Identification and Functional Analysis of Novel Micrornas in Rat Dorsal Root

Journal of Neuroscience Research 90:791–801 (2012)

Identiﬁcation and Functional Analysis of Novel Micro-RNAs in Rat Dorsal Root Ganglia After Sciatic Nerve Resection

Shiying Li, Bin Yu, Shanshan Wang, Yun Gu, Dengbing Yao, Yongjun Wang, Tianmei Qian, Fei Ding, and Xiaosong Gu* Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, People’s Republic of China

Peripheral nerve injures are quite common in clinical and Srivastava, 2007; Hwang and Mendell, 2007). Many practice, and many studies have tried to explore the miRNAs are expressed in a tissue-specific manner (Sood underlying molecular mechanisms. This study focuses et al., 2006) and thus play important roles in regulating on the identification and functional analysis of novel tissue specificity of gene expression, the function of tis- miRNAs in rat dorsal root ganglia (DRGs) following sci- sue-specific genes, or both. In neurons, miRNAs are atic nerve resection, which is a classic model for study- expressed at all development stages, and miRNA-de- ing peripheral nerve injury and regeneration. By using pendent posttranscriptional gene regulation plays a piv- Solexa sequencing, computational analysis, Q-PCR ver- otal role at all stages of neural development, including ification, and Dicer knockdown assay, 114 novel miR- neural differentiation, morphogenesis, and plasticity NAs in rats were identified, of which 51 novel miRNAs (Pillai et al., 2007; Vo et al., 2010). Recent studies have were first reported in rat DRGs, and 63 novel miRNAs also revealed the roles of miRNAs in axonal biology as were produced at days 1, 4, 7, and 14 following sciatic well as in the control of synaptic function and plasticity nerve resection. We further predicted target genes of (Kim et al., 2004; Fiore and Schratt, 2007; Schratt, these miRNAs and analyzed the biological processes in 2009a,b; Natera-Naranjo et al., 2010). which they were involved. The identified biological A single miRNA can affect, albeit modestly, the processes were consistent with the time frame of pe- levels of thousands of proteins (Baek et al., 2008; Sel- ripheral nerve injury and regeneration, revealing that bach et al., 2008); thus, miRNAs form a complex regu- these miRNAs were genuine miRNAs related to nerve latory network affecting the majority of genes. Although regeneration. Our data provide an important resource miRNA expression in central and peripheral nerve for the future study of function and regulation of these injury has been studied (Liu et al., 2009; Nakanishi miRNAs and contribute to elucidation of tyhe molecular et al., 2010), there has been no miRNA research in pe- mechanisms responsible for peripheral nerve injury and ripheral nerve injury using large-scale sequencing tech- regeneration. VC 2011 Wiley Periodicals, Inc. nology. High-throughput technology is being applied increasingly to examine signal pathways and regulatory Key words: micro-RNAs; DRGs; rat; solexa mechanisms. Here we sought to find novel miRNAs and detect their expression levels in rat dorsal root gan- Peripheral nerve injury is quite common in clinical practice, resulting from accidental trauma, deliberate surgery, or acute compression. It is important to understand Additional Supporting Informtion may be found in the online version of the molecular mechanisms involved to provide the best this article. treatment. During peripheral nerve injury and regenera- S. Li and B. Yu contributed equally to this work. tion, the levels of many genes and proteins are changed Contract grant sponsor: Hi-Tech Research and Development Program of (Hama et al., 1995; Angelov et al., 1998; Costigan et al., China (863 Program); Contract grant number: 2006AA02A128; Contract 1998; Zhang et al., 2006; Chen et al., 2010). Despite grant sponsor: National Natural Science Foundation of China; Contract the existence of several robust candidate pathways, these grant number: 30870811; Contract grant sponsor: Jiangsu Provincial Nat- molecular mechanisms remain largely elusive, so thera- ural Science Foundation; Contract grant number: BK2008010; Contract peutic and preventative strategies for peripheral nerve grant sponsor: Priority Academic Program Development of Jiangsu injury and regeneration present a significant challenge. Higher Education Institutions (PAPD). miRNAs are a novel regulatory class of noncoding, *Correspondence to: Xiaosong Gu, 19 Qixiu Road, Nantong, Jiangsu single-stranded RNAs consisting of approximately 22 Province, China. E-mail: [email protected] nucleotides, and they are implicated in a wide range of Received 27 January 2011; Revised 31 May 2011; Accepted 6 genetic regulatory mechanisms under physiological and September 2011 pathological conditions (Ambros, 2004; Kloosterman and Published online 22 December 2011 in Wiley Online Library Plasterk, 2006; Jovanovic and Hengartner, 2006; Zhao (wileyonlinelibrary.com). DOI: 10.1002/jnr.22814

' 2011 Wiley Periodicals, Inc. 792 Li et al. glia (DRGs) following sciatic nerve resection. We iden- with a free energy higher than –20 kcal/mol in accordance tified some candidate miRNAs by Solexa sequencing, a with referenced standards (Wang et al., 2005; Chen et al., method known to reveal novel miRNAs with high rates 2009). Novel miRNAs were separated from those previously of accuracy and efficiency (Chen et al., 2009). We vali- reported using miRBase 16.0 (Griffiths-Jones et al., 2006, dated the candidate miRNAs by using bioinformatics 2008); miRAlign (Wang et al., 2005) was used to identify con- tools, reverse transcription-polymerase chain reaction served miRNAs; MiPred (Jiang et al., 2007) was used to distin- (RT-PCR), and Dicer knockdown assay. Subsequently, guish functional nonconserved miRNA precursors (pre-miR- we predicted the target genes of the novel identified NAs) from dysfunctional pseudohairpins. miRNAs based on the TargetScan algorithm and performed functional annotation of the target genes. Quantitative RT-PCR Assay MATERIALS AND METHODS The same RNA samples as described for the RNA iso- Construction of Animal Models and Sample lation and Solexa sequencing were used. An amount of 10 ng Preparation total RNAs was reverse transcribed using a TaqMan Micro- RNA Reverse Transcription Kit (Applied Biosystems, Carls- Thirty adult, male Sprague-Dawley rats (180–220 g) bad, CA) and stem-loop RT primers according to the manu- were provided by the Experimental Animal Center of Nantong facturer’s instructions. Real-time PCR was performed using University and randomly divided into five groups (six rats per SYBR Green PCR Master Mix (Applied Biosystems) on an group) to undergo sciatic neurectomy. Animal experimentation Applied Biosystems Stepone real-time PCR System. All pri- was carried out in accordance with the NIH Guidelines for the mers are given in Table 6 of Supplement 1, and all reactions care and use of laboratory animals. All rats were anesthetized by were run in triplicate. The cycle threshold (Ct) values were injection of a narcotics complex (10 mg/kg xylazine, 95 mg/kg analyzed by the comparative Ct (DDCt) method. miRNAs ketamine, 0.7 mg/kg acepromazine), and the sciatic nerve was levels were normalized against those of rRNA U6. The identified and lifted through an incision on the lateral aspect of expression levels at each time point were compared with those the midthigh of the left hind limb. A 1-cm segment of sciatic at 0 hr. nerve was then resected at the site just proximal to the division of tibial and common peroneal nerves. After the surgical inci- sions were closed, animals were housed in temperature- and Analysis of the Dicer Knockdown humidity-controlled cages maintained under a 12:12-hr light- To generate expression constructs, pre-miRNA hairpins, dark cycle and were allowed free access to water and food. One together with the surrounding upstream and downstream group of rats was euthanized immediately as a control group regions (either of about 100 nt) on each side of the hairpins, without sciatic nerve resection (0 h), and the other groups were were amplified from rat genomic DNA. The PCR products euthanized on days 1, 4, 7, and 14, with surgery to obtain L4–6 were cleaved with BamH I and Xho I and inserted into the DRGs following euthanasia. 1 BamH I and Xho I fragment of pcDNA3.1 vector. Then, the ligation products were transformed into Top10 cells. Posi- RNA Isolation and Solexa Sequencing tive clones were selected by colony PCR and were Total RNA was isolated from the DRGs using Trizol sequenced. Plasmid DNAs from the confirmed clones were (Invitrogen, Carlsbad, CA) according to the manufacturer’s extracted for transfection using the Plasmid Mini Kit (Qiagen, protocol. Solexa sequencing was performed by the Beijing Valencia, CA). Genomics Institute at Shenzhen. Briefly, small RNA mole- C6 glioma cells in 12-well plates were reverse tranfected cules ranging from 18 to 30 nt were gel purified and ligated 0 0 with 120 pmol/well of Dicer siRNA using Lipofectamine with a pair of Solexa adaptors to their 5 and 3 ends. The li- RNAiMax (Invitrogen). Cells were harvested at 48 hr post- gation products were purified, reverse transcribed, and ampli- transfection for the analysis of Dicer influence on endogenous fied by using adaptor primers. The amplified products were miRNAs. To evaluate Dicer dependence of the overexpressed 1 gel purified and sequenced on an Illumina Genome Analyzer hairpins, constructed pcDNA3.1 -pre-miRNA (pPre- (Illumina, San Diego, CA). The image files obtained by miRNA) plasmid DNA (1 lg each plasmid) was transfected sequencing were then converted to digital-quality data. After into C6 glioma cells using Lipofectamine 2000 (Invitrogen) at 1 the removal of adaptor sequences and contaminated reads, 12 hr after transfection of Dicer siRNA. pcDNA3.1 vector clean reads were processed for computational analysis. was also transfected as control. Cells were harvested at 36 hr after the second transfection. Total RNAs were extracted Novel miRNA Prediction from the harvested cells, and reverse transcribed using oli- After Solexa sequencing, bioinformatics analysis was per- go(dT) primers for analysis of Dicer and pre-miRNA or stem- formed to predict and identify the novel miRNAs. The cleaned loop RT primers and U6 reverse primers for analysis of small RNA sequences from Solexa sequencing were mapped to miRNA, respectively. In the stem-loop reverse transcription the rat genome using SOAP (Li et al., 2008). Only those reaction, four miRNAs were merged for endogenous miRNA mapped perfectly to the rat genome were considered candidate assay, whereas only two miRNAs with their pre-miRNA miRNAs for further analysis. MIREAP (Chen et al., 2009; Liu plasmids were contransfected were merged for the overexpres- et al., 2010) was used to analyze the hairpin structures to deter- sion system. RT-PCR assays were performed using cDNA as mine genuine miRNAs. We subsequently filtered out miRNAs a template. The endogenous miRNAs were amplified for

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TABLE I. Number of Novel miRNAs Sequenced in Rat DRGs After Sciatic Nerve Resaction by Solexa Technology miRNA No. Total miRNAs 114 Mature miRNAs 104 Mature miRNA*s 11 Conserved miRNAs 14

data sets. The context score for each micRNA target site was also calculated. To annotate further the biological function of these target genes, we first screened novel miRNAs that demonstrated a significant change in expression level among five time points using the v2 test. The false discovery rate (FDR) was calculated to correct the P value. Then, the filtered miRNAs (FDR <0.05) were used for expression pattern clustering using the Short Time-Series Expression Miner (STEM; Ernst and Bar-Joseph, 2006). Five expression profiles were applied to perform the Gene Ontology (GO) enrichment analysis with a context score <–0.3 for the target genes. The GO Biological Processes (<0.05) were attributed to several general categories to statistical analysis according to the GO website (http://www.geneontology.org/).

RESULTS Discovery of Novel miRNAs by Solexa Sequencing The strategy used to identify novel micro-RNAs in rat DRGs following sciatic nerve resection is shown schematically in Figure 1. We collected the small RNA fraction and prepared small RNA sequencing libraries from rat DRGs following sciatic nerve resection. The libraries were sequenced using deep sequence technology. The raw data generated by Solexa sequencing are available at Gene Expression Omnibus [GEO: GSE25863]. Intriguingly, the length distribution peaked at 22 nucleotides, and almost half of the clean reads were 22 nucleotides in length (data not shown), consistent with the typical common size of miRNAs.

Identification of Genuine Novel miRNAs by Fig. 1. Step-by-step schematic description of the strategy for the Computational Analysis novel miRNA discovery and validation. By mapping the cleaned small RNA sequences from Solexa sequencing to the rat genome sequences 29–33 cycles, whereas the overexpression system used only using SOAP, scanning canonical stem-loop hairpin struc- 26–29 cycles. tures using MIREAP, removing 19 loci (Supp. Info. Table 4 of Supplement 1) with free energy greater than –20 kcal/mol, and filtering out the reported miRNAs miRNA Target Prediction and Biological Functional by using miRBase 16.0, Solexa sequencing generated Analysis of Target Genes 251 loci that were considered candidate miRNAs. We miRNA targets in animals are usually located in the then used miRAlign to identify rat miRNAs that are 30UTR region of mRNAs. We downloaded the 30UTR paralogs or orthologs to known miRNAs, and detected sequences of all the rat genes and TargetScan source code 14 conserved miRNAs among the 251 (Tables I, II). from TargetScan (http://www.targetscan.org/). miRNA tar- Although the hairpin structure is a necessary feature gets were predicted by complementary matches between the for the computational classification of genuine pre- 2–8-bp seed region (from the 50 end) of miRNAs and 30UTR miRNAs, many random inverted repeats (pseudohairpins)

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TABLE II. Read Counts of Novel miRNAs at All of the Time Points* Read counts Read counts miRNA genes miRNA genes 0 hr 1 day 4 days 7 days 14 days 0 hr 1 day 4 days 7 days 14 days rno-miR-3075 13 8 13 10 12 rno-miR-s49 7 rno-miR-3099 111 128 130 96 67 rno-miR-s50 7 rno-miR-3102-5p.2 47 59 43 43 42 rno-miR-s51 6 rno-miR-1247* 8 38 24 49 24 rno-miR-s52 5 rno-miR-s1 5 8 7 6 7 rno-miR-s53 6 rno-miR-s2 42 36 37 35 24 rno-miR-s54-1 5 rno-miR-s3 16 27 25 33 10 rno-miR-s54-2 5 rno-miR-4646b 6 7 10 11 9 rno-miR-s54-3 5 rno-miR-s4 14 17 31 34 33 rno-miR-s55 6 rno-miR-s5 34 25 31 30 15 rno-miR-s56 5 rno-miR-s6 4 17 7 14 10 rno-miR-s57 8 rno-miR-s7 34 25 41 40 22 rno-miR-s58 19 rno-miR-s8 47 53 35 30 15 rno-miR-s59 6 rno-miR-s9 308 178 246 289 231 rno-miR-s60 5 rno-miR-s10 94 74 115 119 163 rno-miR-s61 7 rno-miR-s11 61 56 66 69 59 rno-miR-s62 5 rno-miR-s12 10 rno-miR-s63 5 rno-miR-s13 4 rno-miR-s64 9 rno-miR-s14 6 rno-miR-s65 5 rno-miR-s15 5 rno-miR-s66 6 rno-miR-s16 10 rno-miR-s67 12 rno-miR-s17 6 rno-miR-s68 5 rno-miR-s18 9 rno-miR-s69 5 rno-miR-s19 11 rno-miR-s70 9 rno-miR-s20 6 rno-miR-s71 5 rno-miR-3082b 5 rno-miR-s72 18 rno-miR-1956 5 rno-miR-s73 7 7 rno-miR-s21 6 rno-miR-s74 28 43 23 rno-miR-s22 8 rno-miR-s75 4 17 8 rno-miR-s23 5 rno-miR-s76 5 7 8 21 rno-miR-s24 5 rno-miR-s77 190 116 140 144 rno-miR-s25 9 rno-miR-s78 8 23 19 12 rno-miR-s26 5 rno-miR-344h-3p 6 8 5 rno-miR-s27 9 rno-miR-466q 7 5 rno-miR-s28 5 rno-miR-s79 5 5 rno-miR-s29 5 rno-miR-s80 4 10 rno-miR-s30 5 rno-miR-s81-1 7 5 rno-miR-s31 5 rno-miR-s81-2 7 5 rno-miR-s32 5 rno-miR-s82 4 5 rno-miR-s33 5 rno-miR-s83 8 7 rno-miR-s34 10 rno-miR-s84 6 7 rno-miR-s35 5 rno-miR-s85 513 545 rno-miR-s36 8 rno-miR-s86 6 6 rno-miR-s37 14 rno-miR-s87 6 8 rno-miR-s38 9 rno-miR-s88 6 6 5 rno-miR-3561b-1 5 rno-miR-s89 10 5 9 rno-miR-3561b-2 5 rno-miR-s90 21 23 24 rno-miR-s39 9 rno-miR-297d-1 18 5 9 5 rno-miR-s40 5 rno-miR-297d-2 18 5 9 5 rno-miR-s41 9 rno-miR-297e 18 5 9 5 rno-miR-s42 5 rno-miR-s91-1 17 8 23 5 rno-miR-s43 7 rno-miR-s91-2 17 8 23 5 rno-miR-s44 5 rno-miR-s91-3 17 8 23 5 rno-miR-s45 5 rno-miR-s91-4 17 8 23 5 rno-miR-s46 5 rno-miR-s91-5 17 8 23 5 rno-miR-s47 6 rno-miR-s91-6 17 8 23 5 rno-miR-s48 5 rno-miR-s92 11 9 5 11 *The miRNAs gene is named by conservation or according to the order of miRNAs in this table; the ‘‘s’’ before number was use to distinguish the known miRNAs. The read counts come from Solexa sequencing results after normalization.

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Fig. 2. Characterization of the novel miRNAs. A: Number of novel miRNAs identified in DRGs. B: Composition of the novel miRNAs of various lengths (in nucleotides [nt]). C: Number of novel miRNAs at each time point. Single time point means the miRNAs were expressed only at this time point; multiple time points means the miRNAs were expressed at multiple time points. in eukaryotic genomes can also fold into dysfunctional Figure 3A, all of the 16 miRNAs could be readily hairpins (Ng and Mishra, 2007; Jiang et al., 2007). To detected with clear bands at the expected molecular overcome this problem, we adopted an ab initio predic- weights. In addition, the expression of 11 miRNA*s tion method, MiPred, to distinguish pre-miRNAs from identified by Solexa sequencing were also confirmed. other similar segments in the rat genome (Jiang et al., Among 11 miRNA*s, eight could be clearly detected 2007). Among the 251 pre-miRNA-like hairpins, 137 (Fig. 3B) and three could not be detected possibly were classified as pseudo-pre-miRNAs (Supp. Info. Table because of their low abundance caused by in vivo degra- 5 of Supplement 1) and 114 as genuine novel miRNAs dation or other reasons. These validations revealed that (Tables I, II, Supp. Info. Tables 1, 2 of Supplement 1), of the results from semiquantitative RT-PCR were basi- which 51 novel miRNAs were first discovered in rat cally consistent with those from sequencing. We there- DRGs, and 63 novel miRNAs were produced at days 1, fore concluded that these were authentic miRNAs and 4, 7, and 14 following sciatic nerve resection (Fig. 2A). that Solexa sequencing was capable of successfully dis- The length ranged from 20 to 24 nucleotides, with a peak covering novel miRNAs with high accuracy and effi- at 22 nucleotides that accounted for 45.7% of the miR- ciency. NAs (Fig. 2B). The novel miRNAs and their read counts The expression levels of the newly identified rat are listed in Table II. The 114 novel miRNAs expressed miRNAs were also analyzed by quantitative real-time 104 mature miRNAs, because five had multiple copies RT-PCR. We selected six miRNAs that showed the distributed on the same or separate chromosomes that homogenous band without background for further anal- produced identical mature miRNAs (Tables I, II). Simul- ysis and demonstrated that the relative changes in taneously, 11 miRNA*s were detected (Table I, Supp. miRNA expression assayed using Solexa sequencing and Info. Figs. 1, 2). Among the 114 novel miRNAs, 16 real-time PCR were generally consistent (Fig. 3C). miRNAs were expressed at all five time points, and 67 miRNAs were expressed at only one time point (Table II). Furthermore, the largest number (62) of novel miR- Experimental Evaluation of the Biogenesis NAs was expressed at 7 days, in which 34 miRNAs were of miRNAs expressed only at this time point, whereas the fewest (34) To investigate Dicer dependence of novel miRNA novel miRNAs were expressed at 14 days (Fig. 2C). biogenesis, we determined the change of miRNAs expressed at all time points by temporary knockdown of Dicer with siRNA (Fig. 4A). The results showed that Validation of Novel miRNAs by Quantitative Dicer knockdown led to an obvious decrease in the RT-PCR expression of 15 mature miRNAs (Fig. 4B). The To verify the accuracy and efficiency of Solexa miR-s8 missed from endogenous measure probably sequencing, the same RNA preparation used for the Sol- because they were not expressed in the C6 glioma cells. exa sequencing was subjected to stem-loop quantitative Given that most authentic miRNAs are processed from RT-PCR assay (Chen et al., 2005). The expression of heterologous transcripts that include the full hairpin 16 miRNAs in rat DRGs at all time points following flanked by about 100 nt of genomic sequence on each sciatic nerve resection was confirmed by photographic side of the hairpin (Chiang et al., 2010), we constructed images of the semiquantitative RT-PCR. As shown in pre-miRNA hairpins together with about 100 nt of their

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Fig. 3. Validation of novel miRNAs and miRNA*s by quantitative miRNA, so here rno-miR-1247 was detected. C: Expression levels RT-PCR. A: Existence and speciﬁcity analysis of the indicated novel of the six miRNAs at each time point were calculated relative to the miRNAs by semiquantitative RT-PCR. B: Validation of miRNA*s amount of U6 rRNA after normalization. using semiquantitative RT-PCR. rno-miR-1247* was a novel

1 upstream and downstream regions into pcDNA3.1 vec- (Fig. 4C). Collectively, our data revealed that Dicer tor and transfected the constructed plasmids into C6 gli- inﬂuenced the maturity of these miRNAs and the proc- oma cells to determine whether these pre-miRNAs essing from pre-miRNA to mature miRNA. were processed by Dicer. The results showed that the expression levels of pre-miRNAs and mature miRNAs were signiﬁcantly increased when constructed pre- Chromosomal Location of Pre-miRNAs and miRNA plasmids were overexpressed. In addition, under Cluster Analysis the Dicer knockdown conditions, pre-miRNAs were We BLAST searched chromosome positions for all accumulated, whereas mature miRNAs were decreased 114 novel pre-miRNAs and determined the exact posi-

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Fig. 4. Experimental evaluation of the biogenesis of miRNAs. A: Expressions of Dicer in C6 glioma cells were knocked down by siRNA. En, endogenous; Con, control; 1–6 represent six groups. B: Expressions of miRNAs were downregulated under Dicer knockdown conditions. C: Expression changes of pre-miRNAs and miRNAs in the pre-miRNA overexpression and Dicer knockdown cells. tion of each miRNA. We observed that miRNA-s81-2 novel pre-miRNAs into clusters (at least two pre-miR- occurred 10 times in chromosome 1, miRNA-s44 six NAs in a cluster) if their interdistance was less than a times in chromosome 13, miRNA-s27 three times, deﬁned maximum interdistance (MID) value. Five clus- miRNA-s81-1, s91-1, s91-2, s91-3 and s91-4 twice, and ters including 10 novel pre-miRNAs were identiﬁed other miRNAs only once. In addition, chromosome 1 with MID 5 3 kb, along with nine clusters including 33 encoded 24 miRNAs, the largest number, whereas chro- novel pre-miRNAs with MID 5 10 kb and 14 clusters mosome 20 revealed only one novel pre-miRNAs including 40 novel miRNAs with MID 5 50 kb (Supp. (Supp. Info. Fig. 3). Info. Table 3 of Supplement 1). Altuvia et al. (2005) As is known, miRNAs are often present as clusters demonstrated that 42% of known human miRNA genes in the genome where multiple miRNAs are aligned in are arranged in clusters in the genome with a 3-kb the same orientation and transcribed as a polycistronic threshold between two miRNA genes. However, the structure, allowing them to function synchronously and clustering of novel pre-miRNAs appears less likely, as cooperatively (Bartel, 2004). Therefore, we grouped evidenced by a lower percentage of clustering. A more

Journal of Neuroscience Research 798 Li et al. detailed analysis of novel pre-miRNA clusters in rats tion or biogenesis were significantly activated, whereas awaits the further development of the rat genome in metabolic process and biological regulation were down- order to find all the hallmarks of pre-miRNAs. regulated at 1 day following sciatic nerve resection. Then, metabolic process and biological regulation were miRNA Target Prediction obviously unregulated. At 7 days following sciatic nerve resection, all the biological processes appeared to be Because miRNAs function by regulating target extremely active. By 14 days following sciatic nerve genes, we next predicted target genes of novel miRNAs. resection, however, most of the biological processes In animals, miRNA targets are preferentially located in 0 went silent (Fig. 5B, Supp. Info. Tables 1–5 of Supple- the 3 UTR region, where the silencing complex can ment 3). easily interact with the initiation complex and promote attenuation of translation (Pillai, 2005). Typical methods for recognizing miRNA binding sites rely on searching DISCUSSION for complementary matches between an miRNA sequence and phylogenetically conserved portions in the Compelling evidence shows that complex gene 30UTR of target mRNAs. Many online prediction pro- regulatory programs orchestrate all stages of neuronal grams that can be used to predict target genes of regeneration by following the classical flow of genetic reported miRNAs cannot be applied to novel miRNAs. information, including gene transcription, splicing, Here we followed a canonical approach of searching for mRNA translation and stability, and posttranslational complementary matches between the ‘‘seed’’ region of modifications (Schratt, 2009a,b). When the class of an miRNA and the 30UTR of genes. According to the RNA regulatory genes known as miRNAs was discovered, a whole new layer of gene regulation in eukaryotes TargetScan algorithm (Lewis et al., 2003), we downloaded all the 30UTR sequences of all rat genes and pre- was uncovered (Bartel, 2004). Increasing attention has dicted the target genes of novel miRNAs using BLAST. recently focused on the study of miRNAs in an attempt to explain the molecular mechanisms of gene regulation, There were 157,236 target sites and 9,843 target genes for the novel miRNAs that we identified (data not pathological processes, and drug action. To date, 1,424 shown). miRNAs in humans and 720 miRNAs in mice have been reported, whereas only 408 miRNAs have been reported in rats, implying that many miRNAs in rats Functional Annotation remain undiscovered. miRNAs are particularly plentiful First, we determined that 95 novel miRNAs dem- in neurons, and, together with the fact that a given onstrated significant changes in expression levels among miRNA usually regulates the expression of hundreds of the five time points (Supp. Info. Table 1 of Supplement target mRNAs, neuronal miRNA pathways create an 2). Then, expression pattern clustering was analyzed extremely powerful mechanism for dynamically adjusting using the 95 miRNAs, and five expression profiles were the protein content of neuronal compartments without selected to perform GO biological process analysis the need for new gene transcription (Schratt, 2009a,b). (Supp. Info. Table 2 of Supplement 2). A single miRNA To look for these regulatory factors, such as the regula- has the potential to regulate hundreds of distinct target tory roles of miRNAs and regulatory networks between genes (Lewis et al., 2005). To increase accuracy, we miRNA and mRNA in peripheral nerve injury and raised the threshold value of the context score of the tar- regeneration, we constructed the nerve injury model and get genes. According to the cumulative distribution of selected five time points for investigation according to context score (Supp. Info. Fig. 4) and a suitable number pathological process of sciatic nerve resection. The dis- of target genes for analysis, a context score <–0.3 was covery and identification of novel miRNAs, as the first selected as the selection threshold. The 866, 911, 1,101, important step, helped to identify a group of novel rat 2,235, and 783 target genes included in the five expres- miRNAs specifically expressed in DRGs or induced by sion profiles (Fig. 5A, Supp. Info. Tables 3–7 of Supple- sciatic nerve resection and to find gene regulatory net- ment 2) were used to perform GO biological process works involved in the survival of injured peripheral analysis. nerves. The results indicated the emergence and disap- To gain a global view of major biological process pearance of novel miRNAs at all the time points, sug- that miRNA regulated in rat DRGs after sciatic nerve gesting that these novel miRNAs were involved in the resection, we classified the GO biological processes with regulation of this pathological process. obvious significance (FDR <0.01 and <0.05) into sev- The peripheral nervous system is able to regenerate eral general categories, including response to stimulus, spontaneously after injury, and nerve regeneration is death, developmental process, cellular component orga- associated with the expression of new genes and proteins nization or biogenesis, cellular process, metabolic pro- (Vogelaar et al., 2004). The function of an miRNA is cess, biological regulation, immune system process, and ultimately defined by the genes it targets, and the pre- others. The data showed that all the biological processes diction of target genes is a prerequisite for experimental but response to stimulus were evenly distributed at 0 hr. validation of target genes. We predicted target genes of Response to stimulus, death, immune system process, novel miRNAs and further analyzed the biological proc- developmental process, and cellular component organiza- esses in which target genes were involved. Robo2, a

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Fig. 5. Statistical analysis of biological process that novel miRNA target genes were involved in. A: Numbers of miRNAs and their target genes for the analysis of GO biological process at each time point. B: Statistical results of GO biological process in which novel miRNA target genes were involved. molecule that promote axonal growth, was highly superfamily, was highly expressed in gastrocnemius expressed in rat DRGs at 7 and 14 days following sciatic muscles at 14 d following sciatic nerve resection (Liu nerve resection (Yi et al., 2006), and myostatin et al., 2007). In this study, Robo2 and MSTN were found (MSTN), a member of the transforming growth factor-b to be the most plausible target genes of rno-miR-s75 and

Journal of Neuroscience Research 800 Li et al. rno-miR-s77, respectively, in terms of the context score many regulatory processes, and the study of miRNAs value; the expression of rno-miR-s75 was not detected contributes to the understanding of disease etiology. The in DRGs at 7 and 14 days, and rno-miR-s77 was not identification and functional analysis of novel miRNAs detected at 7 days following sciatic nerve resection. in rat DRGs, described here, might be helpful in clinical Because miRNAs inhibit the expression of target genes, management of peripheral nerve injury, because the rat the relative low expressions of miRNAs were consistent model was selected in this study to allow interspecies with high expression of target genes. In addition, many scaling up to humans. In addition, our study could pro- genes that play an important biological role were likely vide an important basis for further studying the function the target genes of these novel miRNAs, such as and regulation of miRNAs and the molecular mecha- CAPON, BNDF, PDE1,3,4, BMP7, SOCS3, and nisms underlying peripheral nerve regeneration. CCL2, but these genes were regulated by several novel miRNAs as well as known miRNAs based on predic- ACKNOWLEDGMENTS tion. Therefore, it could not be deduced which miR- We thank Professor Jie Liu for help in manuscript NAs regulated these genes before experiment definition. revision and Associate Professor Xi Chen for assistance However, we noticed that the expression change of in data analysis. stress-related genes, such as CAPON, NOS (Shen et al., 2008), and HSP27 (Costigan et al., 1998), generally occurred during the first 2 days following peripheral REFERENCES nerve injury, whereas the expression change of regenera- Altuvia Y, Landgraf P, Lithwick G, Elefant N, Pfeffer S, Aravin A, tion-related genes, such as NCAM (Zelano et al., 2006), Brownstein MJ, Tuschl T, Margalit H. 2005. Clustering and conserva- BNDF (Funakoshi et al., 1993), and meltrin (Wakatsuki tion patterns of human microRNAs. Nucleic Acids Res 33:2697–2706. et al., 2009), generally occurred beyond 4 days, espe- Ambros V. 2004. The functions of animal microRNAs. Nature 431: 350–355. cially at 7 days, following peripheral nerve injury. 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