the miRNAs of interest ( Figure 3A-C). These results suggest that leptin may influence miR-383, miR-384-3p, and miR-488 expression though different mechanism of action.

Pathway Analysis of miRNAs Targeting POMC
To further study the functions and underlying mechanisms of these three miRNAs of interest, KEGG (http://diana.imis.athena-innovation.gr/DianaTools/index.php) was used to examine the signaling pathways of specified miRNA target genes. We found that the 3 miRNAs are involved in several pathways ( Figure 4A). According to enrichment score, long-term depression (LTD), glutamatergic transmission, cocaine addiction and cGMP-PKG signaling pathway are the main signaling pathways associated with miR-488 ( Figure 4A). The pathways most correlated with miR-384-3p and miR-383 were glutamatergic synapse, mTOR, and insulin signaling pathways ( Figure 4A). In addition, we also examined the signaling pathways associated with simultaneously at least 2 miRNAs. This analysis revealed that the target genes of at least 2 miRNAs are associated with neurotransmission, addiction, and different signaling pathways ( Figure 4B). inhibition of JAK2-STAT3, PI3K-Akt, and MEK 1/2/ERK pathways. The data are expressed as the mean ± SEM of at least three independent experiments. The values are presented as means ± SEM. ** p < 0.01.

Pathway Analysis of miRNAs Targeting POMC
To further study the functions and underlying mechanisms of these three miRNAs of interest, KEGG (http://diana.imis.athena-innovation.gr/DianaTools/index.php) was used to examine the signaling pathways of specified miRNA target genes. We found that the 3 miRNAs are involved in several pathways ( Figure 4A). According to enrichment score, long-term depression (LTD), glutamatergic transmission, cocaine addiction and cGMP-PKG signaling pathway are the main signaling pathways associated with miR-488 ( Figure 4A). The pathways most correlated with miR-384-3p and miR-383 were glutamatergic synapse, mTOR, and insulin signaling pathways ( Figure 4A). In addition, we also examined the signaling pathways associated with simultaneously at least 2 miRNAs. This analysis revealed that the target genes of at least 2 miRNAs are associated with neurotransmission, addiction, and different signaling pathways ( Figure 4B).

Gene Ontology Analysis of miRNAs Targeting POMC
Using GO-analysis, the functional categories associated with the miRNAs of interest were identified. We explored three domains: biological process (BP), cellular component (CC), and molecular function (MF). This analysis revealed that the genes targeted by miR-383 and miR-488 are involved in several important biological pathways ( Figure 5). In contrast, genes targeted by miR-384-3p are involved only in CC domain ( Figure 5). Finely, the genes targeted by the 3 miRNAs are involved in a large panel of biological functions ( Figure 5).

Gene Ontology Analysis of miRNAs Targeting POMC
Using GO-analysis, the functional categories associated with the miRNAs of interest were identified. We explored three domains: biological process (BP), cellular component (CC), and molecular function (MF). This analysis revealed that the genes targeted by miR-383 and miR-488 are involved in several important biological pathways ( Figure 5). In contrast, genes targeted by miR-384-3p are involved only in CC domain ( Figure 5). Finely, the genes targeted by the 3 miRNAs are involved in a large panel of biological functions ( Figure 5).

Discussion
POMC neurons are essential for the integration of peripheral (nutrients and hormones) and central (neuropeptides and neurotransmitters) signals [1,2,[33][34][35]. Recently, pharmacogenetic and optogenetic techniques have further proved that POMC neuronal activation reduces food intake and increases energy expenditure [36,37]. The important aim of current research is to clarify the molecular mechanism involved in the integration by POMC neurons of these multiple peripheral metabolic signals such as leptin. In this study, we also confirmed for that leptin modulates the expression of POMC in the mHypoA-POMC/GFP cell line which can be thus a good model for the mechanistic studies. This model has been validated in other studies [29,[38][39][40].
It is essential to identify the intracellular mediators that allow these POMC neurons to respond to energy status modifications. In this context, we and others observed that miRNAs can be important intracellular mediators in the modulation of POMC neurons activity [1,[13][14][15][16][17]. So far, only two teams

Discussion
POMC neurons are essential for the integration of peripheral (nutrients and hormones) and central (neuropeptides and neurotransmitters) signals [1,2,[33][34][35]. Recently, pharmacogenetic and optogenetic techniques have further proved that POMC neuronal activation reduces food intake and increases energy expenditure [36,37]. The important aim of current research is to clarify the molecular mechanism involved in the integration by POMC neurons of these multiple peripheral metabolic signals such as leptin. In this study, we also confirmed for that leptin modulates the expression of POMC in the mHypoA-POMC/GFP cell line which can be thus a good model for the mechanistic studies. This model has been validated in other studies [29,[38][39][40].
It is essential to identify the intracellular mediators that allow these POMC neurons to respond to energy status modifications. In this context, we and others observed that miRNAs can be important intracellular mediators in the modulation of POMC neurons activity [1,[13][14][15][16][17]. So far, only two teams have identified mir-488 as miRNA that physically interact with the 3 UTR of POMC mRNA [29,32]. For instance, they revealed that miR-488 can modulate the expression of POMC mRNA for animal coat control [32]. In our study, we demonstrated that mir-383 and miR-384-3p bind physically with 3 UTR of POMC mRNA. Many studies have depicted an important role of mir-383 in cancer and cell viability and proliferation [41][42][43]. Interestingly, Wang et al. suggested that miR-383 can protect against cognitive impairment and hippocampal neurons apoptosis induced by propofol [44]. In accordance with these studies, KEEG and GO analyses revealed that miR-383 targeting genes are important for important cell process. It also been suggested that miR-383 suppressed the PI3K-AKT-mTOR signaling pathway to inhibit development of cervical cancer via down-regulating Poly (ADP-ribose) polymerase-2 [45]. The KEEG analysis confirm that miR-383 target several genes involved in mTOR signaling pathways. In the context of feeding behavior, it has been clearly demonstrated that mTOR signaling pathway is essential for POMC neuron activity [46,47]. To date, few studies clarified the function of miR-384-3p. For instance, Xia et al. showed that miR-384-3p inhibited retinal neovascularization during diabetic retinopathy [48]. KEEG analysis also revealed that the miRNAs of interest control activity-dependent synaptic plasticity, such as long-term potentiation (LTP) and LTD. Interestingly, several studies highlighted that energy state may control the LTP as well as LTD [49,50]. Particularly, a tetanic stimulation protocol induced LTD at POMC neurons in fed mice but weak LTP in food-deprived mice [50]. Then, it is conceivable that miRNAs may be involved in the control of LTP and LTD in POMC neurons. Additional experiments could clarify this last point. Subsequently, a GO analysis was added to our study. The GO analysis included biological process, molecular function and cellular components. GO term analysis revealed that miR-383 and miR-488 were involved in a large number of process and functions as cell cycle or morphogenesis as well as organelle function. In accordance with this observation, the importance of miRNAs in the development of POMC neurons has been highlighted by the specific deletion of Dicer in POMC-expressing cells which led to a postnatal ablation of POMC neurons [15,16]. The GO analysis also revealed that miR-384-3p is involved in only few cellular components functions suggesting a minor role for this miRNA.
In a previous study, we have described that leptin modulated the expression of miR-383, miR-384-3p, and miR-488 [13]. Since the miRNAs can be activated or repressed by TFs, we explored the pathways involved in the inhibitory effect of leptin. The study of miRNA regulation by TFs has been relatively limited [27,51]. However, TFs such as p53, MYC, ZEB1 and ZEB2, and myoblast determination protein 1 (MYOD1) positively or negatively regulate miRNA expression [51][52][53]. By pharmacological approach, we showed that leptin modulates by different pathways the expression of miR-383, miR384-3p, and miR-488. The JAK2-STAT3 pathway is involved in the regulation of miR-384-3p and miR-488 by leptin while this signal modulates miR-383 via PI3K-Akt pathway. Interestingly, it has been shown that STAT3 down-regulated the expression of miR-383 in a skin cancer cell line [54]. This different observation can be explained by the fact that the cell line used in this study is different. Our pioneering study highlights the fact that leptin via the JAK2-STAT3 and PI3K-Akt pathways might modulate the expression of miRNAs targeting POMC. However, we need to deepen these observations. In particular, locations of the miRNA promoters have not yet been mapped for miR-383, miR-384-3p, and miR-488 genes but can be inferred from collective analysis of CpG islands and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data [55].

Conclusions
Taken together, these results allowed us to highlight the miRNA involvement in the regulation of POMC expression downstream of the leptin signaling and satiety signal integration.