brain research 1611 (2015) 65–73
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Research Report
MicroRNA-30b promotes axon outgrowth of retinal ganglion cells by inhibiting Semaphorin3A expression F. Han, Y. Huo, C.-J. Huang, C.-L. Chen, J. Yen Department of Ophthalmology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China
art i cle i nfo
ab st rac t
Article history:
Semaphorin3A (Sema3A) is a major inhibitory factor of optic nerve (ON) regeneration post-
Accepted 6 March 2015
injury. Many microRNAs (miRNAs) are expressed specifically in the mammalian brain and
Available online 17 March 2015
retina and are dynamically regulated during development, suggesting that this group of
Keywords:
miRNAs may be associated with neural development. We found that microRNA-30b (miR-
miR-30b
30b) bound to the three prime untranslated region (30 UTR) of Sema3A and inhibited the
Sema3A
expression of Sema3A mRNA. The mRNA expression level of miR-30b and the protein
RGCs
expression levels of Sema3A, Neuropilin1 (NRP1), PlexinA1 (PlexA1), phosphorylated
Regeneration
p38MAPK (p-p38MAPK), and active caspase-3 were all upregulated in retinas from rats with a damaged ON relative to those with an intact ON. Transfection of cultured retinal ganglion cells (RGCs) with an miR-30b mimic led to decreased levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression, as well as axon elongation and reduced levels of apoptosis. These findings provide evidence that miR-30b inhibits Sema3A expression. Decreased Sema3A expression promotes axon outgrowth in RGCs due to reduced levels of Sema3A binding to NRP1 and PlexA1 and simultaneously reduces apoptosis by inhibiting the p38MAPK and caspase-3 pathways. Our findings provide the first evidence that miR-30b-mediated Sema3A downregulation may serve as a new strategy for the clinical treatment of ON injury. & 2015 Elsevier B.V. All rights reserved.
1.
Introduction
of the most significant problems associated with CNS regeneration include glial scar formation, neurotrophic factor
The optic nerve (ON) is a part of the central nerve system
deficits, and the presence of inhibitory proteins. Among
(CNS). The axons of retinal ganglion cells (RGCs) comprise the
them, the inhibitory influences are the most important
ON. The regeneration and functional recovery of a damaged
contributing factors (Fawcett, 2009; Tuszynski and Gage,
ON remain challenging due to many regulatory factors. Some
1995). Semaphorin3A (Sema3A) is a very potent repulsive
n
Corresponding author. Fax: þ86 23 68767066. E-mail address: [email protected] (J. Ye).
http://dx.doi.org/10.1016/j.brainres.2015.03.014 0006-8993/& 2015 Elsevier B.V. All rights reserved.
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axon guidance cue and an important inhibitory factor involved in CNS repair following damage (Luo et al., 1993). Sema3A binds to the Neuropilin1 (NRP1) receptor and then associates with the PlexinA1-A4 (PlexA1-A4) receptor to transmit a signal that induces growth cone collapse (He and Tessier-Lavigne, 1997; Rosenzweig et al., 2010; Takahashi et al., 1999; Zylbersztejn et al., 2012). Sema3A activates p38MAPK phosphorylation and promotes neural progenitor cell apoptosis (Bagnard et al., 2004). Furthermore, Sema3A can reduce myelin lipid phagocytosis by macrophages, which in turn inhibits myelin regeneration of the CNS (Piaton et al., 2011; Syed et al., 2011) and blocks neurotrophic factor transport and recovery of the damaged nerves (Maione et al., 2009). MicroRNAs (miRNAs) are endogenous small molecules that reduce the expression levels of proteins by binding to the three prime untranslated region (30 UTR) of the targeted mRNAs and inhibiting or degrading the mRNAs (Luciano, 2004; Vermeulen et al., 2007), allowing them to regulate cell proliferation, differentiation, and apoptosis. Many miRNAs are expressed specifically in the mammalian brain and retina and are dynamically regulated during development, suggesting that this group of miRNAs may be associated with neural development (Arora et al., 2007, 2010; Krichevsky et al., 2006; Miska et al., 2004; Sempere et al., 2004). Further investigation into Sema3A and retina-associated miRNAs may provide a new window of opportunity for exploring the treatment of ON damage via Sema3A inhibition. After searching the public databases using prediction algorithms, such as TargetScan, miRanda, and Pictar, we identified microRNA-30b (miR-30b) as a candidate modulator of Sema3A expression and a potential mechanism for targeting Sema3A expression.
expression was detected in the inner nuclear layer (INL) and the ganglion cell layer (GCL) (Fig. 1c). Sema3A expression levels in the INL and GCL increased significantly at 7 d in retinas associated with ON injury compared to control retinas.
2.2.
Following ON damage, rAAV-miR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS was injected into the vitreous. The expression levels of miR-30b and Sema3A were analyzed at 3 d and 7 d post-injury. As predicted, miR-30b mRNA expression levels were higher in the miR-30b mimic group and lower in the miR-30b inhibitor group than in the other three groups. In contrast, the expression patterns of Sema3A mRNA were somewhat unexpected. The expression levels of miR-30b mRNA decreased by approximately 5-fold (Po0.01, Fig. 2a) and 10-fold (Po0.05, Fig. 2a) in the miR-30b inhibitor group relative to the PBS group at 3 d and 7 d post-injury, respectively, but the expression levels of Sema3A mRNA increased by only approximately 4-fold (Po0.01, Fig. 2a) and 2-fold (Po0.01, Fig. 2a) in the miR-30b inhibitor group compared to the PBS group at 3 d and 7 d postinjury, respectively. Although the expression levels of miR-30b mRNA increased by approximately 3-fold (Po0.01, Fig. 2a) and 23-fold (Po0.01, Fig. 2a) in the miR-30b mimic group relative to the PBS group at 3 d and 7 d post-injury, respectively, the expression levels of Sema3A mRNA decreased by approximately 3-fold (Po0.05, Fig. 2a) and 5-fold (Po0.01, Fig. 2a) in the miR-30b mimic group compared to the PBS group at 3 d and 7 d postinjury, respectively. At 3 d and 7 d post-injury, the miR-30b mimic group had the lowest levels of Sema3A protein expression, and the miR-30b inhibitor group had the highest levels of Sema3A protein expression (Fig. 2b).
2.3.
2.
Sema3A is regulated by miR-30b in vivo
Sema3A is the target gene of miR-30b
Results
2.1. Analysis of the expression levels of miR-30b, Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 in the retinas of rats with or without ON injury The results of qRT-PCR experiments showed that miR-30b was expressed in the retinas of normal control rats. After the ON of the left eye was damaged with an arterial clamp, miR-30b expression levels in the left retina increased and peaked at 3 d relative to miR-30b expression levels in control retinas (Po0.01, Fig. 1a). However, the peak in miR-30b expression levels in retinas associated with ON injury declined rapidly at 7 d, after which there was no significant difference between miR-30b expression levels in the post-injury and control retinas. Sema3A mRNA expression levels peaked at 7 d post-injury and declined quickly in retinas associated with ON injury, but remained higher than in control retinas (Po0.05, Fig. 1a). Sema3A protein expression levels increased significantly in retinas associated with ON injury relative to control retinas, reaching a peak value at 7 d and slowly declining at subsequent time points (Fig. 1b). The protein expression levels of NRP1, PlexA1, p-p38MAPK, and active caspase-3 also increased significantly in retinas associated with ON injury relative to control retinas (Fig. 1b). There was no significant change in their expression levels at any time point in all sham retinas. In the undamaged retina, Sema3A protein
The miR-30b binding site located in the Sema3A 30 UTR has the sequence UGUUUACA (Fig. 3a), which was identified by searching public databases (TargetScan) using prediction algorithms. Results of the dual-luciferase reporter assay showed that the relative luciferase activity was significantly lower in the treatment group relative to the control or mutation groups (Po0.01, Fig. 3b). In vitro cultures of rat RGCs were transfected with rAAV-miR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS. Seven days later, the changes in NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression levels were similar to those of Sema3A, i. e., lower protein expression levels were detected in the miR30b mimic group relative to the remaining three groups, and higher protein expression levels were detected in the miR-30b inhibitor group relative to the remaining three groups (Fig. 3c). Three different Sema3A siRNAs were used to examine the potential functions of miR-30b. The efficacy of siRNAmediated inhibition of Sema3A expression was determined in rat RGCs by immunoblotting at 5 d after plating. The RGCs transfected with siRNA2-Sema3A exhibited the most efficient knockdown of Sema3A expression (Fig. 3d). Therefore, this siRNA was selected for use in subsequent experiments. Rat RGCs were transfected with siRNA2-Sema3A at 24 h after plating. Subsequently, the RGCs were transfected with
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Fig. 1 – Expression patterns of miR-30b, Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 in the rat retina are shown. (a) miR-30b and Sema3A expression levels were measured by qRT-PCR: retinas from normal rats (control), or retinas associated with an injured left ON (Treatment) or with a sham-operated right ON (Sham) at 1, 3, 7, 14, and 21 days post-operation. (Mean7SEM, n¼ 6, One-way ANOVA, *Po0.05, **Po0.01). (b) Immunoblot analysis of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression in retinas from normal rats (control) or in retinas from rats with a damaged ON (Treatment) or a sham-operated ON (Sham) at 1, 7, 14, and 21 days post-operation. (c) In the retina, Sema3A protein expression was detected in the inner nuclear layer (INL) and the ganglion cell layer (GCL) using immunohistochemistry. IPL: inner plexiform layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PR: photoreceptors. Scale bar: 40 μm. rAAV-miR-30b mimic or rAAV-miR-30b inhibitor at 5 d after plating. Total proteins were extracted at 9 d after plating, and the expression levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 were not significantly different between the rAAV-miR-30b mimic and the rAAV-miR-30b inhibitor groups (Fig. 3e). These results provide evidence that Sema3A is the target gene of miR-30b.
The rate of apoptosis was the lowest in the miR-30b mimic group and highest in the miR-30b inhibitor group (Po0.05, Fig. 4b). These results showed that overexpression of the miR-30b mimic significantly inhibits RGC apoptosis. The length of RGC axons was also determined for the four groups. RGC axon length was longest in the miR-30b mimic group and shortest in the miR-30b inhibitor group at 7 d after plating (Po0.01, Fig. 5).
2.4. Apoptotic rate and axon length of RGCs after miR-30b transfection
3. In order to further characterize the role of miR-30b, the levels of apoptosis were determined by flow cytometry in RGCs at 5 d after plating. The apoptotic cells were identified as being Annexin V-positive and propidium iodide-negative (Fig. 4a).
Discussion
Sema3A is an important repulsive axon guidance cue that plays a crucial role in CNS regeneration and recovery. In a previous report (Shirvan et al., 2002), RGCs underwent
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Fig. 2 – Analysis of miR-30b and Sema3A expression following transfection with miRNA negative control (NC), miR-30b mimic, or miR-30b inhibitor in vivo. Immediately after the ON was damaged, PBS, rAAV-miRNA NC, rAAV-miR-30b mimic, or rAAVmiR-30b inhibitor was injected into the vitreous. The expression levels of miR-30b and Sema3A were analyzed at 3 d and 7 d post-injury. (a) Results of qRT-PCR experiments to examine miR-30b and Sema3A mRNA expression levels. (Mean7SEM, n¼ 6, One-way ANOVA, *Po0.05, **Po0.01). (b) Results of immunoblot experiments to examine Sema3A protein expression levels.
Fig. 3 – MiR-30b directly targets the Sema3A gene. (a) The miR-30b binding site in the 30 UTR of Sema3A was predicted by searching the public database (TargetScan). (b) Cultured HEK293 cells were co-transfected with a luciferase reporter containing the wild-type Sema3A 30 UTR (wild-type pMIR-REPORT-Sema3A 30 UTR [WT-Sema3A]) together with either the miRNA NC (control group) or with the miR-30b mimic (treatment group), or they were co-transfected with a luciferase reporter containing a mutant version of the Sema3A 30 UTR (MT-Sema3A) together with the miR-30b mimic (mutant group). Luciferase activities were measured and the results were compared between the three groups (Mean7SEM, n¼ 4, One-way ANOVA, **Po0.01). (c) In vitro cultures of rat RGCs were transfected with rAAV-miR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS at 24 h after plating. Protein expression levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 were measured at 7 d after the initial plating by immunoblotting. (d) Rat RGCs were transfected with one of three different siRNA duplexes targeting Sema3A (siRNA1-Sema3A, siRNA2-Sema3A, or siRNA3-Sema3A) or with siRNA-NC at 24 h after plating. Sema3A protein expression levels were determined at 5 d post-plating by immunoblotting. (e) Rat RGCs were transfected with siRNA2Sema3A at 24 h after plating, followed by transfection with the rAAV-miR-30b mimic or rAAV-miR-30b inhibitor at 5 d postplating. Protein expression levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 were determined at 9 d postplating by immunoblotting. apoptosis following intravitreal injection of Sema3A. Injection of anti-Sema3A antibodies blocked RGC apoptosis after the axon microsurgery. Blocking Sema3A signaling has also
been shown to reduce the collapse of the dorsal root ganglion growth cone following spinal cord injury, and to promote the regeneration of the corticospinal tract (Kikuchi et al., 2003).
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Fig. 4 – Overexpression of miR-30b inhibits RGC apoptosis. Rat RGCs were transfected with rAAV-miR-30b mimic, rAAV-miR30b inhibitor, rAAV-miRNA NC, or PBS after 24 h. Rates of apoptosis were determined by flow cytometry at 5 d post-plating. (Mean7SEM, n ¼ 3, One-way ANOVA, *Po0.05, **Po0.01).
Fig. 5 – Representative images of RGCs with GAP-43 staining in the indicated groups. Rat RGCs were transfected with rAAVmiR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS after 24 h. The RGCs were stained with anti-GAP-43 antibodies and axon lengths were analyzed at 7 d post-plating. (Mean7SEM, One-way ANOVA, **Po0.01). Scale bar: 80 μm. Therefore, inhibition of Sema3A not only improves the survival of damaged neurons in the CNS, but also promotes axon elongation. This is beneficial for promoting axon connectivity and for the functional reconstruction of damaged nerves (Kaneko et al., 2006). Following ON damage induced by transection injury in rats, Sema3A expression in RGCs significantly increased and peaked at day 3 post-injury (Nitzan et al., 2006; Shirvan, 2002). In our study, Sema3A expression peaked at day 7 following ON damage induced by crush injury. The different results may be due to the fact that transection injuries are more serious than crush injuries. Previous studies have shown that miR-30b is involved in physiological and pathological processes in a variety of tissues (Altmae et al., 2012; Balderman et al., 2012; Gaziel-Sovran et al., 2011; Le Guillou et al., 2012). It is worth noting that aberrant expression of miR-30b has also been correlated with many neurological disorders (Mellios et al., 2012; Quintavalle et al., 2012; Song et al., 2011). Herein, we demonstrate that miR-30b regulates Sema3A expression in HEK293 cells by using a dualluciferase reporter assay, and further confirmed that miR-30b bound to Sema3A at the sequence UGUUUACA.
We found that miR-30b was expressed in the normal uninjured rat retina, and that its expression increased in response to ON damage. The increase in miR-30b expression promoted selfhealing in the animals after injury. However, the increase in miR-30b expression was only maintained for a short period of time. The expression of miR-30b decreased significantly at day 7 post-injury and showed no significant difference when compared to that of controls after this point. The decrease in miR-30b expression that occurred over time post-injury may be due to blocked autocrine or paracrine pathways associated with miR30b; however, further studies are needed to identify the specific mechanism. In contrast, Sema3A mRNA and protein expression levels were maintained at higher levels in the treatment group relative to controls and the regeneration of the nerve was inhibited. These results are supported by the findings of a previous study involving ON axotomy (Nitzan et al., 2006). Interestingly, despite the fact that Sema3A is the target gene of miR-30b, both showed increased expression following ON damage. Additional experiments showed that the changes in miR-30b and Sema3A expression were disproportionate following transfection of miR-30b in vivo (Fig. 2). This
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may be due to differences in secretory volumes of miR-30b and Sema3A at different times following ON damage. In addition, although miR-30b is a candidate regulator of Sema3A, it is not solely responsible for Sema3A regulation. Sema3A binds to the NRP1/PlexA1 receptor complex to inhibit nerve growth (Zylbersztejn et al., 2012). The binding of Sema3A to NRP1 induces a structural alteration in PlexA1 leading to the release of PlexA1 from a self-inhibited status and the initiation of downstream signaling, which in turn induces growth cone collapse and inhibits axon elongation (Antipenko et al., 2003; Takahashi et al., 1999). We found that the changes in NRP1 and PlexA1 expression patterns were the same as for Sema3A in the rat retina post-injury. After RGCs were transfected with the miR-30b mimic, reduced Sema3A expression was accompanied by reduced NRP1 and PlexA1 expression and significant elongation of RGC axons. The opposite effects were observed when RGCs were transfected with the miR-30b inhibitor. These results indicate that miR30b promotes axon elongation by inhibiting Sema3A and reducing the binding of Sema3A to NRP1 and PlexA1. The p38MAPK signaling pathway is involved in apoptosis in a variety of cell types (Sutter et al., 2003; Tamagno et al., 2003; Walton et al., 1998). Overexpression of proNGF can activate the p38MAPK signaling pathway, resulting in the phosphorylation of p38MAPK and induction of apoptosis in RGCs in diabetic rats (Al-Gayyar et al., 2011). In the ON crush model used in our study, the expression patterns of pp38MAPK and active caspase-3 were similar to that of Sema3A, suggesting that Sema3A induced apoptosis of RGCs via the p38MAPK signaling to activate caspase-3. These results are consistent with an earlier report (Nitzan et al., 2006). We found that overexpression of miR-30b in RGCs inhibited the expression of p-p38MAPK and active caspase-3, which were expressed at significantly lower levels than in the miR-30b inhibitor group. The apoptotic rate of RGCs showed the same results. These findings indicate that miR-30b reduces RGC apoptosis by inhibiting Sema3A and p38MAPK signaling via the downregulation of caspase-3. In conclusion, we found that miR-30b bound to the Sema3A 30 UTR. The expression profiles of miR-30b and Sema3A in the retinas of rats following ON damage were observed. We found that miR-30b inhibited Sema3A expression in RGCs, promoted axon outgrowth by reducing the binding capability of Sema3A to NRP1/PlexA1, and decreased RGC apoptosis by inhibiting p-p38MAPK and active caspase-3. Our findings provide a new strategy for the clinical treatment of ON damage and an experimental basis for gene therapy development. The in vivo effects of the miR-30b mimic and the recovery of function of damaged ONs should be a focus of further investigation.
4.
Experimental procedures
4.1.
Materials and animals
Antibodies against Sema3A (sc-28867), NRP1 (sc-7239), and PlexA1 (sc-25639) were purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Antibodies targeting GAP-43 (ab131482),
p-p38MAPK (ab47363), and active caspase-3 (ab2302) were purchased from Abcam (CA, USA). Neonatal Sprague–Dawley (SD) rats of either sex at postnatal day one and adult SD rats of either sex weighing 160– 200 g were provided by the Animal Experimental Center (Institute of Surgery Research, Daping Hospital, Third Military Medical University, China). The Animal Research Committee of the Third Military Medical University approved the study protocol.
4.2.
ON injury model in rats
The left eyes of the rats were selected as the ON crush models and were injured with an ON forceps, and the right eyes of the same rats were designated as the shams. Rats were anesthetized by intraperitoneal injections of pentobarbital sodium (30 mg/kg). A 1-cm horizontal incision was made 3 mm above the margin of the upper eyelid. Part of the superior rectus muscle was cut to expose the ON. The ON was grasped vertically 2 mm from the globe with a small arterial clamp for 10 s, avoiding injury to the ophthalmic artery. The ON of the sham-operated group was exposed but not crushed. The normal control was a healthy ON from a rat that had not undergone surgery.
4.3. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from the retinas using Trizol (Invitrogen, NY, USA). The reverse transcription reaction and qRT-PCR of miR-30b were carried out using a Taqman MicroRNA Reverse Transcription Kit and gene-specific primers/probes (Assay ID: 000602; Applied Biosystems, NY, USA). The reactions were performed according to the company's instructions. A reverse transcription kit (Bio-Rad Laboratories, Inc., CA, USA) was used for the reverse transcription of Sema3A. The primers for Sema3A were 50 -AGCACAGCTTCCTCTACACC-30 (forward) and 50 -TCTCTGTGACTTCGGACTGC-30 (reverse). The primers for β-actin were 50 -TCAGGTCATCACTATCGGCAAT-30 (forward) and 50 -AAAGAAAGGGTGTAAAACGCA-30 (reverse). qRT-PCR was carried out using the SYBR-green method (TOYOBO, Japan). The reaction conditions were prepared according to the manufacturer's instructions.
4.4.
Immunoblotting
Proteins were extracted from the cultured RGCs and the retinas of the rats using RIPA buffer. Then protein samples were loaded onto denaturing 10% sodium dodecyl sulfate (SDS) polyacrylamide gels, subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes. Each membrane was then incubated in 5% skim milk diluted with Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature, followed by overnight incubation with the appropriate primary antibodies (anti-Sema3A [1:200], antiNRP1 [1:200], anti-PlexA1 [1:200], anti-p-p38MAPK [1:500], antiactive caspase-3 [1:500], or anti-β-actin [1:2000], diluted in TBST with 5% skim milk). The membrane was then incubated for 1 h at room temperature with rabbit–anti-goat or goat–anti-rabbit secondary antibodies conjugated with horseradish peroxidase
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(HRP; 1:5000, IgG). The HRP signals were visualized using a Pierce ECL-Plus kit (Thermo Fischer Scientific, IL, USA) according to the manufacturer's instructions. Finally, the membranes were exposed to an autoradiography film.
4.5.
Immunohistochemistry
Rats were anesthetized by intraperitoneal injections of pentobarbital sodium (30 mg/kg) and then perfused with 4% paraformaldehyde. The eyeballs of the rats were surgically removed and fixed with 4% paraformaldehyde for 24 h at 4 1C. Serial 3-μm sections of the eyeball were cut from paraffin blocks. The sections were incubated with primary antibodies (anti-Sema3A at 1:150) at 4 1C overnight. The sections were washed three times with PBS and then incubated with a goat– anti-rabbit secondary antibody (1:200) for 30 min at 37 1C. After three washes with PBS, the sections were visualized using the avidin–biotin complex (ABC, Beijing ZSGB-BIO Co. Ltd.) technique and counterstained with hematoxylin.
4.6.
Dual-luciferase reporter assay
The Sema3A 30 UTR was amplified by polymerase chain reaction (PCR) using rat cDNA as the template, and then subcloned in the pMIR-RTPORT miRNA expression vector (Shanghai SBO Medical Biotechnology Co. Ltd.) to generate WT-Sema3A. The mutation (lack of UGUUUACA) was introduced into the possible miR-30b binding site located in the Sema3A 30 UTR, and then sub-cloned into the pMIR-REPORT miRNA expression vector to generate MT-Sema3A. HEK293 cells were cultured in 24-well plates and transfected with 200 ng WT-Sema3A or MT-Sema3A together with 8 ng pRLCMV (Promega, WI, USA) containing Renilla luciferase and 10 μM synthetic miR-30b mimic or miRNA NC using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the dual-luciferase reporter assay system (Promega). The Renilla luciferase values were normalized to firefly luciferase values.
4.7.
RGC culture and axonal outgrowth assay
SD rats less than 24 h old were sterilized in 75% ethanol and their eyes were enucleated. The retinas were dissociated under a microscope. The retinas were incubated in 0.25% trypsin solution for 30 min. Dulbecco's modified Eagle's medium (DMEM)/F12 containing 10% FBS was added for termination of the digestion. After filtration, DMEM/F-12 culture medium containing 10% FBS, 100 kU/L penicillin, and 100 mg/L streptomycin was added and mixed into a fine suspension. The cells were then seeded on a 24-well plate that was pretreated overnight with poly-D-lysine. The cell density was adjusted to 5–6 105 cells/ml, and they were maintained in an incubator (37 1C, 5% CO2). The RGCs were stained with anti-GAP-43 after 7 days. Sixty neurons from each experimental group were selected and the lengths of their axons were measured using Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA).
4.8.
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Virus production and purification
The miR-30b mimic, miR-30b inhibitor, and miRNA NC were synthesized by RiboBio Co. (Guangzhou, China). The sequences of the miR-30b mimic and miR-30b inhibitor were 30 -UCGACUCACAUCCUACAAAUGU-50 and 50 -AGCUGAGUGUAGGAUGUUUACA30 , respectively. Large-scale recombinant adeno-associated virus (rAAV) production and purification were performed by SBO Medical Biotechnology Co. (Shanghai, China).
4.9.
Virus transduction and intra-ocular injections
The rAAV-miR-30b mimic (1.0 1010 vg), rAAV-miR-30b inhibitor (1.0 1010 vg), rAAV-miRNA NC (1.0 1010 vg), or PBS was injected into the vitreous after the ON was damaged. The injections were made through the sclera, thereby avoiding injury to the lens and retina.
4.10.
siRNA production and transfection
Three small interfering RNA (siRNA) duplexes targeting Sema3A were used: siRNA1: 50 -CCAACAAUGUGAUCACUUUdTdT-30 ; siRNA2: 50 -GCAAUGGAGCUUUCUACUA-dTdT-30 ; and siRNA3: 50 -GGAAGUCAUCGAUACAGAA-dTdT-30 . BLAST analysis revealed that these three sequences exhibited no homology to any rat genes other than Sema3A. RNA duplexes were transfected into RGCs at 24 h after incubation using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). The rAAV-miR-30b mimic or the rAAV-miR-30b inhibitor was added to the cultured RGCs at 5 d post-plating. Total proteins were extracted from the cultured RGCs at 9 d post-plating.
4.11.
Flow cytometric analysis of apoptosis
The rAAV-miR-30b mimic (1.0 104 vg/cell), rAAV-miR-30b inhibitor (1.0 104 vg/cell), rAAV-miRNA NC (1 104 vg/cell), or PBS was added to RGCs at 24 h post-incubation. After the cells were cultivated for 5 days, 1 106 cells were collected, stained with Annexin V-FITC and propidium iodide (IM3546, Beckman Coulter, CA, USA), and subjected to flow cytometric analysis (Beckman Coulter) to quantify rates of apoptosis.
4.12.
Statistical analysis
SPSS 13.0 (IBM, IL, USA) software was used for the statistical analyses. The results are expressed as mean7SEM. Multiplegroup statistical analyses were performed using one-way ANOVA, followed by least significant difference post hoc tests. Po0.05 was considered statistically significant. All experiments were repeated at least three times.
Acknowledgments We thank professor Yuanguo Zhou for his instruction to the study. This study was supported by the National Nature Science Foundation of China (No. 81371006) and the National Natural Science Foundation of China (No. 31070968).
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Appendix A.
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Supplementary materials
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.03.014.
references
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Research Report
MicroRNA-30b promotes axon outgrowth of retinal ganglion cells by inhibiting Semaphorin3A expression F. Han, Y. Huo, C.-J. Huang, C.-L. Chen, J. Yen Department of Ophthalmology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China
art i cle i nfo
ab st rac t
Article history:
Semaphorin3A (Sema3A) is a major inhibitory factor of optic nerve (ON) regeneration post-
Accepted 6 March 2015
injury. Many microRNAs (miRNAs) are expressed specifically in the mammalian brain and
Available online 17 March 2015
retina and are dynamically regulated during development, suggesting that this group of
Keywords:
miRNAs may be associated with neural development. We found that microRNA-30b (miR-
miR-30b
30b) bound to the three prime untranslated region (30 UTR) of Sema3A and inhibited the
Sema3A
expression of Sema3A mRNA. The mRNA expression level of miR-30b and the protein
RGCs
expression levels of Sema3A, Neuropilin1 (NRP1), PlexinA1 (PlexA1), phosphorylated
Regeneration
p38MAPK (p-p38MAPK), and active caspase-3 were all upregulated in retinas from rats with a damaged ON relative to those with an intact ON. Transfection of cultured retinal ganglion cells (RGCs) with an miR-30b mimic led to decreased levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression, as well as axon elongation and reduced levels of apoptosis. These findings provide evidence that miR-30b inhibits Sema3A expression. Decreased Sema3A expression promotes axon outgrowth in RGCs due to reduced levels of Sema3A binding to NRP1 and PlexA1 and simultaneously reduces apoptosis by inhibiting the p38MAPK and caspase-3 pathways. Our findings provide the first evidence that miR-30b-mediated Sema3A downregulation may serve as a new strategy for the clinical treatment of ON injury. & 2015 Elsevier B.V. All rights reserved.
1.
Introduction
of the most significant problems associated with CNS regeneration include glial scar formation, neurotrophic factor
The optic nerve (ON) is a part of the central nerve system
deficits, and the presence of inhibitory proteins. Among
(CNS). The axons of retinal ganglion cells (RGCs) comprise the
them, the inhibitory influences are the most important
ON. The regeneration and functional recovery of a damaged
contributing factors (Fawcett, 2009; Tuszynski and Gage,
ON remain challenging due to many regulatory factors. Some
1995). Semaphorin3A (Sema3A) is a very potent repulsive
n
Corresponding author. Fax: þ86 23 68767066. E-mail address: [email protected] (J. Ye).
http://dx.doi.org/10.1016/j.brainres.2015.03.014 0006-8993/& 2015 Elsevier B.V. All rights reserved.
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brain research 1611 (2015) 65–73
axon guidance cue and an important inhibitory factor involved in CNS repair following damage (Luo et al., 1993). Sema3A binds to the Neuropilin1 (NRP1) receptor and then associates with the PlexinA1-A4 (PlexA1-A4) receptor to transmit a signal that induces growth cone collapse (He and Tessier-Lavigne, 1997; Rosenzweig et al., 2010; Takahashi et al., 1999; Zylbersztejn et al., 2012). Sema3A activates p38MAPK phosphorylation and promotes neural progenitor cell apoptosis (Bagnard et al., 2004). Furthermore, Sema3A can reduce myelin lipid phagocytosis by macrophages, which in turn inhibits myelin regeneration of the CNS (Piaton et al., 2011; Syed et al., 2011) and blocks neurotrophic factor transport and recovery of the damaged nerves (Maione et al., 2009). MicroRNAs (miRNAs) are endogenous small molecules that reduce the expression levels of proteins by binding to the three prime untranslated region (30 UTR) of the targeted mRNAs and inhibiting or degrading the mRNAs (Luciano, 2004; Vermeulen et al., 2007), allowing them to regulate cell proliferation, differentiation, and apoptosis. Many miRNAs are expressed specifically in the mammalian brain and retina and are dynamically regulated during development, suggesting that this group of miRNAs may be associated with neural development (Arora et al., 2007, 2010; Krichevsky et al., 2006; Miska et al., 2004; Sempere et al., 2004). Further investigation into Sema3A and retina-associated miRNAs may provide a new window of opportunity for exploring the treatment of ON damage via Sema3A inhibition. After searching the public databases using prediction algorithms, such as TargetScan, miRanda, and Pictar, we identified microRNA-30b (miR-30b) as a candidate modulator of Sema3A expression and a potential mechanism for targeting Sema3A expression.
expression was detected in the inner nuclear layer (INL) and the ganglion cell layer (GCL) (Fig. 1c). Sema3A expression levels in the INL and GCL increased significantly at 7 d in retinas associated with ON injury compared to control retinas.
2.2.
Following ON damage, rAAV-miR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS was injected into the vitreous. The expression levels of miR-30b and Sema3A were analyzed at 3 d and 7 d post-injury. As predicted, miR-30b mRNA expression levels were higher in the miR-30b mimic group and lower in the miR-30b inhibitor group than in the other three groups. In contrast, the expression patterns of Sema3A mRNA were somewhat unexpected. The expression levels of miR-30b mRNA decreased by approximately 5-fold (Po0.01, Fig. 2a) and 10-fold (Po0.05, Fig. 2a) in the miR-30b inhibitor group relative to the PBS group at 3 d and 7 d post-injury, respectively, but the expression levels of Sema3A mRNA increased by only approximately 4-fold (Po0.01, Fig. 2a) and 2-fold (Po0.01, Fig. 2a) in the miR-30b inhibitor group compared to the PBS group at 3 d and 7 d postinjury, respectively. Although the expression levels of miR-30b mRNA increased by approximately 3-fold (Po0.01, Fig. 2a) and 23-fold (Po0.01, Fig. 2a) in the miR-30b mimic group relative to the PBS group at 3 d and 7 d post-injury, respectively, the expression levels of Sema3A mRNA decreased by approximately 3-fold (Po0.05, Fig. 2a) and 5-fold (Po0.01, Fig. 2a) in the miR-30b mimic group compared to the PBS group at 3 d and 7 d postinjury, respectively. At 3 d and 7 d post-injury, the miR-30b mimic group had the lowest levels of Sema3A protein expression, and the miR-30b inhibitor group had the highest levels of Sema3A protein expression (Fig. 2b).
2.3.
2.
Sema3A is regulated by miR-30b in vivo
Sema3A is the target gene of miR-30b
Results
2.1. Analysis of the expression levels of miR-30b, Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 in the retinas of rats with or without ON injury The results of qRT-PCR experiments showed that miR-30b was expressed in the retinas of normal control rats. After the ON of the left eye was damaged with an arterial clamp, miR-30b expression levels in the left retina increased and peaked at 3 d relative to miR-30b expression levels in control retinas (Po0.01, Fig. 1a). However, the peak in miR-30b expression levels in retinas associated with ON injury declined rapidly at 7 d, after which there was no significant difference between miR-30b expression levels in the post-injury and control retinas. Sema3A mRNA expression levels peaked at 7 d post-injury and declined quickly in retinas associated with ON injury, but remained higher than in control retinas (Po0.05, Fig. 1a). Sema3A protein expression levels increased significantly in retinas associated with ON injury relative to control retinas, reaching a peak value at 7 d and slowly declining at subsequent time points (Fig. 1b). The protein expression levels of NRP1, PlexA1, p-p38MAPK, and active caspase-3 also increased significantly in retinas associated with ON injury relative to control retinas (Fig. 1b). There was no significant change in their expression levels at any time point in all sham retinas. In the undamaged retina, Sema3A protein
The miR-30b binding site located in the Sema3A 30 UTR has the sequence UGUUUACA (Fig. 3a), which was identified by searching public databases (TargetScan) using prediction algorithms. Results of the dual-luciferase reporter assay showed that the relative luciferase activity was significantly lower in the treatment group relative to the control or mutation groups (Po0.01, Fig. 3b). In vitro cultures of rat RGCs were transfected with rAAV-miR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS. Seven days later, the changes in NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression levels were similar to those of Sema3A, i. e., lower protein expression levels were detected in the miR30b mimic group relative to the remaining three groups, and higher protein expression levels were detected in the miR-30b inhibitor group relative to the remaining three groups (Fig. 3c). Three different Sema3A siRNAs were used to examine the potential functions of miR-30b. The efficacy of siRNAmediated inhibition of Sema3A expression was determined in rat RGCs by immunoblotting at 5 d after plating. The RGCs transfected with siRNA2-Sema3A exhibited the most efficient knockdown of Sema3A expression (Fig. 3d). Therefore, this siRNA was selected for use in subsequent experiments. Rat RGCs were transfected with siRNA2-Sema3A at 24 h after plating. Subsequently, the RGCs were transfected with
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Fig. 1 – Expression patterns of miR-30b, Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 in the rat retina are shown. (a) miR-30b and Sema3A expression levels were measured by qRT-PCR: retinas from normal rats (control), or retinas associated with an injured left ON (Treatment) or with a sham-operated right ON (Sham) at 1, 3, 7, 14, and 21 days post-operation. (Mean7SEM, n¼ 6, One-way ANOVA, *Po0.05, **Po0.01). (b) Immunoblot analysis of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression in retinas from normal rats (control) or in retinas from rats with a damaged ON (Treatment) or a sham-operated ON (Sham) at 1, 7, 14, and 21 days post-operation. (c) In the retina, Sema3A protein expression was detected in the inner nuclear layer (INL) and the ganglion cell layer (GCL) using immunohistochemistry. IPL: inner plexiform layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PR: photoreceptors. Scale bar: 40 μm. rAAV-miR-30b mimic or rAAV-miR-30b inhibitor at 5 d after plating. Total proteins were extracted at 9 d after plating, and the expression levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 were not significantly different between the rAAV-miR-30b mimic and the rAAV-miR-30b inhibitor groups (Fig. 3e). These results provide evidence that Sema3A is the target gene of miR-30b.
The rate of apoptosis was the lowest in the miR-30b mimic group and highest in the miR-30b inhibitor group (Po0.05, Fig. 4b). These results showed that overexpression of the miR-30b mimic significantly inhibits RGC apoptosis. The length of RGC axons was also determined for the four groups. RGC axon length was longest in the miR-30b mimic group and shortest in the miR-30b inhibitor group at 7 d after plating (Po0.01, Fig. 5).
2.4. Apoptotic rate and axon length of RGCs after miR-30b transfection
3. In order to further characterize the role of miR-30b, the levels of apoptosis were determined by flow cytometry in RGCs at 5 d after plating. The apoptotic cells were identified as being Annexin V-positive and propidium iodide-negative (Fig. 4a).
Discussion
Sema3A is an important repulsive axon guidance cue that plays a crucial role in CNS regeneration and recovery. In a previous report (Shirvan et al., 2002), RGCs underwent
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Fig. 2 – Analysis of miR-30b and Sema3A expression following transfection with miRNA negative control (NC), miR-30b mimic, or miR-30b inhibitor in vivo. Immediately after the ON was damaged, PBS, rAAV-miRNA NC, rAAV-miR-30b mimic, or rAAVmiR-30b inhibitor was injected into the vitreous. The expression levels of miR-30b and Sema3A were analyzed at 3 d and 7 d post-injury. (a) Results of qRT-PCR experiments to examine miR-30b and Sema3A mRNA expression levels. (Mean7SEM, n¼ 6, One-way ANOVA, *Po0.05, **Po0.01). (b) Results of immunoblot experiments to examine Sema3A protein expression levels.
Fig. 3 – MiR-30b directly targets the Sema3A gene. (a) The miR-30b binding site in the 30 UTR of Sema3A was predicted by searching the public database (TargetScan). (b) Cultured HEK293 cells were co-transfected with a luciferase reporter containing the wild-type Sema3A 30 UTR (wild-type pMIR-REPORT-Sema3A 30 UTR [WT-Sema3A]) together with either the miRNA NC (control group) or with the miR-30b mimic (treatment group), or they were co-transfected with a luciferase reporter containing a mutant version of the Sema3A 30 UTR (MT-Sema3A) together with the miR-30b mimic (mutant group). Luciferase activities were measured and the results were compared between the three groups (Mean7SEM, n¼ 4, One-way ANOVA, **Po0.01). (c) In vitro cultures of rat RGCs were transfected with rAAV-miR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS at 24 h after plating. Protein expression levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 were measured at 7 d after the initial plating by immunoblotting. (d) Rat RGCs were transfected with one of three different siRNA duplexes targeting Sema3A (siRNA1-Sema3A, siRNA2-Sema3A, or siRNA3-Sema3A) or with siRNA-NC at 24 h after plating. Sema3A protein expression levels were determined at 5 d post-plating by immunoblotting. (e) Rat RGCs were transfected with siRNA2Sema3A at 24 h after plating, followed by transfection with the rAAV-miR-30b mimic or rAAV-miR-30b inhibitor at 5 d postplating. Protein expression levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 were determined at 9 d postplating by immunoblotting. apoptosis following intravitreal injection of Sema3A. Injection of anti-Sema3A antibodies blocked RGC apoptosis after the axon microsurgery. Blocking Sema3A signaling has also
been shown to reduce the collapse of the dorsal root ganglion growth cone following spinal cord injury, and to promote the regeneration of the corticospinal tract (Kikuchi et al., 2003).
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Fig. 4 – Overexpression of miR-30b inhibits RGC apoptosis. Rat RGCs were transfected with rAAV-miR-30b mimic, rAAV-miR30b inhibitor, rAAV-miRNA NC, or PBS after 24 h. Rates of apoptosis were determined by flow cytometry at 5 d post-plating. (Mean7SEM, n ¼ 3, One-way ANOVA, *Po0.05, **Po0.01).
Fig. 5 – Representative images of RGCs with GAP-43 staining in the indicated groups. Rat RGCs were transfected with rAAVmiR-30b mimic, rAAV-miR-30b inhibitor, rAAV-miRNA NC, or PBS after 24 h. The RGCs were stained with anti-GAP-43 antibodies and axon lengths were analyzed at 7 d post-plating. (Mean7SEM, One-way ANOVA, **Po0.01). Scale bar: 80 μm. Therefore, inhibition of Sema3A not only improves the survival of damaged neurons in the CNS, but also promotes axon elongation. This is beneficial for promoting axon connectivity and for the functional reconstruction of damaged nerves (Kaneko et al., 2006). Following ON damage induced by transection injury in rats, Sema3A expression in RGCs significantly increased and peaked at day 3 post-injury (Nitzan et al., 2006; Shirvan, 2002). In our study, Sema3A expression peaked at day 7 following ON damage induced by crush injury. The different results may be due to the fact that transection injuries are more serious than crush injuries. Previous studies have shown that miR-30b is involved in physiological and pathological processes in a variety of tissues (Altmae et al., 2012; Balderman et al., 2012; Gaziel-Sovran et al., 2011; Le Guillou et al., 2012). It is worth noting that aberrant expression of miR-30b has also been correlated with many neurological disorders (Mellios et al., 2012; Quintavalle et al., 2012; Song et al., 2011). Herein, we demonstrate that miR-30b regulates Sema3A expression in HEK293 cells by using a dualluciferase reporter assay, and further confirmed that miR-30b bound to Sema3A at the sequence UGUUUACA.
We found that miR-30b was expressed in the normal uninjured rat retina, and that its expression increased in response to ON damage. The increase in miR-30b expression promoted selfhealing in the animals after injury. However, the increase in miR-30b expression was only maintained for a short period of time. The expression of miR-30b decreased significantly at day 7 post-injury and showed no significant difference when compared to that of controls after this point. The decrease in miR-30b expression that occurred over time post-injury may be due to blocked autocrine or paracrine pathways associated with miR30b; however, further studies are needed to identify the specific mechanism. In contrast, Sema3A mRNA and protein expression levels were maintained at higher levels in the treatment group relative to controls and the regeneration of the nerve was inhibited. These results are supported by the findings of a previous study involving ON axotomy (Nitzan et al., 2006). Interestingly, despite the fact that Sema3A is the target gene of miR-30b, both showed increased expression following ON damage. Additional experiments showed that the changes in miR-30b and Sema3A expression were disproportionate following transfection of miR-30b in vivo (Fig. 2). This
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may be due to differences in secretory volumes of miR-30b and Sema3A at different times following ON damage. In addition, although miR-30b is a candidate regulator of Sema3A, it is not solely responsible for Sema3A regulation. Sema3A binds to the NRP1/PlexA1 receptor complex to inhibit nerve growth (Zylbersztejn et al., 2012). The binding of Sema3A to NRP1 induces a structural alteration in PlexA1 leading to the release of PlexA1 from a self-inhibited status and the initiation of downstream signaling, which in turn induces growth cone collapse and inhibits axon elongation (Antipenko et al., 2003; Takahashi et al., 1999). We found that the changes in NRP1 and PlexA1 expression patterns were the same as for Sema3A in the rat retina post-injury. After RGCs were transfected with the miR-30b mimic, reduced Sema3A expression was accompanied by reduced NRP1 and PlexA1 expression and significant elongation of RGC axons. The opposite effects were observed when RGCs were transfected with the miR-30b inhibitor. These results indicate that miR30b promotes axon elongation by inhibiting Sema3A and reducing the binding of Sema3A to NRP1 and PlexA1. The p38MAPK signaling pathway is involved in apoptosis in a variety of cell types (Sutter et al., 2003; Tamagno et al., 2003; Walton et al., 1998). Overexpression of proNGF can activate the p38MAPK signaling pathway, resulting in the phosphorylation of p38MAPK and induction of apoptosis in RGCs in diabetic rats (Al-Gayyar et al., 2011). In the ON crush model used in our study, the expression patterns of pp38MAPK and active caspase-3 were similar to that of Sema3A, suggesting that Sema3A induced apoptosis of RGCs via the p38MAPK signaling to activate caspase-3. These results are consistent with an earlier report (Nitzan et al., 2006). We found that overexpression of miR-30b in RGCs inhibited the expression of p-p38MAPK and active caspase-3, which were expressed at significantly lower levels than in the miR-30b inhibitor group. The apoptotic rate of RGCs showed the same results. These findings indicate that miR-30b reduces RGC apoptosis by inhibiting Sema3A and p38MAPK signaling via the downregulation of caspase-3. In conclusion, we found that miR-30b bound to the Sema3A 30 UTR. The expression profiles of miR-30b and Sema3A in the retinas of rats following ON damage were observed. We found that miR-30b inhibited Sema3A expression in RGCs, promoted axon outgrowth by reducing the binding capability of Sema3A to NRP1/PlexA1, and decreased RGC apoptosis by inhibiting p-p38MAPK and active caspase-3. Our findings provide a new strategy for the clinical treatment of ON damage and an experimental basis for gene therapy development. The in vivo effects of the miR-30b mimic and the recovery of function of damaged ONs should be a focus of further investigation.
4.
Experimental procedures
4.1.
Materials and animals
Antibodies against Sema3A (sc-28867), NRP1 (sc-7239), and PlexA1 (sc-25639) were purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Antibodies targeting GAP-43 (ab131482),
p-p38MAPK (ab47363), and active caspase-3 (ab2302) were purchased from Abcam (CA, USA). Neonatal Sprague–Dawley (SD) rats of either sex at postnatal day one and adult SD rats of either sex weighing 160– 200 g were provided by the Animal Experimental Center (Institute of Surgery Research, Daping Hospital, Third Military Medical University, China). The Animal Research Committee of the Third Military Medical University approved the study protocol.
4.2.
ON injury model in rats
The left eyes of the rats were selected as the ON crush models and were injured with an ON forceps, and the right eyes of the same rats were designated as the shams. Rats were anesthetized by intraperitoneal injections of pentobarbital sodium (30 mg/kg). A 1-cm horizontal incision was made 3 mm above the margin of the upper eyelid. Part of the superior rectus muscle was cut to expose the ON. The ON was grasped vertically 2 mm from the globe with a small arterial clamp for 10 s, avoiding injury to the ophthalmic artery. The ON of the sham-operated group was exposed but not crushed. The normal control was a healthy ON from a rat that had not undergone surgery.
4.3. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from the retinas using Trizol (Invitrogen, NY, USA). The reverse transcription reaction and qRT-PCR of miR-30b were carried out using a Taqman MicroRNA Reverse Transcription Kit and gene-specific primers/probes (Assay ID: 000602; Applied Biosystems, NY, USA). The reactions were performed according to the company's instructions. A reverse transcription kit (Bio-Rad Laboratories, Inc., CA, USA) was used for the reverse transcription of Sema3A. The primers for Sema3A were 50 -AGCACAGCTTCCTCTACACC-30 (forward) and 50 -TCTCTGTGACTTCGGACTGC-30 (reverse). The primers for β-actin were 50 -TCAGGTCATCACTATCGGCAAT-30 (forward) and 50 -AAAGAAAGGGTGTAAAACGCA-30 (reverse). qRT-PCR was carried out using the SYBR-green method (TOYOBO, Japan). The reaction conditions were prepared according to the manufacturer's instructions.
4.4.
Immunoblotting
Proteins were extracted from the cultured RGCs and the retinas of the rats using RIPA buffer. Then protein samples were loaded onto denaturing 10% sodium dodecyl sulfate (SDS) polyacrylamide gels, subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes. Each membrane was then incubated in 5% skim milk diluted with Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature, followed by overnight incubation with the appropriate primary antibodies (anti-Sema3A [1:200], antiNRP1 [1:200], anti-PlexA1 [1:200], anti-p-p38MAPK [1:500], antiactive caspase-3 [1:500], or anti-β-actin [1:2000], diluted in TBST with 5% skim milk). The membrane was then incubated for 1 h at room temperature with rabbit–anti-goat or goat–anti-rabbit secondary antibodies conjugated with horseradish peroxidase
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(HRP; 1:5000, IgG). The HRP signals were visualized using a Pierce ECL-Plus kit (Thermo Fischer Scientific, IL, USA) according to the manufacturer's instructions. Finally, the membranes were exposed to an autoradiography film.
4.5.
Immunohistochemistry
Rats were anesthetized by intraperitoneal injections of pentobarbital sodium (30 mg/kg) and then perfused with 4% paraformaldehyde. The eyeballs of the rats were surgically removed and fixed with 4% paraformaldehyde for 24 h at 4 1C. Serial 3-μm sections of the eyeball were cut from paraffin blocks. The sections were incubated with primary antibodies (anti-Sema3A at 1:150) at 4 1C overnight. The sections were washed three times with PBS and then incubated with a goat– anti-rabbit secondary antibody (1:200) for 30 min at 37 1C. After three washes with PBS, the sections were visualized using the avidin–biotin complex (ABC, Beijing ZSGB-BIO Co. Ltd.) technique and counterstained with hematoxylin.
4.6.
Dual-luciferase reporter assay
The Sema3A 30 UTR was amplified by polymerase chain reaction (PCR) using rat cDNA as the template, and then subcloned in the pMIR-RTPORT miRNA expression vector (Shanghai SBO Medical Biotechnology Co. Ltd.) to generate WT-Sema3A. The mutation (lack of UGUUUACA) was introduced into the possible miR-30b binding site located in the Sema3A 30 UTR, and then sub-cloned into the pMIR-REPORT miRNA expression vector to generate MT-Sema3A. HEK293 cells were cultured in 24-well plates and transfected with 200 ng WT-Sema3A or MT-Sema3A together with 8 ng pRLCMV (Promega, WI, USA) containing Renilla luciferase and 10 μM synthetic miR-30b mimic or miRNA NC using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the dual-luciferase reporter assay system (Promega). The Renilla luciferase values were normalized to firefly luciferase values.
4.7.
RGC culture and axonal outgrowth assay
SD rats less than 24 h old were sterilized in 75% ethanol and their eyes were enucleated. The retinas were dissociated under a microscope. The retinas were incubated in 0.25% trypsin solution for 30 min. Dulbecco's modified Eagle's medium (DMEM)/F12 containing 10% FBS was added for termination of the digestion. After filtration, DMEM/F-12 culture medium containing 10% FBS, 100 kU/L penicillin, and 100 mg/L streptomycin was added and mixed into a fine suspension. The cells were then seeded on a 24-well plate that was pretreated overnight with poly-D-lysine. The cell density was adjusted to 5–6 105 cells/ml, and they were maintained in an incubator (37 1C, 5% CO2). The RGCs were stained with anti-GAP-43 after 7 days. Sixty neurons from each experimental group were selected and the lengths of their axons were measured using Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA).
4.8.
71
Virus production and purification
The miR-30b mimic, miR-30b inhibitor, and miRNA NC were synthesized by RiboBio Co. (Guangzhou, China). The sequences of the miR-30b mimic and miR-30b inhibitor were 30 -UCGACUCACAUCCUACAAAUGU-50 and 50 -AGCUGAGUGUAGGAUGUUUACA30 , respectively. Large-scale recombinant adeno-associated virus (rAAV) production and purification were performed by SBO Medical Biotechnology Co. (Shanghai, China).
4.9.
Virus transduction and intra-ocular injections
The rAAV-miR-30b mimic (1.0 1010 vg), rAAV-miR-30b inhibitor (1.0 1010 vg), rAAV-miRNA NC (1.0 1010 vg), or PBS was injected into the vitreous after the ON was damaged. The injections were made through the sclera, thereby avoiding injury to the lens and retina.
4.10.
siRNA production and transfection
Three small interfering RNA (siRNA) duplexes targeting Sema3A were used: siRNA1: 50 -CCAACAAUGUGAUCACUUUdTdT-30 ; siRNA2: 50 -GCAAUGGAGCUUUCUACUA-dTdT-30 ; and siRNA3: 50 -GGAAGUCAUCGAUACAGAA-dTdT-30 . BLAST analysis revealed that these three sequences exhibited no homology to any rat genes other than Sema3A. RNA duplexes were transfected into RGCs at 24 h after incubation using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). The rAAV-miR-30b mimic or the rAAV-miR-30b inhibitor was added to the cultured RGCs at 5 d post-plating. Total proteins were extracted from the cultured RGCs at 9 d post-plating.
4.11.
Flow cytometric analysis of apoptosis
The rAAV-miR-30b mimic (1.0 104 vg/cell), rAAV-miR-30b inhibitor (1.0 104 vg/cell), rAAV-miRNA NC (1 104 vg/cell), or PBS was added to RGCs at 24 h post-incubation. After the cells were cultivated for 5 days, 1 106 cells were collected, stained with Annexin V-FITC and propidium iodide (IM3546, Beckman Coulter, CA, USA), and subjected to flow cytometric analysis (Beckman Coulter) to quantify rates of apoptosis.
4.12.
Statistical analysis
SPSS 13.0 (IBM, IL, USA) software was used for the statistical analyses. The results are expressed as mean7SEM. Multiplegroup statistical analyses were performed using one-way ANOVA, followed by least significant difference post hoc tests. Po0.05 was considered statistically significant. All experiments were repeated at least three times.
Acknowledgments We thank professor Yuanguo Zhou for his instruction to the study. This study was supported by the National Nature Science Foundation of China (No. 81371006) and the National Natural Science Foundation of China (No. 31070968).
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Appendix A.
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Supplementary materials
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.03.014.
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