effects of mir-541 on neurite outgrowth during neuronal differentiation

Effects of miR-541 on neurite outgrowth during neuronaldifferentiation

Jun Zhang 1*,y, Jing Zhang 2y, Li-hong Liu 2, Yang Zhou1, Yi-ping Li 1, Zhi-hua Shao 1, You-jun Wu2,Meng-jie Li 2, Ying-ying Fan 1 and Hong-jun Shi 1

1Department of Regenerative Medicine, Tongji University School of Medicine, Shanghai, China2School of Life Science and Technology, Tongji University, Shanghai, China

MicroRNA (miRNAs) are short non-coding RNA molecules that downregulate gene expression at post-transcriptional level. miRNAs arepost-transcriptional regulators of gene expression important for neuron development and function. This report demonstrated that a putativeand chemically synthesized miRNA rno-mir-541 played an important role in the neuron development. Differentiation of PC12 cells with nervegrowth factor (NGF) is associated with neurite outgrowth, a process that involves upregulation of Synapsin I. We predicted, detected andassessed the expression levels of a number of possible miRNAs for synapsin I in rats and our outcomes showed that rno-mir-541 wasassociated with rat synapsin I expression. miR-541, a brain specificmiRNA, plays an important role in repressing neurite extension in culturedPC12 neurons. The neurites of PC12 cells was shortened drasticly as a result of the overexpression of rno-mir-541. In contrast, the neurites ofPC12 cell developed well after the knockdown of rno-mir-541 by RNA interference. Our study showed that rno-mir-541 played an importantrole in neuron-cell proliferation and neurite outgrowth through suppressing the expression of its target gene synapsin I. Furthermore, theintroduction of NGF causes downregulation of miR-541, de-repression of its target, Synapsin-I and allows for neuritogenesis. Thus, miR-541functions in neuronal precursors as an endogenous conditional component between NGF and Synapsin-I. Copyright # 2011 John Wiley &Sons, Ltd.

key words—miRNA; Synapsin I; rno-mir-541; PC12; neurite

INTRODUCTION

MicroRNAs (miRNAs) are small endogenous non-codingRNA with a length of 19–23 nt, which can negativelyregulate gene expression at the post-transcriptional level bybinding to the complementary sequence in the 30untranslatedregion (30UTR) of target message RNA (mRNA), inducingmRNA cleavage or translational repression.1–4 Hundredsof miRNA genes have been found in animal and plantgenomes. Recent studies revealed that miRNA played animportant role in multiple biological pathways in a varietyof animals. In mammals, miRNAs have been implicatedin a broad range of processes, including hematopoieticcell lineage differentiation,5 developmental patterning,6,7

left/right asymmetric neural cell fate,8,9 apoptosis,10 heartand skeletal muscle differentiation and function,11,12 insulinsecretion,13,14 fat metabolism and immune function.15,16

miRNAs appear to have important roles in the mammaliancentral nervous system (CNS) as well. Numerous miRNAs

are expressed in the CNS, and many of them are expressed insuch spatial and/or temporal patterns that strongly suggest theirimportant roles in the regulation of CNS development.17–22

PC12 are rat adrenal pheochromocytoma cells and havebeen widely used as a mature neural cell model of neuralproliferation and differentiation, ion channels, receptors,neurotransmitter release and neurotoxicity. A number ofproteins have been associated with PC12 cell differentiationand neurite outgrowth, including growth associated protein43 (GAP-43) and pre-synaptic membrane-associatedproteins such as synapsin.23 Differentiation of PC12 cellswith nerve growth factor (NGF) treatment is associatedwith upregulation of Synapsin I, in parallel with neuritegrowth.23,24 To further understand the mechanism ofmiRNA in the regulation of neuron genes, we investigatedthe involvement of miRNAs in mediating PC12 cellsphenotype by controlling the expression level of synapsin I.In this study, we demonstrated that miR-541 was associatedwith the expression of synapsin I by software predictionand expression profile analysis. After the investigation of theeffect of miR-541 on cells proliferation, neurite outgrowth,synapse formation and altered expression level of synapsin I,we concluded that miR-541 can control the PC12 celldifferentiation through regulating synapsin I expression.

cell biochemistry and function

Cell Biochem Funct 2011; 29: 279–286.

Published online 30 March 2011 in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/cbf.1747

*Correspondence to: J. Zhang, Department of Regenerative Medicine,Tongji University School of Medicine, 1239 Siping Road, Shanghai200092, P.R.China E-mail: [email protected] authors contributed equally to this work.

Copyright # 2011 John Wiley & Sons, Ltd.

Received 10 November 2010Revised 6 February 2011

Accepted 10 February 2011

MATERIALS AND METHOD

PC12 cell culture and induction of cell differentiation

PC12 cells were bought from Chinese Academy of Sciencescell bank. The cells were grown in 25mm2 tissue cultureflasks with complete Dulbecco’s modified Eagle’s medium(DMEM) containing 5% heat-inactivated fetal bovineserum (FBS), 10% horse serum, 100mgml�1 of penicillinand 100mgml�1 of streptomycin. Cultures were maintained,according to standard protocols, at 37 8C in a 95% humi-dified incubator with 5% CO2.To induce the PC12 cells to differentiate, cells were plated

onto 6-well tissue culture plates at a relatively low density(2� 103 cells cm�2) in DMEM medium with 5% FBS and10% horse serum. After plating cells for 24 h, a concentratedstock of NGF (human recombinant NGF, Sigma, Missouri,USA) was then added to above culture medium till finalconcentration of 50 ngml�1, and the cells were incubated fortransfection. Cells exposed to vehicle alone (5% FBS and10% horse serum culture medium) were used as controls.

Real-time RT-PCR

Total RNA was isolated from PC12 cells using TRIzol1

reagent (Invitrogen, Carlsbad, CA, USA) following themanufacturer’s instruction, and the first-strand cDNA wasprepared using 1mg of RNA and the PrimerScript ReverseTranscriptase Kit (Takara, Dalian, China). The primers,designed in this laboratory using a primer designing soft-ware package.synapsin I: sense 50-CAGGGTCAAGGCCGCCAGTC-30

and antisense 50-CACATCCTGGCTGGGTTTCTG-30.b-actin: sense 50-TTACTGCCCTGGCTCCTA-30 and

antisense 50-ACTCATCGTACTCCTGCTTG-30.SYBR Green quantitative PCR amplifications were

performed on the Rotor-Gene 3000 Real-Time PCR System.Reactions were carried out in a 25-ml volume containing12�5ml of 2� SYBR Premix Ex TaqTM (Takara). Theconditions for real-time PCR were: 95 8C for 5min,followed by 45 cycles of 95 8C for 15 s, 60 8C for 15 sand 72 8C for 20 s. All reactions were run in triplicate. Thethreshold cycle (Ct) was defined as the fractional cyclenumber at which the fluorescence passed the fixed threshold.The average DCt of each group was calculated usingthe following formula: DCt¼Ct gene�Ct reference RNA.DDCt was calculated as DDCt¼DCt of experimentalgroup�DCt of negative control group. The relativeexpression level of gene was calculated using the formulaof 2�DDCt .

Western-blot analysis

Cells were cultured in 25 cm2 tissue culture flasks andinduced to differentiate as described above. First, the cellswere washed twice by centrifugation in phosphate-bufferedsaline (PBS) 1000� g for 5min at 4 8C. The supernatantwas decanted and cell pellets were incubated for 20min inice-cold lysis buffer. The cell lysate was centrifuged at

10 000� g for 10min at 4 8C and the resulting supernatantwas saved for Western blot. The total protein wasresolved by SDS-PAGE on 10% polyacrylamide gels andwas transferred onto Hybond-P polyvinylidene difluoride(PVDF) membrane (Millipore, USA). The membranewas then incubated with 1:10 000 dilution anti-Synapsin Iprimary antibody at 4 8C (Cell Signaling, CA, USA) over-night. Protein bands were visualized using IR Dye 800conjugated secondary antibody of Rabbit IgG (Rockland,Philadelphia, USA). Images were documented and banddensity was analysed using Odyssey Infrared ImagingSystem (LI-COR Biosciences, USA).

miRNA prediction and expression analysis

The rno-mir-541 miRNA and its target genes were predictedwith bioinformatics based on the evidence that target genehas the conserved or non-conserved sites matching the seedregion of corresponding candidate miRNA to form RNAsecondary structure.25 We applied microCosm software inpredicting the miRNAs for synapsin I. RNA extracted fromculture cells, RNA-tailing, primer extension and real-timequantification of miRNAs were conducted as describedpreviously. Briefly, the total RNA (1mg) was polyadenylatedwith ATP and poly(A) polymerase (PAP) at 37 8C for 1 h ina 20ml reaction mixture following the manufacturer’srecommendation of the Poly(A) Tailing Kit (Ambion, USA).After phenol-chloroform extraction and ethanol precipi-tation, the RNAs were reverse-transcribed using specificRT primer and PrimerScript Reverse Transcriptase (Takara)as previously described26 and the quantitative RT-PCRwas performed using 2� SYBR Premix Ex TaqTM mixture(Takara) with rat 5S ribosome RNA as the internalreference RNA. The relative expression level of miRNAwas calculated using the formula of 2�DDCt as describedabove. The primers were designed in this laboratory using aprimer designing software package.MiR-541: sense 50-AAGGGATTCTGATGTTGGTCA-

CAC-30 and antisense 50-GCTGTCAACGATACGCTACG-TAACG-30,5S ribosome RNA: sense 50-GTCTACGGCCATAC-

CACCCTGAAC-30 and antisense 50-GCTGTCAACGA-TACGCTACGTAACG-30.

miRNA overexpression plasmid and 20-O-methyloligonucleotide: preparation and transfection

According to the DNA sequence of pre-miR-541 and themultiple clone site of pSuper EGFP1, the PCR primersdesigned to amplify this miRNA gene were as follows:sense 50-GCGGATCCAGTTTCCAGAACACCCAGAT-30;and antisense 50-GCAAGCTTAAAAAACGCACAAG-CACGTCATAGA-30.The PCR fragments, after BamH I and Hind III digestion,

were subcloned into the pSuper EGFP1 vector and the re-combinant was named pSuper-541. pSuper EGFP1 was usedas the internal control for the subsequent transfectionexperiments. 20-O0-Methyl oligonucleotide, complementary

Copyright # 2011 John Wiley & Sons, Ltd. Cell Biochem Funct 2011; 29: 279–286.

280 j. zhang ET AL.

to mir-541 and composed entirely of 20-O0-Methyl bases,was chemically synthesized by ShengGong Company(Shanghai, China).

For transfection, PC12 cells were plated onto 6-well tissueculture plates with 3ml of cell suspension to each well atthe cell density of 105 cells per ml without antibiotics.After 24 h, the culture medium was replaced with a freshmedium with added NGF as described above. Transfectionwas performed with the pSuper-541, pSuper EGFP1 and20-O0-Methyl oligonucleotide by Lipofectamine 2000(Invitrogen Life Technologies Inc.) following the manu-facturer’s protocol. Analyses of the effects of miRNAs onPC12 cell differentiation were examined on the 2nd, 4th and6th days, respectively, after transfection.

STATISTICAL ANALYSES

For all statistical analysis including the results of thequantitative RT-PCR and Western blots, the analyses soft-ware SAS (8�2) was used for performing analysis ofvariance. p-Value below 0�05 was considered statisticallysignificant.

RESULTS

Expression level of Synapsin I during PC12 celldifferentiation

It has demonstrated that NGF, epidermal growth factor(EGF), fibroblast growth factor (FGF) or KCl can inducesPC12 cell differentiation and neurite formation, and thatNGF is able to lead to a significant neurite outgrowth.27 Inthe absence of NGF, the cells are relatively rounded and havelittle visible neuritis (Figure 1; Day 0). When PC12 cellswere cultured on six-well plates in DMEM containing 5%FBS, 10% horse serum and 50 ngml�1 NGF, neuritisoutgrowth was observed 2 days after induction (Figure 1;Day 2). The treatment of PC12 cells with NGF induces the

cells differentiation, leading to the smaller cell bodies,neurites formation and proliferation decrease. Neuritesformation can be observed as early as 24 h after the cellswere treated with 50 ngml�1 NGF, the proportion ofneurites-exhibiting cells increased to >90% within 4 days(Figure 1; Day 4), and the neurites length achieved themaximal extension at days 6 (Figure 1; Day 6).

Growth-associated Synapsin I is a useful indicator ofPC12 cell differentiation.23 Our study indicated thatSynapsin I expression levels increased during NGF-induced differentiation of PC12 cells. After the additionof 50 ngml�1 NGF, the mRNA levels of synapsin I increasedat 2, 4 and 6 days (Figure 2A). Within 48 h after NGFtreatment, relative mRNA levels of synapsin I approxi-mately doubled, as compared to the undifferentiated PC12cells. Western blot analysis showed that the expressionof Synapsin I increased significantly at 2, 4 and 6 days aftercells were exposed to NGF, suggesting the protein levelsincreased to more than 40-fold compared to the undiffer-entiated cells (Figure 2B). These results correlated quitewellwith previously published data.23

Prediction of miRNAs for synapsin I

Prediction of miRNAs for synapsin I was performed byusing microCosm software. We applied microCosm tosearch the most probable miRNAs against the 30UTR ofsynapsin I in databases of rodent transcriptome. In thesepredicted miRNAs, those from rat were selected as theputativemiRNAs for next-step study. The prediction result ispresented in Table 1.

Expression analysis and identification of putativemiRNA in PC12 cell

The expression analysis of those predicted miRNAs showedthat a decreased expression of rno-mir-541 rno-mir-540, and

Figure 1. Morphological changes of PC12 cells after NGF treatment. Phase-contrast images were photographed by a digital CCD camera equipped in aninverted microscope. The top row shows phase-contrast images at magnification 200�; as the bottom row shows phase-contrast images at magnification 400�.PC12 cells cultured without NGF (day 0) display little visible neuritis. PC12 cells were treated with 50 ngml�1 NGF for 2, 4 and 6 days. As shown in the figure,both neuritis length and the number of neuritis-presenting cells gradually increased under the effect of NGF


EFFECTS OF MIR-541 ON NEURITE OUTGROWTH 281

rno-mir-28 had been coincident with an increased expres-sion of synapsin I during PC12 cell differentiation(Figure 3A–C). All of the three putative miRNAs showeda downregulation in paralled with upregulation of synapsin I,and miR-541 displayed a more obvious decline trend thanthe other two miRNAs–miR-540 and miR-28. Hence, miR-541 was chosen as the candidate miRNA for synapsin I infurther study and the relationship between miR-541 andneurites outgrowth was explored.

Effect of mir-541 on PC12 cells and expression ofSynapsin I

To explore the roles of the candidate miRNA in PC12cells, we constructed an ectopic vector pSuper-541 formiR-541 overexpression with the goal to downregulatethe early expression of synapsin I and test its effect oncells phenotype. The overexpression of miR-541 dramatic-ally reduced PC12 cell differentiation, as evidenced by a

significant reduction of neurites growth and cell differen-tiation. Previous studies indicated that oligonucleotideswith 20-O-methyl (20-O-Me) were effective inhibitors ofmiRNAs in cell culture.28–30 These molecules complemen-tary to the miRNAs can specifically bind to miRNAsand disrupt miRNA functions. We chemically synthesized20-O-Me oligonucleotides complementary to miR-541 andassessed the effect of the loss-of-function of miR-541 onPC12 cell differentiation. In order to determine the effect ofmiR-541 on PC12 cells, we evaluated multiple parametersincluding cell proliferation, neurite length, and Synapsin Iexpression level at different time after the transfection.As shown in Figure 4A, this analysis revealed that there

was a gradual decrease in neurite length after transfectionwith pSuper-541, and some cells even became roundedand without neurite outgrowth. No visible neurite formationwas observed in PC12 cells with miR-541 over-expressedat 24, 48 and 72 h after transfection. Similarly, the numberof differentiated cells gradually decreased in miR-541overexpression cells. In contrast, cells differentiation andneurites outgrowth were increased significantly aftertransfection 24, 48 and 72 h, when treated with anti-miR-541 20-O-methyl oligonucleotides.In order to quantify the effect of miR-541 on cells

differentiation and neurite outgrowth, neurite lengthwere measured by camera software 72 h after transfection(Figure 4B). The neurite length of untransfected cellsand mock-transfected cells was 43�76� 7�53mm and40�07� 6�97mm, respectively, while cells with transfectedpSuper-541 were 1�53� 3�00mm and the proportion ofneurite-expressing cells was significantly reduced. Incontrast, the neurite length increased to 63�45� 7�87mmwhen treated with the anti-miR-541 20-O-Me oligonucleo-tides.We examined the effect of miR-541 overexpression on

the expression level of Synapsin I. As shown inFigure 4C, overexpression of miR-541 caused a significantlydecrease on the mRNA levels of synapsin I to approximatelyone-fifth compared with cells untransfected or pSuperEGFP1-tansfected. Similarly, the expression level of Syna-psin I protein was also decreased approximately to one-thirdafter the miR-541 overexpression compared with controlcells. Compared to the untransfected or pSuper EGFP1-transfected cells, the mRNA and protein levels of synapsin Iin cells that were transfected with anti-miR-541 20-O-Meoligonucleotides increased approximately two- and four-fold, respectively.

DISCUSSION

miRNAs are a group of small non-coding RNA and involvedin the post-transcriptional regulation of gene expressionduring development, differentiation and proliferation. Theyalso play an important role in ESC differentiation to specificneural cell line and cell morphology maintenance. Duringthe neural system development, the changes of expressionprofile of miRNAs parallel with neural cell growth anddevelopment.17–20 Therefore, comprehensive analysis of the

Figure 2. The expression level of Synapsin I during PC12 cell differen-tiation following NGF treatment (A) Cells were fed with NGF for 0, 2, 4 and6 days. Relative mRNA level of synapsin I were calculated at these fourdifferent stages by quantitative RT-PCR. Data represent mean�SE of threedifferent experiments. �p< 0�05 (B) Western Blot was performed to analysethe Synapsin I protein level at the indicated time points. GAPDH was usedas the internal control, as data were presented as means� S.E. (n¼ 3).�p< 0�05 as compared to the control


282 j. zhang ET AL.

spatial and/or temporal patterns of miRNA expression isof vital importance to understand the complex nature of theregulation of gene expression involved in NSC formation.

PC12 cells have been widely used as a model system ofmature neural cells for neurobiological research and can beinduced to differentiate and to grow spreading neurites withNGF treatment. The Synapsin I protein is a member of thesynapsin family that are neuronal phosphoproteins which ispresent in the nerve terminal of axons, specifically in the

membranes of synaptic vesicles and is thought to modulateneurotransmitters release and correlate with synapseformation. Growth-associated Synapsin I is a useful markerof PC12 cell differentiation.23 The expression of Synapsin Iincreases during the differentiation of PC12 cells, and thisincrease correlates with the neurite outgrowth and theappearance of synaptic vesicles in nerve terminals.31

miRNAs play an important role in regulation of thegene expression of neural system and affecting the neuron

Table 1. Candidated miRNAs for synapsin I

RfamID Score Energy Start End Aligment

mo-mi R-540 17�4927 �19�31 308 327

mo-mi R-28 17�4235 �20�16 559 580

mo-mi R-327 17�3766 �27�13 511 529

mo-mi R-146a 16 8013 �19�16 151 172

mo-mi R-29G 16 5939 �22 29 40

mo-mi FMB8 16�3864 �15�41 641 663

mo-mi R-541 16�172 �14�46 63 87

mo-mi R-837 15�9448 �16�43 284 304



cell differentiation, formation and maintenance.17–20

To better understand the regulation of miRNAs on neural-cell development, it was therefore of interest to studymiRNAs in neural cells and its possible effect on neuritesand synapses plasticity. In this report, we analysed therelationship between miRNAs and PC12 cells. In orderto explore the regulation mechanisms of miRNAs onsynapsin I during the PC12 cell differentiation, we appliedthe bioinformatics method and expression-level analysisto find the putative miRNAs for synapsin I. miRNAs aresmall non-coding RNAs that are involved in down-regulation the gene expression. So it has been suggestedthat simultaneous expression profiling of miRNAs andmRNAs could be an effective strategy for miRNA targetidentification.32 Based on this research evidence, we carriedout real-time quantification to identify the reliably miRNAsfor synapsin I, according to the reverse relationshipbetween miRNA and its target genes. We first detectedthe expression level of neuron-related gene synapsin Iand the result confirmed that synapsin I was a growth-associated gene and useful indicator for PC12 celldifferentiation. We then applied the bioinformaticsapproach to predict the candidate miRNAs for synapsin Iand analyse the expression level of these candidatemiRNAs during PC12 cell differentiation. Pictar, TargetScanand microCosm are algorithm for the identification ofmicroRNA targets. Pictar didn’t find any miRNA forsynapsin I and the candidate miRNAs for synapsin I

predicted by microCosm from rat were also identified byTarget Scan. So, according to the microCosm predictionresult, rno-miR-541 was a putative miRNA for synapsin I,which had a reverse expression level compared withsynapsin I.miR-541 is a member of miR379–410, a large cluster

of brain-specific miRNAs.33 miR-541 has been found tobe abundant in distal axons as compared with the somasof primary sympathetic neurons.34 It has been reportedthat miR-541 exhibited decrease expression level inhippocampus after traumatic brain injury.35 All of theseresearches demonstrated that miR-541 may be implicated inregulation of neuronal gene. But the cellular role of miR-541is not clear. Here we analyse the effect of miR-541 onsynapsin I expression level and neurite outgrowth. Furtherresearch indicated that in PC12 cells, overexpression ofmiR-541 reduced cell differentiation, neurite formationand extension, while transfection of 20-O-Me oligonucleo-tides complementary to miR-541 increased neuritelength. As a target gene of miR-541, synapsin I expressionlevel decreased after overexpression of miR-541 andincreased following transfection of 20-O-Me oligonucleo-tides, paralleling with the neurites outgrowth. Theseobserved phenotype changes demonstrated the specificfunctions of miR-541 that mediate the expression of agrowth-associated gene synapsin I and play an importantrole in axon maintenance and function during neurocytedifferentiation.

Figure 3. (A–C) The expression level of rno-mir-541, rno-mir-540 and rno-mir-28 in PC12 cells after the addition of 50 ngml�1 NGF for 0, 2, 4 and 6 days.RNA extraction and tailing were conducted as described in Section Materials and Methods. miRNA expression levels were calculated at the indicated timepoints by quantitative RT-PCR. Data were represented as mean�SE of three different experiments. �p< 0�05


284 j. zhang ET AL.

Figure 4. The effect of mir-541 on PC12 cells differentiation, neurite growth and expression of Synapsin I. PC12 cells were transfected with mir-541overexpression vector pSuper-541, pSuper EGFP1 control vector and 20-O-Me oligonucleotide complementary to mir-541, and then treated with NGF for 24, 48and 72 h, respectively. (A) Phase-contrast images and EGFP protein fluorescence were captured with a digital CCD camera equipped in an inverted microscopeat 200� magnification. Cells transfected with pSuper-541 displayed a markedly reduction in cell differentiation and neurite outgrowth, compared to theuntransfected cells and pSuper EGFP-transfected cells. In contrast, cells with 20-O-Me of mir-541 showed a significantly increase in differentiation andneurite length. (B) 72 h after transfection, neurite length of cells was analysed by camera software. Data are means�S.E. (n¼ 3). �p< 0�05 compared withcontrol. (C) 72 h after transfection, relative mRNA level of synapsin I was calculated by quantitative RT-PCR. Data were presented as means� SE of threedifferent experiments. �p< 0�05 compared with control. (D) Total protein was extracted and subjected to western blot analysis with Synapsin I primaryantibodies. Bands of proteins were quantified using scanning densitometry, and the relative ratio to GAPDH was calculated. GAPDH was used as the internalcontrol. Data were expressed as fold increase of relative protein expression to that in cells without transfection, and were presented as mean� SE of threedifferent experiments. �p< 0�05



CONFLICT OF INTEREST

The authors have declared that there is no conflict of interest.

ACKNOWLEDGEMENTS

The authors would like to thank all members of the labora-tory of Dr. Jun Zhang and Dr. Jing Zhang for support andcooperation. We also thank Dr. Chang-jie Cheng for hisassistance in article edit. This work was supported by theNatural Science Foundation of China (30570690 and81041069) and Natural Science Foundation of Shanghai(08ZR1420700).

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effects of mir-541 on neurite outgrowth during neuronal differentiation

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