Department of Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei, China
Center for Medical Research, Wuhan University School of Medicine, Wuhan, Hubei, China
Address correspondence and reprint requests to Yu Wan, Center for Medical Research and Department of Physiology, Wuhan University School of Medicine, Wuhan, Hubei 430071, P. R. China. E-mail: email@example.com
microRNAs are a class of small non-coding RNA molecules negatively regulating gene expression at post-transcriptional level in many tissues including the central nervous system. cAMP response element binding protein (CREB) is a key nuclear factor highly expressed in hippocampal neurons on which many signal pathways converge. Recent studies have found that microRNA-181a is rich in mature nerve cells, and bioinformatics analysis shows that the CREB1 mRNA 3′-untranslated region (3′UTR) contains complementary sequence to the miR-181a seed region. In this study, we investigated whether miR-181a is a negative regulator for CREB1 expression in neurons. It was found that the expression of miR-181a was negatively correlated with Insulin-like growth factor-1 (IGF-1) and CREB1 in the Lewis rat hippocampus. miR-181a bound to CREB1 mRNA through a specific binding site in the 3′UTR sequence. The expression of CREB1 in PC12 cells was down-regulated by transfection with a miR-181a mimic and up-regulated by a miR-181a inhibitor. A down-regulated miR-181a and an up-regulated CREB1 were observed in IGF-1-stimulated PC12 cells. And miR-181a inhibited dendritic growth of cultured hippocampus neurons. These suggest that miR-181a is involved in IGF-1-regulated CREB1 expression by targeting its mRNA 3′UTR.
microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are involved in the central nervous system development. Here, we demonstrate that miR-181a can inhibit the expression of the transcription factor CREB1 by specifically targeting its mRNA 3′UTR and inhibit the development of hippocampus neurons. Repressed expression of miR-181a is involved in IGF-1-mediated up-regulation of CREB1 in vivo and in vitro. These findings indicate that miR-181a could be a potential target for preventing neurodegenerative diseases.
Insulin-like growth factor-1 (IGF-1), also called somatomedin C, is one of the most important stimulators of cell growth (Yakovchenko et al. 1996), and a potent inhibitor of programmed cell death in both neuronal and non-neuronal cells (Morales et al. 2000; Tu et al. 2010). Although liver is the primary source of circulating IGF-1, significant expression of this growth factor is observed in various organs such as brain where it is known to exhibit autocrine and paracrine functions (D'Ercole et al. 1996). IGF-1 exerts its cellular effects by binding to its type I receptor, which subsequently activates two main downstream signaling pathways, namely the RAF-MEK-extracellular signal-regulated kinase phosphorylation cascade and the phosphatidylinositol 3-kinase-AKT pathway (Zheng et al. 2000; Leinninger and Feldman 2005).
The cAMP response element binding protein (CREB) is one of the common nuclear targets of the extracellular signal-regulated kinase pathway and the PI 3-kinase signaling pathway (Johannessen et al. 2004). It is a 43-kDa nuclear transcription factor belonging to the CREB/ATF family that regulates the transcription of the downstream genes (Kulik et al. 1997). There is a large amount of evidence to suggest that CREB plays a significant role in memory formation, stabilization, and retention (Yin et al. 1994; Bartsch et al. 1995). CREB controls the transcriptional responses of neurons to many extracellular stimuli, such as IGF-1 (Balschun et al. 2003) and also participates in synaptic plasticity and memory consolidation (Mozzachiodi and Byrne 2010). Hippocampal CREB gene transfer increases CREB expression and significantly improves the learning and memory function in aging rats (Mouravlev et al. 2006). PC12 cell line is a commonly used model of neuron derived from a transplantable rat adrenal phaeochromocytoma. It has been showed that IGF-1 mediated phosphorylation and transcriptional activation of CREB in PC12 cells and IGF-1 leading to CREB1-dependent neuronal specific gene expression (Pugazhenthi et al. 1999).
The microRNAs (miRNAs) are a group of small non-coding RNAs that are single stranded chains consisting of 19–25 nucleotides (~ 22 nucleotides) and transcribed by RNA polymerase II or III in the nucleus (Lee et al. 2004). More and more studies showed that miRNAs play an important role in gene regulations by binding imperfectly to the 3′-untranslated region (3′-UTR) of the target mRNAs, which leads to either translational repression or target mRNA cleavage (Lee et al. 1993).
microRNA-181 family, including miR-181a, miR-181b, miR-181c, and miR-181d, is one of the identified miRNAs that widely exists in vertebrate cells. miR-181a was found highly expressed in adult mouse brain tissues (Miska et al. 2004). miR-181a, showing different expression profiles at different stages of human neurodevelopment, was highly expressed in mature human neurons (Smith et al. 2010). These studies strongly suggest that miR-181a is closely related to brain cell development and differentiation. The bioinformatics analysis showed that CREB1 mRNA 3′UTR contains complementary sequence to the miR-181a seed region. In addition, IGF-1 regulated the expression of CREB to delay the arrival of cognitive and memory dysfunction with the aging (Anderson et al. 2002).
To understand whether miR-181a is a negative regulator for CREB1 expression in neurons, the expression of IGF-1, CREB1, and mature miR-181a in the hippocampus of Lewis rat were analyzed in this study, with the effect of miR-181a on dendrites growth of neurons and the mechanism of miR-181a in IGF-1 induced activation of CREB1 in PC12 cells investigated.
Materials and methods
Adult male (9–11 months old) and pregnant female (in gestation days 14–16) Lewis rats were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The male animals were acclimated for 5–7 days under standard conditions and the pregnant females were kept for generation of newborns. Sixteen males and sixty newborns (0–2 days after birth) were killed and the brains were quickly removed. Fresh hippocampi were then dissected from the brain on a chilled glass plate on ice according to the procedures described in reference (Sun et al. 2005). The hippocampi were stored at −70°C until the day of assay, and those from newborns were used for the neural cell preparation immediately. All animal experiments in this study were approved by Medical Ethics Committee of Wuhan University, and carried out in accordance with the guiding principles for the care and use of laboratory animals published by the U.S. National Institutes of Health (NIH Publication No.85-23, revised 1996) and the ARRIVE guidelines.
PC12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (High glucose/DMEM, Invitrogen, Ontario, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 5% heat inactivated horse serum (HyClone), 1% penicillin-streptomycin at 37°C. HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C.
The cells (1 × 105) were seeded in six-well plates prior to treatment with IGF-1 (25 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, R & D Systems, Minneapolis, MN, USA) for 24 h, and the control group was treated with 1× phosphate-buffered saline (PBS) alone. After the treatment, total RNA of the cells was collected for western blot analysis and total RNA isolated for miRNA analysis.
Microarray analysis was performed by Kangcheng Bio-tech Inc. (Shanghai, China). Briefly, total RNA was harvested using Trizol (Invitrogen, Carlsbad, CA, USA) and miRNeasy mini kit (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. After RNA quantity measurement with the NanoDrop 1000, the samples were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.16.0, Exiqon, Skelstedet, Vedbaek, Denmark). After hybridization, scanning was performed with the Axon GenePix 4000B microarray scanner (Molecular Devices, Downingtown, PA, USA). The raw intensity of the image was read with GenePix pro V6.0 (Molecular Devices) and the intensity of green signal was calculated after background subtraction. Four replicated spots of each probe on the same slide were averaged. Median Normalization Method was used to obtain ‘Normalized Data’: Normalized Data = (Foreground − Background)/median, where the median was the 50% quantile of miRNA intensity, which was larger than 50 in all samples after background correction.
Vector construction and transfection
Total RNA was extracted using Trizol reagent (Invitrogen). cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Dalian, China). A 622 bp fragment of the rat CREB1 mRNA 3′UTR (corresponding to nt 1003–1624 of the Entrez PubMed transcript FQ211925) was amplified by PCR from CREB1 full length cDNA. The PCR products were cloned into pGL3-control luciferase reporter vector (Promega, Madison, WI, USA) via an XbaI (Takara) restriction site, immediately downstream of the luciferase gene, named pGL3C-CREB1 3′UTR and pGL3C-CREB1 3′UTR mutant. The primers selected were as the following:
The miR-181a mimic, inhibitor, and negative control were purchased from RiboBio (RiboBio Co. Ltd., Guangdong, China). Transfection of microRNA or microRNA inhibitor was performed using the X-tremeGENE HP DNA Transfection Reagent (Roche, Mannheim, Germany) according to the manufacturer's instruction.
HEK293 cells (1 × 104) were seeded in triplicates in 96-well plates and allowed to settle for 24 h. A co-transfection of the cells was performed with 100 ng luciferase reporter plasmids of pGL3C-CREB1 3′UTR (wt) + 1 ng pRL-TK renilla plasmid (Promega) + PBS, 100 ng of luciferase reporter plasmids of pGL3C-CREB1 3′UTR (wt) + 1 ng of pRL-TK renilla plasmid + scramble miRNAs, 100 ng of luciferase reporter plasmids of pGL3C-CREB1 3′UTR (wt) + 1 ng of pRL-TK renilla plasmid + miR-181a mimics (100 ng) or 100 ng of luciferase reporter plasmids of pGL3C-CREB1 3′UTR (mu) + 1 ng of pRL-TK renilla plasmid + miR-181a mimics (100 ng) using the X-tremeGENE HP DNA Transfection Reagent (Roche). Luciferase and renilla signals were measured 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega). Three independent experiments were performed and the data are presented as means ± SEM.
Quantitative RT-PCR detection
Total RNA was isolated and converted into cDNA by same way as described above. Detection of the mature form of miR-181a was performed using Quantitect SYBR Green PCR Kit (Takara) and quantitative RT-PCR Primer Sets (Ribobio) with the U6 small nuclear RNA as an internal control.
Western blot analysis
PC12 cells (1.5 × 105) were seeded in six-well plates and allowed to settle for 24 h. miR-181a mimic (50 nM) or inhibitor (100 nM) was transfected into the cells using the X-tremeGENE HP DNA Transfection Reagent (Roche). The cells were collected at 24 h after transfection. Hippocampus and cells were homogenized in radioimmune precipitation assay buffer (25 mM Tris-HCl pH7.6, 150 mM NaCl, 1%NP-40, 1% Sodium deoxycholate and 0.1% sodium dodecyl sulfate) containing complete EDTA-free protease inhibitor (Roche). Western blotting was conducted according to (Khanna et al. 2011). The following primary antibodies were used: rabbit monoclonal antibodies (mAb) against CREB1 and Phospho-CREB (Ser133) (#9197, #9198; Cell Signaling Technology Inc, Danvers, MA, USA) and mouse mAb against IGF-1 (ab36532; Abcam Inc., Cambridge, MA, USA). The reaction was visualized by corresponding horseradish peroxidase-conjugated secondary antibodies. Bands were detected using Super Signal Femto chemiluminescent reagent (Pierce, Rockford, IL, USA) and quantified by the Chemi-doc gel quantification system (Bio-Rad Laboratories, Hercules, CA, USA). All data were normalized to β-actin.
Hippocampal neurons were isolated from newborn Lewis rats and seeded onto poly-L-lysine coated coverslips in 24-well plates as described by (Brewer 1997). The neurons were cultured in Neurobasal A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% B27 (Invitrogen), 0.5% l-glutamine (Invitrogen), 20 ng/mL epidermal growth factor (Peprotech, Rocky Hill, NJ, USA), 10 ng/mL basic fibroblast growth factor (Peprotech). The neurons were cultured 6 days with a half of the medium replaced every 2 days. After that, scramble miRNA, miR-181a mimic (50 nM) and miR-181a inhibitor (100 nM) were transfected into cells using the X-tremeGENE HP DNA Transfection Reagent (Roche). At 48 h after transfection, the cells were fixed with 4% paraformaldehyde in PBS buffer (0.01 M, pH 7.3) for 10 min and then incubated with a mouse monoclonal antibody against β-III tubulin (1 : 100, Millipore, Billerica, MA, USA) at 4°C overnight to specifically mark the neurons. After additional washes in PBS, the cells were stained in a rabbit anti-mouse IgG-FITC (1 : 200; Santa Cruz, Dallas, TX, USA) at (25°C) for 1 h and digitally photographed under Olympus BX51 inverted microscope (Tokyo, Japan) with an UplanFI (40x/0.75) objective (Olympus Optical, Tokyo, Japan). Morphometric measurements of dendrite were performed by using Openlab software (Improvision, Lexington, MA, USA). Dendritic length and branch number were analyzed as described in reference (Wayman et al. 2006). Each experiment was repeated with at least three independent preparations.
All data were analyzed statistically with Prism (PrismGraphPad Software, Graphpad Software Inc, La Jolla, CA, USA) using the Student's t-test or one-way anova followed by Bonferroni-corrected pairwise comparisons. Unless indicated otherwise, a p < 0.05 was considered statistically significant.
Microarray analysis for expression of miRNAs in hippocampus of newborn and adult Lewis rats
Microarray analysis showed that out of 680 detected miRNAs 148 displayed distinct expression in the hippocampus, with 44 differentially expressed between the newborn and adult Lewis rats (up or down over 1.5 times; Fig. 1) and verified by qPCR. Among these differentially expressed miRNAs, the miR-181 family (miR-181a, miR-181b, miR-181c, miR-181d) were significantly down-regulated with miR-181a displaying the highest expression level in the hippocampus. Bioinformatics analysis predicted that the miR-181a and other members in its family all have binding sites located on CREB1 mRNA 3′UTR sequences (Fig. 5a). These data provide a clue that miR-181 family might be involved in regulation of CREB1 expression, with the miR-181a as a most potential candidate. As shown in Fig. 2, miR-181a expression was much lower in hippocampus of adult Lewis rats than that in the newborns.
Expression of IGF-1 and CREB1 in hippocampus of newborn and adult Lewis rats
Western blot analysis revealed that IGF-1 protein levels were significantly higher in hippocampus of adult Lewis rats than that in the newborns (Fig. 3). Similar expression patterns of total CREB1 and p-CREB1 proteins were also found between the two animal groups with the total-CREB1 and P-CREB1 levels approximately tripled in the adult rats (Fig. 4). This result suggests that the expression of both IGF-1 and CREB1 increases with the development of hippocampus, and is in line with the previous evidence that IGF-1 regulates the expression of CREB (Anderson et al. 2002).
Analysis of miR-181a binding site on mRNA 3′UTR of CREB1
Target Scan identified 896 predicted target genes for miR-181a, including CREB1; and 90 candidate miRNAs that have the seed matches with CREB1 mRNA 3′UTR were retrieved from the Target Scan database, in which miR-181a was included. Similar results were obtained from MicroCosm and ClustalW2. Manual alignment between the miR-181a seed region and the seed matches on CREB1 mRNA 3′UTR region revealed that they are highly complementary and highly conservative in human, mouse and rat (Fig. 5a).
To confirm the direct binding of miR-181a to CREB1 mRNA 3′UTR region, a luciferase report vector containing the mRNA 3′UTR of rat CREB1 was transfected into HEK293 cells. The luciferase activity was significantly repressed when miR-181a mimic was co-transfected with the report gene (Fig. 5b). In contrast, the luciferase activity was not changed significantly when the scramble miRNA was co-transfected. Similarly, miR-181a mimic co-transfection with pGL3C-CREB1 3′UTR mutant did not reduce the luciferase activity notably.
Effects of miR-181a on expression of CREB1
To evaluate the effect of miR-181a on expression of CREB1, miR-181a mimic was transfected into PC12 cells. As demonstrated by western blot analysis, the expression of CREB1 by the cells was significantly repressed (0.73 ± 0.11 vs. 1.00 ± 0.07, p < 0.05) (Fig. 6a). In contrast, a transfection of the cells with miR-181a inhibitor significantly up-regulated the expression of CREB1 product (1.67 ± 0.09 vs. 1.00 ± 0.21, p < 0.005) (Fig. 6b).
Effect of IGF-1 on expression of CREB1
To confirm the effect of IGF-1 on expression of CREB1, PC12 cells were treated with different concentrations of IGF-1 (25 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL,respectively) for 24 h. The expression of CREB1 and p-CREB1 was evaluated by western blot. As seen in Fig. 7, IGF-1 significantly up-regulated both CREB1 protein expression and phosphorylation with a maximal effect at the concentration of 50 ng/mL (2.21 ± 0.13 vs. 1.00 ± 0.21, p < 0.001).
Effect of IGF-1 on miR-181a expression
To investigate the effect of IGF-1 on miR-181a expression, PC12 cells were treated IGF-1 for 24 h. As demonstrated by Quantitative RT-PCR (Fig. 8) IGF-1 (50 ng/mL) caused a significant decrease in miR-181a expression to about 45% of the control level (1.00 ± 0.09 vs. 0.45 ± 0.12, p <0.005).
Effect of miR-181a on dendritic growth of hippocampus neuron
To address whether miR-181a regulated the growth and development of hippocampus neuron, the primary-cultured hippocampal neurons from newborn Lewis rats were transfected with miR-181a mimic, miR-181a inhibitor and a scramble miRNA, respectively. After 48 h, the cells were fluorescently labeled with anti-β-III tubulin (a characteristic marker for neurons) and the dendritic length and branching were measured. As shown in Fig. 9, transfection with miR-181a mimic decreased total dendritic length by about 60% and branching by about 70% in comparison to scramble miRNA; while transfection with miR-181a inhibitor increased the total dendritic length by about 65% and branching by about 60%. The scramble miRNA transfection did not affect dendritic growth (Fig. 9) and none of the treatments increased apoptosis as demonstrated by Hoechst staining (data not shown).
IGF-1, as a potent neurotrophic factor, plays a critical role in the development and maturation of the CNS by promoting survival and proliferation of many types of brain cells (Guan et al. 2003). Transgenic and gene knock-out mice study indicates that IGF-1 plays a key role in the CNS development and helps to reduce the risks of gliomas and neurodegenerative diseases (Russo et al. 2005).
miRNA is involved in regulation of the CNS development in a temporal and spatial order; and at the same time, it is also participates in maintenance of various different nerve cells in the normal morphology and functions. miRNA expression has conservative, short-term and tissue-specific features in CNS (Kapsimali et al. 2007). miR-181a was highly expressed in mature neurons and adult mouse brain (Miska et al. 2004; Smith et al. 2010). CREB is the converged point of many signal pathways in hippocampal neurons and plays a crucial role in learning and memory (Disterhoft and Oh 2006; Mozzachiodi and Byrne 2010). Bioinformatics analysis showed that there is a possible target of miR-181a in CREB1 mRNA 3′UTR sequence. At present, the exact function of miRNA-181a in hippocampus has not been cleared and its role in regulation of CREB1 remains to be determined. This study confirmed that CREB1 mRNA 3′UTR is a direct target of miR-181a, suggesting a role of miR-181a in regulation of CREB1 expression.
Previous studies have suggested that in hippocampus or brain, IGF-1 protein expression is maybe growth hormone-independent and its autocrine is an important regulatory factor in hippocampal neurogenesis (Sun 2006). In the present study, the expression of miR-181a, IGF-1, and CREB1 in hippocampus of newborn and adult Lewis rats were examined. The results revealed that IGF-1 protein levels were significantly higher in adult Lewis rats compared with the newborns, and the total and phosphorylated CREB1 were approximately tripled in the hippocampus of adult rats. While miRNA microarray and quantitative RT-PCR showed that miR-181a expression was obviously lower in adult Lewis rats in comparison to the newborns. These findings suggested that an enhanced expression of CREB1t by higher levels of IGF-1 in hippocampus of adult Lewis rats is related to an inhibited miR-181a expression.
To further confirm the relationship between the inhibited miR-181a and up-regulated CREB1, this study then investigated whether miR-181a can bind and affect the predicted target CREB1 mRNA through interactions with its 3′UTR. Accordingly the mRNA 3′UTR of CREB1 was cloned into a reporter vector, downstream of a firefly luciferase cDNA. It was demonstrated that in HEK293 cells, the vector pGL3C-CREB1 3′UTR containing the 3′UTR of CREB1 mRNA displayed a clear reduction of light emission upon co-transfection with miR-181a mimic. On the other hand, co-transfection of scramble miRNA or miR-181a mimic with pGL3C-CREB1 3′UTR mutant did not induce obvious changes in the luciferase activity. These indicate the presence of a direct binding site for miR-181a.
As demonstrated by western blot analysis, when miR-181a mimic or miR-181a inhibitor was transfected into PC12 cells, CREB1 was inhibited by over-expressed miR-181a or enhanced upon miR-181a repression. In primary cultured hippocampal neurons, dendritic growth was obviously induced by transfection of miR-181a inhibitor and blocked, by miR-181a mimic. These findings, together with the previous evidence that dendritic growth is regulated by activation of a transcriptional program that transduces CREB1-mediated signaling into the nucleus (Redmond et al. 2002), support a negative effect of miR-181a on dendritic growth of neurons by regulating CREB1 gene.
From worms to humans, IGF-1 has important roles in CNS development. It promotes the proliferation, survival, and differentiation of all types of brain cells (D'Ercole et al. 1996). Pugazhenthi et al. (1999) reported that IGF-1 mediated transcriptional activation of CREB1 within a time less than 30 min, in rat PC12 cells. Here, this study demonstrated that IGF-1 up-regulated CREB1 protein expression and phosphorylation at 24 h in the same cell line with a maximal effect at a concentration of 50 ng/mL. In addition, this study also demonstrated that IGF-1 had a negative effect on miR-181a expression. These data, together with those mentioned above, suggest that exogenous IGF-1 can markedly up-regulate CREB1, at least partially, by inhibiting miR-181a expression. There are numerous studies demonstrated that other miRNAs participated in regulating CREB1 protein expression (Leone et al. 2011; Kong et al. 2012). And CREB1 plays a key role in learning and memory. All these add a complex non-coding RNA regulation of CREB1 gene to those by traditional cytokines.
IGF-1 and CREB1 activities are critically reduced in the context of aging and of age associated brain diseases (Caccamo et al. 2010; Piriz et al. 2011). As a potent neuroprotective agent, IGF-1 promotes neuronal cell health by triggering genetic programs that are largely dependent on CREB1 (Carro and Torres-Aleman 2004). This research shows that IGF-1 can enhance CREB1 expression but inhibit the generation of mature miR-181a. And in contrast, miR-181a has a negative effect on CREB1 expression, which indicates an important role for miR-181a in neurodegeneration. This find may direct to a new therapeutic target for the prevention of age-associated cognitive impairment and neurodegeneration.
In summary, this study demonstrated for the first time that miR-181a is involved in IGF-1 mediated regulation of CREB1 expression through an interaction with CREB1 mRNA's 3′UTR; and its mimic inhibits dendritic growth of hippocampal neurons. These findings suggest miR-181a as a potential target for preventing neurodegenerative diseases.
This study was funded by The National Natural Science Foundation of China (30870924, Yu Wan).
The authors have no conflict of interest to declare.