lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth

Accepted Manuscript lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth Hao-ran Lv PII:

S0006-291X(17)31276-7

DOI:

10.1016/j.bbrc.2017.06.145

Reference:

YBBRC 38051

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 19 June 2017 Accepted Date: 23 June 2017

Please cite this article as: H.-r. Lv, lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.145. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth

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Hao-ran Lv

Department of Traumatic Orthopedics, the Fifth Affiliated Hospital of GuangZhou

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Medical University, GuangZhou 510700, Guangdong Province, China

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Corresponding author: 621# Harbour Road, Whampoa District, Guangzhou 510700, P R China. Tel. +86-020-82285645; Fax, +86-020-82285645.

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E-mail: [email protected].

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Running title: lncRNA-Map2k4 regulates the miR-199a/FGF1 pathway in neuron.

Key words: Spinal cord injury; neuron; lncRNA; lncRNA-Map2k4; miR-199a;

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FGF1.

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Abstract Spinal cord injury (SCI) is a common critical illness in clinical practice. SCI prevention, treatment and rehabilitation have become important topics in today's

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medical profession. Studies have shown that long noncoding RNAs (lncRNAs) also play an important role in the pathology of SCI. The biology software analysis identified miR-199a binding sites in the lncRNA-Map2k4 and FGF1 sequences,

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which were confirmed by the subsequent dual luciferase reporter assay. When

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lncRNA-Map2k4 expression was down-regulated by siRNA, miR-199a expression in neurons was up-regulated and FGF1 expression was down-regulated. In turn, miR-199a up-regulation inhibited lncRNA-Map2k4 and FGF1 expression. But when lncRNA-Map2k4-m (a lncRNA-Map2k4 overexpression vector with mutated

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miR-199a binding sites) was co-transfected into neuronal cells with miR-199a mimics, lncRNA-Map2k4-m over-expression did not block the inhibition of FGF1 expression by miR-199a. Moreover, lncRNA-Map2k4 and FGF1 promoted the proliferation and

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inhibited the apoptosis of neuronal cells, whereas miR-199a down-regulated the functions

of

lncRNA-Map2k4

and

FGF1;

however,

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aforementioned

lncRNA-Map2k4-m could not block the inhibitory action of miR-199a on proliferation. Thus, lncRNA-Map2k4 regulates neuronal proliferation and apoptosis through a miR-199a/FGF1 pathway. This finding provides more evidence for the role of lncRNAs in SCI.

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Introduction Spinal cord injury (SCI) is a serious traumatic disease that occurs in the central nervous system. The morbidity is high, and the incidence increases yearly [1]. The

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effective control of SCI is a difficult problem in spinal surgery and neuroscience research.

Recent studies have shown that long noncoding RNAs (lncRNAs) are involved

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in the development of neurological diseases at many levels, including brain

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development, neuronal differentiation and functional retention, and neuronal cell apoptosis [2]. The results of Ding et al. [3] indicated for the first time that LncRNAs were differentially expressed in a contusion SCI mouse model. They found that lncRNA ENSMUST00000138093 (which we named lncRNA-Map2k4) had high

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expression level and declined in the SCI pathology, and its expression level had a dynamic relation with fibroblast growth factor 1 (FGF1) mRNA expression [3]. FGF1 was a neurotrophic factor which had high expression level in gliocyte cell and a

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powerful neuroprotective and neuroregenerative factor of the nervous system [4].

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Therefore, lncRNA-Map2k4 may play a role through FGF1. Studies have shown that lncRNA functions are closely related to microRNAs

(miRNAs) [5]. For example, Cai et al. [6] found that ribonuclease P RNA component H1 (Rpph1) upregulated cell division cycle 42 (CDC42) expression and promoted hippocampal neuron dendritic spine formation by competing with miR-330-5p. miRNAs targeting lncRNA-Map2k4 and FGF1 were analyzed using biological software, which identified miR-199a as one of the most important miRNAs. Rane et 3

ACCEPTED MANUSCRIPT al. [7] reported that miR-199a participated in ischemic tolerance in cardiomyocytes by regulating hypoxia-inducible factor 1α (HIF-1 α) and Sirtuin (Sirt) 1 expression. miR-199a-5p has been reported to be specifically expressed in nerve tissues [8], and

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to participate in nerve regeneration, neurite outgrowth, and synaptic plasticity [9]. Therefore, miR-199a is an important miRNA in the nervous system, and lncRNA-Map2k4 may play a role through miR-199a.

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In this study, we investigated the regulation of lncRNA-Map2k4, miR-199a, and

mechanism of SCI.

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Materials and methods

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FGF1 in dorsal root ganglion neurons and provided new evidence for the molecular

Cell culture

Mouse Neurons-spinal cord were purchased from ScienCell Research Laboratories

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(Carlsbad, CA, USA), which were isolated from embryonic day 14 C57BL/6 mouse

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spinal cord. Neurons were cultured in neuronal medium (ScienCell) at 37°C in 5% CO2 incubator.

Vector construction and transfection Neurons were collected and RNA was extracted with Trizol (Thermo Fisher Scientific, USA), then reverse transcription PCR was used to amplify lncRNA-Map2k4. The forward primer was 5’-CGGGGTACCAGTATTCATCTAGAGATCTTCACAGG-3’ 4

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the

reverse

primer

was

5’-CCGCTCGAGATTACTACATCCAAATCCATGAGAAG-3’. The PCR products were digested with Kpn I and Xho I (TaKaRa, China), and then cloned into pcDNA3.1 (Thermo

Fisher

Scientific),

sequenced

and

verified.

The

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vector

pcDNA3.1-lncRNA-Map2k4 (lncRNA-Map2k4-O) plasmid was used to generate mutants (lncRNA-Map2k4-m-O, the miR-199a binding site was mutated) by mutagenesis.

lncRNA-Map2k4

siRNA

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PCR-based

FGF1

siRNA

(sense,

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5’-GGGAGAGTTAAACAGCTTT-3’),

(sense,

5’-AAGUGUUAUAAUGGUUUUCUU-3’), miR-199a mimics and inhibitors were purchased from GenePharma (China). Neurons were seed into 6-well plate at 1 × 105 cells/ml and incubated for 24 h. Transfection was carried out when cell confluence

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reached ~70% with Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. SiRNAs and miRNAs transfection were carried out with a concentration of 50 nM/well, and the concentration of plasmid for transfection was

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4 µg/well. Nontransfected cells were used as the control, and cells transfected with a

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blank vector and siRNA/miRNA negative controls were used as a negative control (NC). After 48 h, transfected cells were subjected to further analysis.

Dual luciferase assay Full-length

FGF1

3’UTR

5’-CCGCTCGAGAGGAGTCTGTTCTGAGTGTTC-3’;

(sense antisense

5’-ATAAGAATGCGGCCGCGATAAGGTGCAGAGTGTAATACAG-3’)

primer, primer, and 5

ACCEPTED MANUSCRIPT lncRNA-Map2k4

(sense

primer,

5’-CCGCTCGAGAGTATTCATCTAGAGATCTTCACAGG-3’;

antisense

primer,

5’-ATAAGAATGCGGCCGCATTACTACATCCAAATCCATGAGAAG-3’)

were

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amplified and cloned into psiCHECK-2 vector (Promega) using Xho I and Not I, resulting in the wild-type luciferase reporter construct. Mutants within the miR-199a binding site were created using the QuikChange II Site-Directed Mutagenesis Kit

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(Stratagene, CA, USA). Vectors and miR-199a mimics were cotransfected, and

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luciferase activity was measured using the Dual Glo Luciferase Assay System (Promega) following the manufacturer’s instructions. The experiment was performed in duplicate in three independent experiments.

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RNA extraction and quantitative real-time PCR (qRT-PCR) Neurons were collected and RNA was extracted using Trizol (Thermo Fisher Scientific) according to manufacturer's protocol. In order to detect the expression of

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miR-199a, cDNA was generated by using the miScript II RT Kit (Qiagen, Hilden,

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Germany) with the provided RT-U6 and miRNA-specific stem-loop primers. miRNA expression levels were determined by using the miScript SYBR Green PCR Kit (Qiagen). In addition, approximately 1 mg of total RNA was reversely transcribed into first-strand cDNA using a QuantiTect Reverse Transcription Kit (Qiagen). On an ABI Prism 7500 Sequence Detection System (Thermo Fisher Scientific), quantitative real time-PCR (qRT-PCR) was carried out to measure the expression of mouse FGF1 (sense

primer,

5’-TGTGATAAAGTGGAGTGAAGAG-3’;

antisense

primer, 6

ACCEPTED MANUSCRIPT 5’-GCTGCGAAGGTTGTGATC-3’)

and

5’-ATCTGCTTGGTGCCATTGAG-3’;

lncRNA-Map2k4

(sense

antisense

primer, primer,

5’-GCTCCTCGTCCAATTTCTCC-3’) using a Brilliant SYBR Green II qRT–PCR kit

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(Strategene, USA) according to manufacturer’s protocol. Fold changes of genes were then quantified using the 2-∆∆CT method and compared to the expression levels under

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control conditions.

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Western blot assay

Neurons were collected and lysed with a Tris-based RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1 protease cocktail; Sigma-Aldrich, USA). Total protein was electrophoresised on 8%

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SDS-PAGE and then transferred to nitrocellulose membranes (Roche Biosciences, Germany). The membranes were blocked by 5% non-fat dry milk and 1% BSA for 2 h, and incubated with anti-FGF1 (1:800, Abcam, USA) or anti-GAPDH (1:10000,

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Abcam) primary antibody overnight at 4 °C. After three washes, membranes were

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incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000, New England Biolabs, USA) for 2 h at room temperature, followed by blotting with an enhanced chemiluminescence system (Pierce, USA) according to manufacturer’s protocol.

RNA binding protein immunoprecipitation (RIP) assay RIP experiment was conducted using the EZ-Magna RIP Kit (Millipore, USA) 7

ACCEPTED MANUSCRIPT according to the manufacturer’s instructions. Briefly, neurons at 80-90% confluence were lysed in complete RIP lysis buffer, following by incubation with RIP buffer including magnetic beads coupled with anti-Argonaute-2 (Ago2) antibody (Abcam).

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Isotype-matched immunoglobulin G (IgG) was used as a negative control. After samples were incubated with proteinase K (Millipore), immunoprecipitated RNA was isolated. qRT-PCR was performed to analyze the levels of lncRNA-Map2k4 and

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miR-199a in the precipitates.

Cell proliferation

Cell proliferation was measured in 96 well plates using a colorimetric immunoassay based on the measurement of BrdU incorporation during DNA synthesis (BrdU

media

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ELISA kit, Roche Diagnostics, Germany). After transfection and incubation for 48 h, was

removed

and

cells

were

labeled

with

BrdU

(10

mM

5-bromo-2'-deoxyuridine) for 3 h at 37°C. Cells were fixed and incubated with

substrate

(3,3',5,5'-tetramethylbenzidine)

was

added,

and

BrdU

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peroxidase

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peroxidase-conjugated anti-BrdU antibody for 90 min at room temperature. Then, the

incorporation was quantitated by differences in absorbance at wavelength 370 minus 492 nm. Cell proliferation was expressed as the mean percentage of the control value (set at 100%).

Apoptosis assay Neuronal apoptosis was assayed by using a FITC-labelled Annexin V (Annexin 8

ACCEPTED MANUSCRIPT V-FITC) apoptosis detection kit (BD Biosciences, China). After transfection for 48 h, neurons were collected and washed twice with cold PBS and then resuspended in 1× binding buffer, followed by staining with Annexin V-FITC and propidium iodide (PI)

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in the darkness. The percentage of apoptotic cells was quantified by flow cytometry (BD FACSCalibur) according to the manufacturer’s instructions.

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Statistical analysis

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In our study, all experiments were repeated in triplicates and the averaged results were presented as mean ± standard error. Statistical analysis was performed using unpaired two-tail Student’s t-test on SPSS17.0 statistical software (Chicago, IL, USA).

Results

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Difference was significant if P < 0.05.

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lncRNA-Map2k4 is the target gene of miR-199a, and its down-regulation

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promotes miR-199a expression in neurons Emerging evidences have confirmed that lncRNAs might function as a competing endogenous RNA (ceRNA) or a molecular sponge in modulating miRNA [10]. The possible miRNA binding sites of lncRNA-Map2k4 was predicted using common bioinformatics algorithms (miRanda and RNAhybrid 2.2), and the analysis showed that miR-199a was one of the most important miRNAs (Figure 1A). Moreover, miR-199a expression was significantly up-regulated when lncRNA-Map2k4 9

ACCEPTED MANUSCRIPT expression was down-regulated by siRNA (Figure 1B). lncRNA-Map2k4 could inhibit the expression of miR-199a, but lncRNA-Map2k4-m (a lncRNA-Map2k4 overexpression vector with mutated miR-199a binding sites) could not (Figure 1B).

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To further investigate whether lncRNA-Map2k4 was a functional target of miR-199a, dual-luciferase reporter assay was performed. Our results showed that the luciferase activity was significantly decreased by the co-transfection of miR-199a mimics and

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lncRNA-Map2k4-Wt rather than the co-transfection of negative control (NC) miRNA

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and lncRNA-Map2k4-Wt (Figure 1C), suggesting that lncRNA-Map2k4 was the target of miR-199a. Meanwhile, co-transfection of miR-199a mimics and lncRNA-Map2k4-Mu (the miR-199a binding sites were mutated) did not change the luciferase activity (Figure 1C), suggesting that the miR-199a binding sites within

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lncRNA-Map2k4 was functional. Although the interaction between miR-199a and lncRNA-Map2k4 was confirmed, the biological behaviors of neurons regulated by

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miR-199a and lncRNA-Map2k4 need to be well confirmed.

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miR-199a targeted regulation of FGF1 expression in neurons The analysis found that FGF1 was an important miR-199a target gene (Figure 2A). Previous work using recombinant aFGF was able to increase the survival of neurons and increase the intrinsic capacity of mature neurons for regrowth [4]. Therefore, studies investigating the relationship and effects of miR-199a and FGF1 in spinal neurons are important. We confirmed the presence of a miR-199a binding site in the FGF1 gene using the dual luciferase reporter assay (Figure 2B). Furthermore, 10

ACCEPTED MANUSCRIPT miR-199a inhibited FGF1 expression in neurons, and FGF1 expression was up-regulated when miR-199a expression was down-regulated (Figure 2C).

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Knockdown of lncRNA-Map2k4 inhibited the FGF1 expression by up-regulating miR-199a

Of note, RIP experiment showed that compared with IgG pellets, lncRNA-Map2k4

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and miR-199a were enriched in the Ago2 pellet (Figure 3A). In line with luciferase

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assay, these data confirmed that lncRNA-Map2k4 is recruited to Ago2-related RNA-induced silencing complexes and functionally interacts with miR-199a. Upon transfection

of

neurons

with

synthetic

miR-199a

mimics,

expression

of

lncRNA-Map2k4 and FGF1 was inhibited (Figure 3B, C, D). Upon co-transfection of

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neurons with the lncRNA-Map2k4 over-expression vector and miR-199a mimics, inhibition of FGF1 expression was reduced relative to the group that was only transfected with miR-199a mimics (Figure 3C, D). Upon co-transfection of neurons

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with lncRNA-Map2k4-m (the miR-199a binding site was mutated) overexpression

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vector and miR-199a mimics, FGF1 expression was not inhibited (Figure 3C, D). Therefore, our data suggest that lncRNA-Map2k4 regulates FGF1 expression through miR-199a.

lncRNA-Map2k4 regulates neuron proliferation and apoptosis through an miR-199a/FGF1 pathway We found that both lncRNA-Map2k4 and FGF1 promoted neuron proliferation and 11

ACCEPTED MANUSCRIPT inhibited apoptosis, whereas miR-199a inhibited neuron proliferation and induced apoptosis (Figure 4). miR-199a was also shown to reduce lncRNA-Map2k4 and FGF1-induced neuron proliferation. Mutation of the miR-199a binding sites on

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lncRNA-Map2k4 (lncRNA-Map2k4-m) did not prevent miR-199a from inhibiting neuron proliferation (Figure 4). These data suggest that lncRNA-Map2k4 regulates

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neuron proliferation and apoptosis through an miR-199a/FGF1 pathway.

Discussion

By studying the biological functions of non-coding RNAs in development, normal physiology and disease, and investigating the regulatory mechanisms, we can

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elucidate the mechanisms underlying the pathogenesis and development of disease more comprehensively and provide insights for the search for new disease diagnostic markers and therapeutic targets [11].

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Recent studies have shown that lncRNAs are involved in the physiological

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functions of neurons [12-13]. For example, Chen et al. [12] indicated that lncRNA TUG1 sponges microRNA-9 to promote neurons apoptosis by up-regulated BCL2 like 11 (BCL2L11) under ischemia. However, limited research has investigated the roles of lncRNAs in SCI. Wang et al. [14] constructed a SCI rat model and detected a series of lncRNA expression changes. They further found that lncSCIR1 knockdown promoted astrocyte proliferation and migration in vitro [14]. Research by Zhang et al. [13] proved that inhibition of long non-coding RNA IGF2AS had profound effect on 12

ACCEPTED MANUSCRIPT inducing neuronal growth and protecting local-anesthetic induced neurotoxicity in dorsal root ganglion neurons. We studied lncRNA-Map2k4 based on the work of Ding et al. [3]. The results showed that lncRNA-Map2k4 promoted the growth of neurons.

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Therefore, a series of lncRNAs play important regulatory roles in the pathology of SCI.

The study also showed that miR-199a regulated lncRNA-Map2k4 expression and

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that lncRNA-Map2k4 expression affected miR-199a expression. Xu et al. [15] found

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that miRNA-199a inhibited Sirtl expression and increased the level of cerebral ischemia induced by 3-nitropropionic acid in rats. Wang et al. [16] indicated that miR-199a-5p expression was up-regulated, SIRT1 levels were decreased, and neuron loss and apoptosis were induced in epilepsy model rats compared with normal

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controls; In vivo knockdown of miR-199a-5p by an antagomir protected against neuron damage, in accordance with up-regulation of SIRT1 and subsequent deacetylation of p53. This work of Park et al. [17] recognized multidrug

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resistance-associated protein 1 (MRP1) as a protective factor against stroke, and the

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underlying mechanism identified MRP1 as a target of miR-199a-5p. Thus, miR-199a is a factor that promotes injury, and lncRNA-Map2k4 may play a role in regulating miR-199a.

We found that lncRNA-Map2k4 up-regulated the expression of its target gene

FGF1 by sequestering miR-199a to promote neuron proliferation and inhibit neuron apoptosis. FGF1, which is a multi-functional peptide growth factor, is mainly distributed in the central nervous system and has obvious nutritional support in 13

ACCEPTED MANUSCRIPT neurons. The functions of FGF1 in the occurrence and development of central nervous system degenerative diseases, such as Parkinson's disease and senile dementia disease, and in central nervous system injury repair have received increasing attention [18-19].

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Huang et al. [20] found that adeno-associated virus-mediated human acidic fibroblast growth factor expression promotes functional recovery of spinal cord-contused rats. And the existing clinical studies showed that the use of aFGF for spinal cord injury

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was safe and feasible in the present trial [21]. Therefore, FGF1 is essential for the

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survival of neurons and plays an important role in central nervous system injury repair. lncRNA-Map2k4 can regulate FGF1 expression, indicating that this lncRNA has an effect similar to FGF1 and may be an important neuroprotective lncRNA. Our findings suggest that lncRNA-Map2k4 can regulate neuron proliferation and

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apoptosis through an miR-199a/FGF1 pathway. This finding may provide targets and

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Figure legends

Figure 1. miR-199a expression and its binding sites in lncRNA-Map2k4. (A)

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Conserved miR-199a binding sites in lncRNA-Map2k4. (B) After transfection with lncRNA-Map2k4 siRNA, pcDNA3.1-lncRNA-Map2k4 (lncRNA-Map2k4-O) and pcDNA3.1-lncRNA-Map2k4-m

(lncRNA-Map2k4-m-O,

a

lncRNA-Map2k4

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overexpression vector with mutated miR-199a sites) respectively, miR-199a

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expression levels were measured by qRT-PCR. Control, cells were not transfected; NC, cells were transfected with pcDNA3.1 empty vector and NC siRNA. (C) Luciferase expression level in cells transfected with cloned lncRNA-Map2k4 wt or mu vector and miR-199a mimics. Each column represents the mean ± SD of 3

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samples. * P < 0.05 vs. control.

Figure 2. Prediction and validation of miR-199a target gene. (A) miR-199a target

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sites in the conserved sequence of FGF1 3' UTR. (B) Luciferase expression level in

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cells transfected with cloned FGF1 wt or mu 3' UTR vector and miR-199a mimics. (C) After transfection with NC miRNA, miR-199a mimics and inhibitors respectively, miR-199a and FGF1 expression levels were measured by qRT-PCR. All values are the means ± SDs (*P < 0.05 vs. Control).

Figure 3. miR-199a, lncRNA-Map2k4 and FGF1 expression in and its binding sites in neurons. (A) Association of lncRNA-Map2k4 and miR-199a with Ago2 was 18

ACCEPTED MANUSCRIPT detected by RIP experiment. Relative levels of lncRNA-Map2k4 and miR-199a were expressed as fold enrichment in Ago2 relative to IgG immunoprecipitates by qRT-PCR. (B) After transfection, lncRNA-Map2k4 expression levels were measured

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by qRT-PCR. (C, D) After transfection, FGF1 expression levels in neurons were assessed by western blots. Each bar represents the mean ± SD of three samples (*P <

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0.05 vs. Control).

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Figure 4. lncRNA-Map2k4 regulated neuron proliferation and apoptosis by promoting the expression of miR-199a target gene. (A) Proliferation measured as BrdU assay in neurons. (B) Representative density plots showing expression of annexin V in apoptotic neurons after transfection. (C) Graphic representation of the

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control.

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data shown in (B). Each column represents the mean ± SD of 3 samples. P < 0.05 vs.

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Highlights lncRNA-Map2k4 is the target gene of miR-199a, and its

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down-regulation promotes miR-199a expression in neurons.

miR-199a targeted regulation of FGF1 expression in neurons.

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Knockdown of lncRNA-Map2k4 inhibited the FGF1 expression by

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up-regulating miR-199a.

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lncRNA-Map2k4 regulates neuron proliferation and apoptosis

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through an miR-199a/FGF1 pathway.