MicroRNA-9 regulates neural apoptosis in methylmalonic acidemia via targeting BCL2L11

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ARTICLE IN PRESSG ModelN 1871 1–6

Int. J. Devl Neuroscience xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Developmental Neuroscience

j ourna l ho me page: www.elsev ier .com/ locate / i jdevneu

icroRNA-9 regulates neural apoptosis in methylmalonic acidemiaia targeting BCL2L11

anfei Lia, Tao Penga, Lin Lib, Xiaohan Wanga, Ranran Duana, Huili Gaoa, Wenjuan Guana

Jingjing Lua, Junfang Tenga, Yanjie Jiaa,∗

Department of Neurology, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, ChinaDepartment of Neurology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450052, China

r t i c l e i n f o

rticle history:eceived 11 March 2014eceived in revised form 17 April 2014ccepted 27 April 2014

eywords:icroRNA-9

CL2L11poptosisethylmalonic acidemia

a b s t r a c t

Methylmalonic acidemia (MMA) is an autosomal-recessive inborn metabolic disorder that results froma deficiency in methylmalonyl-coenzyme A mutase or its cofactor, adenosylcobalamin. Currently, neu-rological manifestations in MMA are thought to be associated with neural apoptosis. BCL2L11, whichis a proapoptotic Bcl-2 family member, is resident in the outer mitochondrial membrane, where thisprotein acts as a central regulator of the intrinsic apoptotic cascade and mediates excitotoxic apoptosis.MicroRNAs (miRNAs) are a class of non-coding RNAs that function as endogenous triggers of the RNAinterference pathway. Currently, little is known regarding the role of miRNA in MMA. In our previousstudy, we preliminarily found that the expression of miR-9 was significantly down-regulated in MMApatient plasma and sensitively changed after VitB12 treatment, which may act as a potential “competi-
tor” of gas chromatography-mass spectrometry for the diagnosis of MMA. In the present study, we firstconfirmed that miR-9 inhibited BCL2L11 expression by directly targeting its 3′-untranslated region, andthe up-regulation of miR-9 reduced neural apoptosis induced by methylmalonate via targeting BCL2L11.Taken together, our results suggested that miR-9 might act as a monitor of changes in MMA and mightprovide new insights into a therapeutic entry point for treating MMA.
© 2014 Published by Elsevier Ltd. on behalf of ISDN.

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. Introduction

Methylmalonic acidemia is an inborn error of intracellularobalamin metabolism with a wide spectrum of clinical mani-estations. This metabolic disease is caused by mutations in the

ethylmalonyl-CoA mutase gene and results in impaired intracel-ular synthesis of adenosylcobalamin and methylcobalamin, whichre cofactors for methylmalonyl-CoA mutase and methionine syn-hase enzymes. Elevated methylmalonic acid and homocysteine,s well as decreased methionine production, are the biochemicalallmarks of this disorder. Providing a timely diagnosis is necessaryo guide the management of affected individuals (Carrillo-Carrascot al., 2012; Carrillo-Carrasco and Venditti, 2012). The pathophy-iology of complications observed in these patients is not fully

Please cite this article in press as: Li, Y., et al., MicroRNA-9 regulates neInt. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014

nderstood; however, reports have confirmed that MMA might benduced by neuronal apoptosis resulting from mitochondrial dys-unction, impaired methyl group metabolism, and oxidative stress

∗ Corresponding author. Tel.: +86 371 66862102; fax: +86 371 66862102.E-mail addresses: [email protected], [email protected] (Y. Jia).

ttp://dx.doi.org/10.1016/j.ijdevneu.2014.04.005736-5748/© 2014 Published by Elsevier Ltd. on behalf of ISDN.

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(Mc Guire et al., 2009; Richard et al., 2007, 2009; Wajner andCoelho, 1997).

MiR-9 is important in development and in various diseasesvia its ability to regulate different targets genes. MiR-9 exertsdiverse effects on the proliferation, apoptosis, migration, and dif-ferentiation of neural progenitor cells (Delaloy et al., 2010; Zhaoet al., 2009). Moreover, the downregulation of miR-9 in post-mitotic neurons is also implicated in some neurodegenerativediseases. As previously reported, MMA diagnosis is dependenton the gas chromatography-mass spectrometry method. In ourprevious study, we found that the expression of miR-9 was signif-icantly down-regulated in MMA patients’ plasma and sensitivelychanged after VitB12 treatment, which suggest that the changein miR-9 expression might act as a potential competitor of gaschromatography-mass spectrometry for the diagnosis of MMA andmight reflect the state of the disease (Li et al., 2014).

MiR-9 has many predicted targets, one of which is BCL2-like

ural apoptosis in methylmalonic acidemia via targeting BCL2L11..04.005

11(BCL2L11), as determined by target gene prediction systemincluding Targetscan, miRWalk and miRbase. BCL2L11 is one of themost important apoptosis regulators that mediate excitotoxic apo-ptosis, mitochondrial depolarization, and apoptosis inducing factor

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dx.doi.org/10.1016/j.ijdevneu.2014.04.005


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ranslocation (Concannon et al., 2010). In the present study, weonducted a series of experiments to elucidate the role of miR-9 inMA. Our results suggest that miR-9 could reduce neural apoptosis

nduced by methylmalonate via targeting BCL11 and thus speculat-ng up-regulation of miR-9 may be a therapeutic entry point for thereatment of MMA.

. Results

.1. MiR-9 suppresses neuronal apoptosis induced byethylmalonate

Neurons were transfected with miR-9 mimic or inhibitor andhen were exposed to 2.5 mM methylmalonate for 24 h. The MTT

ethod, TUNEL, and Annexin V-FITC staining showed that the cellpoptosis index was higher after the methylmalonate treatmenthan before the treatment. In addition, the cell apoptosis index wasower in the miR-9 mimic + MMA group, whereas the cell apoptosisndex was higher in the miR-9 inhibitor + MMA group when com-ared with the MMA group and with the control group. And thereas no significant difference in apoptosis rate of the siRNA nega-

ive control when compared to the control group (Fig. 1). This resultndicated that methylmalonate caused neuronal apoptosis and that

iR-9 may reduce neuronal apoptosis induced by methylmalonate.

.2. MiR-9 specifically reduces the expression level of BCL2L11

To explore the possible mechanism by which miR-9 suppresseseuronal apoptosis, we performed an analysis to search for tar-et genes regulated by miR-9 using bioinformatics software. Afterhe preliminary screen, we chose BCL2L11 as a possible target of

iR-9 and predicted the target sequence. Then, we constructed luciferase reporter plasmid and the psiCHECK2 vector contain-ng the 3′-UTR of BCL2L11 with the binding site of miR-9 directlyownstream of the luciferase reporter gene. Next, 293T cells wereo-transfected with the vector and hsa-miR-9 or the control, andhe relative luciferase activity was determined. The result showedhat, when compared with the control, the relative luciferase activ-ty was significantly decreased (47% reduction) by miR-9, whereashe luciferase activity was not altered by the vector containing the

utant 3′-UTR (Fig. 2A).To directly examine how miR-9 regulates BCL2 L11 levels, we

ransfected the miR-9 mimic and miR-9 inhibitor into neurons.n response, the BCL2L11 level was significantly downregulatedn the miR-9 mimic group and was upregulated in the miR-9nhibitor group when compared with the control group. And there

as no significant difference between control group and siRNAegative control group. Therefore, we confirmed that BCL2L11

evels are inhibited by miR-9 (Fig. 2B and C). We confirmed theffective delivery of the mimic and inhibitor into neurons usingRT-PCR; miR-9 levels were enhanced by approximately 7.2-fold

n the mimic group and were reduced by 5.9-fold in the inhibitorroup (Fig. 2F). Notably, BCL2L11 mRNA levels were also sig-ificantly down-regulated following miR-9 mimic transfection,hereas these levels were up-regulated after miR-9 inhibitor trans-

ection (Fig. 2E).

.3. MiR-9 may reduce neuronal apoptosis in MMA via targetingCL2L11

After the transfection of the miR-9 mimic and inhibitor, the


eurons were treated with methylmalonate. The Western blotesults indicated that the BCL2L11 level was lower in the miR-

mimic + MMA group and higher in the miR-9 inhibitor + MMAroup when compared with the non-transfected group. Therefore,

PRESSence xxx (2014) xxx–xxx

we speculated that miR-9 reduced neuronal apoptosis in MMA bysuppressing the expression of BCL2 L11 (Fig. 3).

2.4. The expression of miR-9 in neurons with the methylmalonatetreatment

As the qRT-PCR results showed, the expression of miR-9 wasreduced 22.3-fold in neurons with the methylmalonate treatmentwhen compared with neurons without the methylmalonate treat-ment (Fig. 4).

3. Discussion

MMA patients present predominantly neurological symptoms,whose pathogenesis is not yet fully established. Currently, neuro-logical manifestations in MMA are thought to be associated withthe accumulation of methylmalonate in tissues and in biologicalfluids with mitochondrial injury occurring through a combinationof the inhibition of specific enzymes and transporters, the limita-tion of the availability of substrates for mitochondrial metabolicpathways, and oxidative stress, which leads to neural apopto-sis (Fernandes et al., 2011; Melo et al., 2011). In addition, ithas been shown that MMA causes brain injury through gluta-matergic mechanisms (de Mello et al., 1996; Malfatti et al., 2003)and through striatal degeneration (Narasimhan et al., 1996). Theoverexcitation of glutamate receptors, particularly N-methyl-d-aspartic acid (NMDA) receptors, has been implicated in neuronalinjury with rapid necrotic death or a more delayed apoptotic injury,which is characterized by cell shrinkage and nuclear condensa-tion (Ward et al., 2007). Additionally, it has been demonstratedthat NMDA receptor activation-induced excitotoxicity is due tonitric oxide (NO) generation. Accordingly, the excessive produc-tion of NO inhibits the mitochondrial respiratory chain, whichleads to oxidative damage in the brain (Stewart and Heales, 2003).A great deal of work has suggested that reactive oxygen species(ROS) generation may underlie the neurotoxic effects of MMA.High levels of ROS induce severe damage in the cell and causechanges in cellular ATP and Ca2+ levels, eventually leading tothe release of cytochrome c and to the induction of apopto-sis.

BCL2L11, which is a proapoptotic Bcl-2 family member, is resi-dent in the outer mitochondrial membrane, where this moleculeacts as a central regulator of the intrinsic apoptotic cascade.BCL2L11 is activated in multiple excitotoxicity paradigms, medi-ating excitotoxic apoptosis, mitochondrial depolarization, andapoptosis inducing factor translocation. Studies have suggestedthat BCL2L11 activation requires the activation of AMPK and thatprolonged AMPK activation is sufficient for increasing BCL2L11gene expression and for triggering NMDA-mediated excitotoxicitythrough BCL2L11. FoxO3a has emerged as an important mediatorof cell fate, including apoptosis. Recent reports show that BCL2L11is a direct target of FoxO3a in A�-treated neurons, which indicatesthat FoxO3a is activated, translocated to the nucleus, and mediatesneuron death via BCL2L11 in response to A� toxicity (Sanphui andBiswas, 2013). Furthermore, FoxO3 activates the overproductionof ROS because of the BCL2L11-dependent impairment of mito-chondrial respiration in neuronal cells, which leads to apoptosis(Hagenbuchner et al., 2012). BCL2L11 has an ability to directly acti-vate a voltage-dependent anion channel (VDAC) that regulates themitochondrial membrane potential and, thus, controls the produc-tion of reactive oxygen species and the release of cytochrome C by


mitochondria, both of which are potent inducers of cell apoptosis(Sugiyama et al., 2002).

Our study found that miR-9 was down-regulated in neuronstreated with methylmalonate, which had a similar trend with

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Fig. 1. MiR-9 reduces neuronal apoptosis induced by methylmalonate. (A) MTT result indicating that the cell survival rate was lower after the methylmalonate treatmentthan before the treatment. In addition, the cell survival rate was higher in the miR-9 mimic + MMA group than in the miR-9 inhibitor + MMA group (n = 3; P < 0.05). (B) TUNELstaining showing that the cell apoptosis index was higher after methylmalonate treatment than before treatment. Additionally, the cell apoptosis index was lower in themiR-9 mimic + MMA group but was higher in the miR-9 inhibitor + MMA group when compared with non-transfected group and with the control group. (C) Annexin V-FITCstaining result was similar to the TUNEL staining. (D) TUNEL staining (scale bar: 100 �m). (E) Flow cytometry results analyzed using the CellQuest software. The apoptosisrate in miR-9 mimic + MMA group, miR-9 mimic group, miR-9 inhibitor +MMA group, miR-9 inhibitor group, MMA group, the control group and the siRNA negative controlg

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roup was 15.36%, 10.59%, 30.9%, 11.18%, 18.89%, 10.91% and 11.02%.

he previous result in MMA plasma (Li et al., 2014). In order toxplore the mechanism of down-regulation of miR-9 in MMA,e constructed a series of experiments and found that the cell

poptosis rate with the methylmalonate treatment was lower inhe miR-9 mimic group than in the non-transfected group. Con-equently, we speculated that miR-9 might suppress apoptosisnduced by methylmalonate. In our vitro study, we found that


he expression of BCL2L11 decreased after the overexpression ofiR-9 and increased after the down expression of miR-9, which

ndicated that BCL2L11 might be regulated by miR-9. These dataere further confirmed by the dual-luciferase assay. Therefore,

our results indicated that miR-9 inhibited neural apoptosis inmethylmalonic acidemia by suppressing the expression of BCL2L11, and we speculated that up-regulated miR-9 might yield newinsights into a therapeutic target of MMA. Additionally, otherunknown factors might also be involved in the role of miR-9in the pathogenesis of MMA, which must be explored in futurestudies.


In the study, miR-9 was observed to reduce neuronal apo-ptosis induced by methymalonic acid through down-regulatingthe expression of the proapoptotic factor BCL2L11 in vitro.However, the mechanism of miR-9 in MMA requires further

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Fig. 2. MiR-9 targeted BCL2L11 by dual luciferase assay. (A) Relative luciferase activity assays of luciferase reporters with BCL2L11 or mutBCL2L11 3′UTR were performedafter co-transfection with the miR-9 mimic, inhibitor or control. Transfections with psiCHECK2–BCL2L11 (blank), co-transfected with the miR-9 mimic (miR-9 mimic), andco-transfected with the miR-9 inhibitor (miR-9-inhibitor), miR-9 negative control (NC), and miR-9-inhibitor negative control (NC inhibitor) were established. The relativeluciferase activity was significantly reduced when BCL2L11 3′UTR reporter vectors containing the BCL2L11 binding site were co-transfected together with hsa-miR-9 comparedwith the blank and NC (n = 3; P < 0.01). This reduction was not observed when mut-BCL2 L11 or control expression vectors were used (n = 3; P > 0.05). Briefly, miR-9 couldinhibit BCL2L11 expression, and the suppression proportion was 47%. (B) The protein levels of BCL2L11 were detected using a Western blot. (C) The IOD ration of BCL2L11.T R-9 inw 0.05).( ssion o

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he BCL2L11 level was lower in the miR-9 mimic + MMA group and higher in the mias no significant difference between control and negative control group (n = 3; P >

E)The expression of BCL2L11 mRNA was determined using qRT-PCR. (F) The expre

iscussion. Our results may prompt further investigation regardinghe possibility of miR-9 as a miRNA-based therapy target for

MA.

. Materials and methods

.1. Cell culture and transfection

Cortex neurons were prepared from newborn BalB/C mice. The mice have beenandled in accordance with appropriated guidelines for the care and use of lab-ratory animals and the protocols have been approved by Zhengzhou Universitynimal Care Committee. The cortex tissue was dissected out in D-Hanks’ balancedalt solution and dissociated via trypsin/EDTA treatment. Cells were plated on cul-ure plates precoated with polylysine and cultured in neurobasal (Gibco) mediumupplemented with B-27(Gibco) and glutamine in a 5% CO2-humidified incubatort 37 ◦C. The medium was changed every 3–4 days.


The miR-9 mimic and miR-9 inhibitor was obtained (Ribobio). MiR-9 mimicas actual miRNA which was synthetized chemically according to miR-9

equence obtained from the miRBase database: ucuuugguuaucuagcuguauga. MiR-9nhibitor was reverse complement of miR-9 sequence which suppress endoge-ous miR-9 function through binding endogenous miR-9 competitively. Neurons

hibitor + MMA group when compared with the control group (n = 3; P < 0.05). There (D) The target site of miR-9 with BCL2L11 was predicted by the Targetscan system.f miR-9 was determined by qRT-PCR.

(2 × 105 cells/well) were seeded in 24-well plates in 500 �l neurobasal mediumbefore transfection. Then, miR-9 mimic or inhibitor labeled with Alexa Fluor 488(Qiagen) were diluted in 100 �l neurons culture medium, 3 �l HiPerFect transfectionreagent was added to the diluted miRNA mimic or inhibitor and mixed by vortexing.The complexes were incubated for 8 min at room temperature, and then the com-plexes were added drop-wise onto the cells to a final miRNA mimic concentration of5 nM and inhibitor concentration of 50 nM. Negative Control miRNA (Ribobio) wasused as a negative control, which has no homology to any known mammalian geneand the sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′ . Cells were incubatedwith transfection complexes under normal growth condition for 48-72 h.

4.2. Methylmalonate treatment

Referring to the previous report (McLaughlin et al., 1998), a 100 mM stock solu-tion of methylmalonate (TCI) was made in neurobasal medium supplemented withB27. The pH was adjusted to 7.4–7.5, and solutions were sterile-filtered through


a 0.22 �m filter. Serial dilutions of this stock were made in sterile media andwere warmed to 37 ◦C. Then, the medium was removed from the coverslips andreplaced with 2.5 mM methylmalonate. Cells were returned to the incubator for24 h. The neurons were divided into groups as follows: miR-9 mimic + MMA group(transfection of the miR-9 mimic and the methylmalonate treatment), miR-9 mimic

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Fig. 3. Western blot results showing that BCL2L11 expression was lower in themiR-9 mimic + MMA group and higher in the miR-9 inhibitor + MMA group whenc

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ompared with the MMA group (n = 3; P < 0.05).

roup (transfection of the miR-9 mimic but without the methylmalonate treatment),iR-9 inhibitor + MMA group (transfection of miR-9 inhibitor and methylmalonate

reatment), miR-9 inhibitor group (transfection of miR-9 inhibitor but withoutethylmalonate treatment), MMA group (neurons with methylmalonate treat-ent), negative control group (transfection of negative control miRNA) and the

ontrol group (neurons without transfection or the methylmalonate treatment).

.3. MTT method

The previous study showed that the significant inhibition of the solubleuccinate dehydrogenase activity was induced by methylmalonate in purified mito-hondrial fractions (Brusque et al., 2002). Mitochondrial succinate dehydrogenasen live cells could deoxidize exogenous MTT into insoluble formazan, whereas deadells could not. Consequently, the MTT assay was chosen to measure cell viabilitys well as methylmalonate toxicity. Neurons in each group were incubated for 4 hith MTT (5.0 mg/ml), and then 200 �l of dimethyl sulfoxide was added. Using an

nzyme-linked immunosorbent assay, the absorbance at 490 nm was determined.


he cell viability (%) for each group was calculated by comparing with the control.

ig. 4. qRT-PCR results showing that the expression of miR-9 was reduced 22.3-old in neurons with the methylmalonate treatment when compared with neuronsithout the methylmalonate treatment (n = 4; P < 0.05).

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4.4. TUNEL assay

A terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling(TUNEL) assay was used to assess neuronal apoptosis with an apoptosis detec-tion kit (Promega) according to the manufacturer’s instructions. Neurons on theslides were rinsed twice with PBS and then were fixed with 4% paraformalde-hyde. After blocking endogenous peroxidase activity using permeability liquid(1 g/L TritonX-100 was dissolved in 0.1% sodium citrate), the TUNEL reactionsolution was added. DAPI staining was used as the final step in the fluores-cent staining procedure to label cell nuclei. For each slide, six nonadjacent fieldswere randomly selected. Apoptotic neurons were quantified using a fluorescentmicroscope in a blinded manner, and the apoptotic index [(number of apoptoticcells/total number counted) × 100%] was used to quantify the number of TUNEL-positive cells. The brown particles in the nucleus were considered apoptosis-positivecells.

4.5. Flow cytometry

Annexin V-FITC staining was performed according to manufacturer’s instruc-tions (KGA106, Keygen). The adherent cells were washed twice with ice-cold PBSand digested in 0.25% trypsin for 5 min. Then, binding buffer was added, and thedensity of the cells was adjusted to 5 × 105/ml. In total, 0.5 ml of the cell suspen-sion was incubated with 5 �l fluorescein isothiocyanate (FITC)-labeled Annexin Vand 5 �l propidium iodide for 15 min at room temperature in the dark. The numberof viable, apoptotic and necrotic cells were quantified using a flow cytometer (BD)and analyzed using the CellQuest software. The apoptosis rate (%) = (the number ofapoptotic cells/the number of total cells observed) × 100%.

4.6. Dual luciferase assay

Several putative target sites for miR-9 in BCL2L11 3′-UTR were predictedusing Targetscan, miRWalk and miRbase prediction programs. The 5′-flankingregions of hsa-miR-9 (MI0000466) were synthesized. According to the miRandasystem, segments in the 3′-UTR of BCL2L11 were amplified by genomicPCR and cloned between the Xhol-Notl sites of psicheck-2 (Promega). Wecloned BCl2L11 3′-UTR that contains two potential miR-9 binding site and islocated at nucleotides 1801–3850. The oligonucleotide sequences used for thePCR are BCL2L11SallF2: 5′-acgcgtcgacAAACAACTTTTAAAAGAAATAACTAAATTTTC-3′; BCL2 L11NotIR2: 5′-ataagaatgcggccgcTGACCAAATGCAGTGACCGTATG-3′ . ThePCR conditions were 30 cycles of 94 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦Cfor 1 min and 40 s. The site-directed mutagenesis was constructed at thebinding site using the following sequences: MutBCL2 L11-F: 5′-CCTGCTGGACACACACATACTGGTTTCTTGTATTTGGATCTGG GCACC-3′; mutBCL2L11-R: 5′-GGTGCCCAGATCCAAATACAAGAAACCAGTATGTG TGTGTCCAGCAGG-3′ .

Next, 293T cells (2 × 104 cells/well) were seeded in 24 well-plates, and transfec-tion was performed in triplicate at 50–60% confluency by adding 100 �l transfectionsolution, which contained 50 nM microRNA, 0.5 �g reporter plasmid in psiCHECK2(Promega), and 1 �l Lipofectamine 2000 (Invitrogen). Then, the cells were incubatedfor 5 h in 5% CO2 at 37 ◦C, and the transfection solution was replaced with completemedium. Two days after transfection, 100 �l Passive Lysis Buffer (Dual-LuciferaseReporter Assay System; Promega) was added. The relative amounts of Renilla andfirefly luciferase were analyzed using a dual luciferase assay. The Renilla/fireflyluciferase ratio was calculated and normalized with the control.

4.7. Real time PCR for the detection of BCL2L11 and miR-9

Total cellular RNA was isolated using TRIzol reagent (Invitrogen) andconventionally quantified following the manufacturer’s instructions. Primerswere acquired from Guangzhou Landbiology Technical Services. MiR-9-F:5′ACACTCCAGCTGGGTCTTTGGTTATCTAGCTG and R: 5′CTCA ACTGGTGTCGTGGA.BCL2L11-F: 5′CTTCCCGTTCACTGCTTTAGG, BCL2L11-R: 5′ACACAGGC ACCAGGCT-GAAT. GAPDH-F: 5′GGCCTCCAAGGAGTAAGAAA. GAPDH-R: 5′GCCCCTCCTGT TAT-TATGG. The isolated RNA was mixed with 0.5 �l oligos (Promega), 0.5 �l randomprimers (Promega), 2 �l dNTPs, 0.5 �l RNase inhibitor, 4 �l 5× buffer and 0.5 �lM-MLV. The mixture was incubated at 30 ◦C for 10 min, 42 ◦C for 60 min and 85 ◦Cfor 10 min. According to the SYBR Green qPCR SuperMix (Invitrogen) instructions,the total PCR volume was 20 �l (5 �l cDNA, 0.5 �l forward primer, 0.5 �l reverseprimer, 10 �l 2× SYBR Green qPCR SuperMix and 4 �l H2O). The GAPDH was usedfor the normalization of samples. The amplification was performed in a LightCy-cler 1.5 Real-Time PCR System (Roche). Relative changes in gene expression werequantified using the comparative threshold method (Ct) after determining the Ctvalues for the reference and target genes in each sample set according to the 2−��Ct

method. All reactions were performed in triplicate.

4.8. Western blot analysis


The cell lysates were collected from each group. An equal amount of pro-tein was separated by SDS-PAGE on an 8% running gel and a 5% stacking gel,and then transferred to a polyvinylidene fluoride (PVDF) membrane (0.45 �m,Millipore). The membranes were blocked with 10% non-fat milk for 2 h and

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ncubated with the primary antibodies BCL2L11 (1:1000, Abcam), and GAPDH pro-ein (rabbit, 1:1000, KangChen bio-tech) overnight at 4 ◦C. Next, membranes werencubated with horseradish peroxidase-conjugated (HRP) anti-rabbit IgG (1:2000)or 2 h at room temperature and visualized by enhanced chemiluminescenceMillipore).

.9. Statistical analysis

All data were recorded as the means ± SD. To analyze significant differences,ormal distribution data between groups were compared using Student’s t-test,iscontinuous variables between groups were compared using a chi-square test,nd abnormal distribution data were compared using the Wilcoxon test. Differencesere considered significant when P values <0.05. All analyzes were performed usingraphPad Prism version 5.0 software.

ncited reference

Ribeiro et al. (2013).

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MicroRNA-9 regulates neural apoptosis in methylmalonic acidemia via targeting BCL2L11