Transcriptomic differential IncRNA expression is involved in neuropathic pain in rat dorsal root ganglion after spared sciatic nerve injury.
After traumatic damage, the peripheral nervous system can regenerate spontaneously by activating the inherent growth ability of neurons, while the central nervous system cannot do so (1, 2). The sciatic nerve is commonly used as a model to study peripheral nerve regeneration. It includes a complex bunch of motor and sensory axons, in which the sensory neurons are situated in the L4-L6 dorsal root ganglion (DRG) (3, 4). After sciatic nerve damage, the damaged neurons initiate a regeneration process but cease having the neurotransmitter status (3, 5). Axon regeneration and pathfinding after damage involves a complex mechanism involving axon cross-talk with neurogliocytes, nerve growth factors, neurotrophic factors, and receptors (6, 7).
Neuropathic pain after traumatic or surgical nerve injury challenges doctors and patients and regulatory noncoding RNAs (ncRNAs) are key molecules for understanding and treating this pain (8, 9). Regulatory ncRNAs are transcribed from protein noncoding genes to interfere in gene expression and they include, but are not limited to, miRNAs (21-24 nt), siRNAs (21-25 bp), piRNAs (26-31 nt), and long non-coding RNAs (IncRNAs, 200 bp to more than 100,000 bp) (8, 10, 11). The role of miRNAs, IncRNAs, and piRNAs in neuropathic pain after nerve injury has been reviewed by Bali and Kuner (8). The role and regulatory mechanisms of IncRNAs in vertebrate central nervous system and human nervous system diseases have been reported in the literature (8, 12-15).
In peripheral nerve injury, IncRNAs play an important role in stress responses, plasticity, and axonal outgrowth of DRG neurons (8, 16-21). Yu et al. (16) investigated the IncRNA transcriptome of DRG neurons after sciatic nerve injury in rat models and found that IncRNAs modulate DRG neurons responses to ligation stimuli. Zhao et al. (17) identified the modulating effect of the IncRNA (Kcna2 antisense RNA) on a voltage-dependent potassium channel mRNA Kcna2 in primary afferent neurons. Yao et al. (18) reported that the IncRNA uc.217 modulates neurite outgrowth of DRG neurons after sciatic nerve injury. IncRNA NONRATT 021972 and IncRNA uc.48 modulate neuropathic pain mediated by the P2X(3/7) purinergic receptors (the cation-permeable ATP-binding ligand-gated ion channels) in the DRG neurons in diabetic rat models, and siRNA therapy alleviate the pain significantly (19-23). However, all these studies are based on microarray analysis (24). A sequencing study to elucidate vital roles of IncRNAs in peripheral nerve pathology will help to understand neuropathic pain.
In this study, we described the IncRNAs expression and mRNA expression in DRGs during nerve regeneration by a transcriptome-level deep sequencing. The results reveal a novel layer of regulation of the inherent growth ability of neurons by IncRNAs.
Material and Methods
Animal surgery and sample preparation
Six male Sprague-Dawley rats (180-220 g) were housed in large cages with sawdust bedding at 25[degrees]C in 12 h/12 h dark/light cycle and allowed free access to food and water in the Animal Center of Beijing China-Japan Friendship Hospital. Rats were randomly divided into test group and sham-operation group, three in each group. Surgery was performed as described in the literature with modifications (16). Briefly, rats were anesthetized by intra-peritoneal injection of 10 wt.% chloral hydrate (3 mL/kg, Tianjin Fuchen Chemical Reagent Factory, China). The sciatic nerves were exposed and lifted through an incision on the right lateral thigh. Sciatic nerve segments were tied at the site proximal to the bifurcation of tibial and common peroneal nerves. Rats in the sham-operation group only had the sciatic nerves exposed without tying. L4-L6 DRGs were harvested from each animal a week later. All animal experiments were performed in accordance with institutional animal welfare and care guidelines and approved by the Animal Ethics Committee of Beijing China-Japan Friendship Hospital.
RNA isolation, cDNA library preparation, and sequencing
Total RNAs were extracted from the L4-L6 DRG tissues using Trizol reagent according to the instructions of the manufacturer (Invitrogen, USA). RNAs were cleaned, including a DNase I digestion step, using RNeasy spin columns (Qiagen, USA). RNA integrity was detected by agarose gel electrophoresis and RNA was quantified using Nanodrop2000 (Bio-Rad, USA). After rRNA was removed, RNA was interrupted into short fragments by adding fragmentation buffer. These short RNA fragments were used as templates to synthesize the first-strand cDNA using FastQuant RT Kit (with gDNAase) (Tiangen, China). Then, double-strand cDNA was obtained.
The cDNA products were purified with QiaQuick PCR extraction kit (Qiagen, Germany) and the purified cDNA were dissolved in EB buffer, followed by end reparation and poly(A) addition. The cDNA fragments were connected to sequencing adaptors. The cDNA fragments at 150-200 bp in size were separated on gel-electrophoresis and were used as the templates for PCR amplification. Two cDNA libraries for the test and sham-operation groups were sequenced using the Illumina HiSeq2500 at Beijing Ori-Gene Science and Technology Co., LTD (China).
Raw images generated by sequencers were converted by Illumina software (USA) to nucleotide sequences, called raw reads. FastQC software package (USA) was used to generate clean reads by removing adaptor reads, low quality reads (QC30), sequences containing fuzzy N bases and sequences less than 60 bp. All the following analyses were based on clean reads. The clean reads were mapped to the genome using Tophat2 software package (USA). RSeQC software package with all default parameters (USA) was used to detect the splice junction sites for evaluating the saturation of sequencing.
Differential expression and enrichment
The RPKM method (reads per kilobase per million mapped reads) was used to calculate the differential expression level using the Cufflinks software package (USA). Cufflinks Cuffdiff was used to screen the differential expression genes (DEGs). The criterion to identify the DEGs between two groups was as follows: sum of mapping reads of two samples [greater than or equal to] 10; |log2RPKM fold change| > 1; P value was corrected by false discovery rate (FDR) to get a Q-value; both P [less than or equal to] 0.05 and Q [less than or equal to] 0.05 were required to determine the significance of differential expression. In order to get biological functions, DEGs were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. R package was used for the analysis. GO terms of DEGs were compared with the genome background and the corrected P value (FDR correction) < 0.05 was set to judge the significantly enriched GO terms. For the KEGG pathway enrichment analysis, P value < 0.05 was the threshold.
Known and novel IncRNA
The RPKM method was used to calculate the known expression level using the Cufflinks software package. Because IncRNAs have no conservation between species, we used rat IncRNA database for annotating known IncRNA in the transcript obtained from sequencing. New transcripts with opening read frame features were aligned with known protein database with CPC scoring to predict either coding or non-coding RNA. The predicted non-coding RNA is a novel IncRNA.
Coexpression network and target gene enrichment analysis
By comparing the differential gene lists, we obtained gene pairs of novel IncRNA and differential mRNA. FPKM values for each pair were used to calculate the Pearson correlation, where we chose significantly correlated gene pairs (correlation coefficient >0.995 or < -0.995, P<0.05 as threshold) to build a coexpression network using Cytoscape software package (USA). GO and KEGG enrichment analyses were performed as described above but the corrected P value threshold was set to <0.1. The correlated mRNA genes that coexpressed with IncRNAs served as the candidate target genes for IncRNAs.
Real-time qPCR quantification
To quantify IncRNA and target mRNAs, real-time qPCR were performed on representative rno-Cntnap2-201 IncRNA gene, rno-Fam171b mRNA gene, rno-Hebp2 mRNA gene, rno-Gde1 mRNA gene, rno-AC111653.1 IncRNA gene, and rno-Hypm mRNA gene in the sham-operation and test DRG groups. Briefly, the first-strand cDNA was synthesized using FastQuant RT Kit (with gDNAase) (Tiangen). Then, double-strand cDNA was synthesized. Real time qPCR was performed on a Roche LightCycler[R] 96 fluorescence quantifying PCR machine. Primer sequences are listed in Table 1.
The reaction system included 1 [micro]L of cDNA, 10 [micro]L of RealStar Green Mixture (2 x), 0.6 [micro]L of primers, and filled up to 20 [micro]L with PCR-grade water. The PCR program included a pre-denaturation at 95[degrees]C for 5 min, 40 cycles (95[degrees]C for 15 s, 60[degrees]C for 20 s, 72[degrees]C for 15 s) for amplification, and a default condition for dissociation. The cycle threshold (Ct) values were obtained. Relative interest/ reference mRNA expression was calculated by the formula: 2-[DELTA]Ct (interest-reference). rno-GAPDH was used as inner reference.
Wistar rat DRG cells were isolated and cultured as reported in the literature (25). Cell assays were performed as reported in the literature with a minor modification (26). Briefly, one-day-old Wistar rat DRG cells were isolated and cultured in 95% Eagle's DMEM feeding medium with 600 mg/mL glucose, 10% fetal bovine serum, 5% horse serum, 20 ng/mL nerve growth factors, and 1 [micro]g/100 mL neurotrophins (25). For RNA silencing, siRNA sequences targeting lnc-AC111653.1 were designed and synthetized (GenePharma, China), and a final concentration of 50 nM was used for transient transfection. For overexpression of lnc-AC111653.1, full-length rat lnc-AC111653.1 cDNA was cloned into the pcDNA3.1 expression vector (Gene-Pharma). Lipofectamine 3000 (Invitrogen) was used for transfection according to manufacturer's instructions (26).
Total proteins were extracted from cell lysates and separated by 10% SDS-polyacrylamide gel. Then, they were electroblotted to PVDF membranes (Beyotime, China). Membranes were incubated in rabbit polyclonal HYPM antibody (Novus Biologicals, China), followed by horse-radish peroxidase-labeled goat anti-rabbit secondary anti-body (Boster, China). Signals were revealed using ECL detection reagent (Beyotime). GAPDH served as control. Images were analyzed by Image-Pro Plus 6 software (Media Cybernetics, USA). The intensity of test protein bands was normalized to the GAPDH bands.
Gene mapping and differential expression genes
The RNA quality and sequencing quality were guaranteed. Clean reads were obtained. The results of gene mapping to the rat genome is shown in Table 2. Known gene expression is shown in Tables 3 and 4.
On differential expression analysis, a total of 18,824 genes were included, of which there were 2643 differential genes between DRG test group and sham-operation group. By comparison of the DRG group with the sham-operation group, 1228 were up-regulated and 1415 down-regulated. On enrichment analysis of DEGs, up-regulated differential genes were attributed to 624 GO terms and 50 KEGG pathways; down-regulated differential genes were attributed to 424 GO terms and 30 KEGG pathways. DEGs were clustered into a heatmap (Figure 1).
Known IncRNA, co-expression network, and target gene enrichment
We found 69 neurite-associated known IncRNA genes linking to 866 target mRNA genes (Table 5). After the GO and KEGG enrichment information was presented at a P value threshold <0.1, the 866 targets were enriched to 737 GO terms and 40 KEGG pathways. They were involved either in the downregulation of neurotransmitter status of neurons or in the upregulation of peripheral neuronal regeneration. The GO terms and KEGG pathways involved in the downregulation effects included, but were not limited to, synaptic vesicle exocytosis, neurotransmitter secretion, voltage-gated potassium channel activity, regulation of synaptic transmission, GABAergic synapse, response to pain, endocytosis, neuronal action potential, detection of mechanical stimulus involved in sensory perception of pain, neurotransmitter transport, the GABAergic synapse pathway, the cholinergic synapse pathway, the neuroactive ligand-receptor interaction pathway, the dopaminergic synapse pathway, and the synaptic vesicle cycle pathway. The GO terms and KEGG pathways involved in the upregulation effects included, but were not limited to, response to mechanical stimulus, regulation of cell growth, positive regulation of cell migration, positive regulation of ERK1 and ERK2 cascade, positive regulation of PI3K signaling, activation of MAPKK activity, cell differentiation, regulation of neuron projection regeneration, regulation of nerve growth factor receptor activity, peripheral nervous system axon regeneration, glial cell differentiation, the AMPK signaling pathway, the calcium signaling pathway, the PI3K-Akt signaling pathway, the glucose metabolism pathway, the MAPK signaling pathway, and the cGMP-PKG signaling pathway.
The target gene from GO enrichment of known IncRNA apparently pointed to ENSRNOG00000006617 (P<0.05), thus we singled out the known IncRNA gene ENSRN0G00000006617 named rno-Cntnap2 (contactin associated protein-like 2). Through serial analyses of molecular network (Figure 2) and GO enrichment on gene rno-Cntnap2, we found 13 credible GO terms at P<0.05 (Table 6).
According to the 13 GO terms, rno-Cntnap2 had a putative gene function that is involved in the cell component of voltage-gated potassium channel complex on cell surface of brain neurites where it has an enzyme binding activity. Considering the condition of the current study, it was assumed that rno-Cntnap2 is involved in the cell component of voltage-gated potassium channel complex on cell surface of sciatic nerve neurites.
We reviewed the differential expression and co-expression network analysis of rno-Cntnap2 (ENSRNOG 00000006617) gene, which was down-regulated (20.34 and 3.94 for sham group vs DRG group, fold change -2.37, P< 0.001, Q=0.0003).
Novel IncRNA, co-expression network, and target gene features
We found 525 novel transcripts containing 26 novel IncRNAs referenced to rat IncRNA database (Table 7). We constructed the co-network of novel IncRNAs with mRNAs, and only 4 IncRNAs were related to 21 mRNAs under the conditions of thresholds of < 0.05 or 0.1 (Figure 3). The 4 IncRNAs were ENSRNOG00000055411, 000000 59555, 00000059564 and 00000057337 (Table 7).
We noticed that the transcript TCONS_00016823 included only one novel IncRNA gene, AC111653.1 (ENSRNOG00000057337), with a sense strand length of 828 nt. Gene AC111653.1 was null expressed in the sham-operation group and upregulated to 0.527889 in the DRG group. Gene AC111653.1 was correlated to the target gene ENSRNOG00000021452 named huntingtin interacting protein M (rno-Hypm). The rno-Hypm gene GO annotations included the molecular function of DNA binding and protein heterodimerization activity, the biological process of chromatin silencing, and the cellular component of nuclear chromatin and nucleosome. Huntingtin is essential for neuron survival, and the lack of huntingtin synthesis may lead to Huntington's disease (27). Up-regulation of both AC111653.1 and rno-Hypm genes after sciatic nerve injury implies a rescue course that triggers the regeneration of injured neurons. However, the function of Hypm gene is not completely understood.
Quantification of several genes
For known IncRNAs detection, we selected rno-Cntnap2 IncRNA gene and three down-regulated gene representatives, Fam171b (ENSRNOG00000004783), Hebp2 (ENSRNOG00000053735), and Gde1 (ENSRNOG00000 050445) from the rno-Cntnap2 gene coexpression network (Figure 2). Real time qPCR was performed to quantify expression levels of the four genes. The quantification results are shown in Table 8. Expression levels of the four genes were down-regulated. This result was consistent with the sequencing outcomes.
For novel IncRNAs identification, we selected AC111653.1 gene (ENSRNOG00000057337) and rno-Hypm gene (ENSRNOG00000021452) from the AC111653.1 gene coexpression network (Figure 3). They had a correlation coefficient of 1 (significance P=0). Up-regulation of both AC111653.1 and rno-Hypm genes after sciatic nerve injury may imply a rescue course that triggers the regeneration of injured neurons, thus the function of Hypm gene deserved to be studied. Real time qPCR was performed to quantify expression levels of the two genes (Table 8), which were up-regulated. This result was consistent with the sequencing outcomes.
To test the biological function of novel IncRNA AC111653.1 gene, we detected AC111653.1 and its target Hypm in primarily cultured Norway rat DRG cells in vitro. QPCR results are shown in Figure 4, and western blots in Figure 5. Novel IncRNA AC111653.1 was overexpressed after pcDNA3.1-lnc-AC111653.1 transfection. At the same time, its target hypm was also upregulated. Expression of AC111653.1 was reduced after siRNA transfection, and at the same time, its target hypm was downregulated. This suggested that novel IncRNA AC111653.1 was positively associated with hypm regulation in rats.
In this study, we used a common sciatic nerve injury model to investigate gene expression conditions in rat DRGs using a high-throughput Illumina HiSeq2500 sequencing. In total, 86 known IncRNAs and 26 novel IncRNAs were altered during nerve regeneration. To understand the functions of the 86 known IncRNAs, we analyzed the molecular network including 866 co-expressed target genes. After sciatic nerve damage, the nerve systems switched from a neurotransmitter status to a neuronal regeneration status (1, 3, 5).
Based on the GO and KEGG enrichment results, we found that the neurotransmitter status of neurons are deregulated by the molecular mechanisms linking to the deregulation of the neuroactive neurotransmitter secretion, transmission, and ligand-receptor interaction pathway, while the neuronal regeneration was activated through the molecular mechanisms linking to the positive regulation of cell migration, cell differentiation, cell growth, PI3K signaling, MAPK cascade activity, nerve growth factor receptor activity, and peripheral nerve regeneration.
Glial cells migration, dedifferentiation, differentiation, proliferation, and growth play important roles in peripheral nerve regeneration (1-4). The results in this study showed the promotion of glial cell migration and growth by multiple signaling pathways. After sciatic nerve damage, local Schwann cells can shed off the myelin sheaths and transform to a neuroblast status, where their proliferation and migration capacities can help to sweep away myelin remnants and generate a conduit for the axonal pathfinding, and consequently form the beneficial conditions for neurite outgrowth (1-3). The same IncRNA-linked nerve regeneration mechanism is identified by Yu et al. (16) and Yao et al. (18).
We singled out the known rno-Cntnap2 IncRNA gene, thought to be involved in the cell component of voltage-gated potassium channel complex on cell surface for the neurites of the sciatic nerve system. We speculated that sciatic injury might trigger a switch from a neurotransmitter status to a regeneration status of neurons. The gene mo-Cntnap2 may be involved in a neurotransmitter delivery process linking to the function of voltage-gated potassium channel complex. Thus, rno-Cntnap2 gene expression was down-regulated because a neurotransmitter status was ceased. The modulation of voltage-gated potassium channels by a IncRNA has been identified in DRG first-order sensory neurons in a spinal nerve ligation rat model (17). In this previous study, peripheral nerve injury increased a conserved IncRNA (Kcna2 antisense RNA) expression in injured DRG through activation of the transcription factor myeloid zinc finger protein 1. This increase of IncRNA downregulates the voltage-dependent potassium channel Kcna2 mRNA, consequently reducing total potassium current. The decrease of potassium current increases the neural excitability, namely neuropathy-induced sensitivity to mechanical stimuli in DRG neurons, resulting in neuropathic pain symptoms. The modulation of rno-Cntnap2 mRNA may also follow this molecular mechanism, though identification is required.
We further selected the transcript TCONS_00016823 containing only one novel IncRNA gene AC111653.1 (ENSRNOG0000005733). This IncRNA's upregulation improved the rno-Hypm gene expression, which promoted huntingtin synthesis regenerating sciatic nerves. We tested the biological function of novel IncRNA AC111653.1 in rat dorsal root ganglion cells. The overexpression of IncRNA AC111653.1 upregulated rno-Hypm gene substantially, indicating that this novel IncRNA is accurately associated with the huntingtin protein regulation.
The time-course factor should be considered a limitation because transcript levels vary depending on the time between the mechanic stimuli of nerve tying until the detection starts (16). Thus, time-dependent gene expression change and more testing on IncRNA functions should be done in the future. In addition, more annotations on genes should be investigated.
In conclusion, a total of 26 novel IncRNAs were found. Both down-regulated rno-Cntnap2 gene and up-regulated rno-Hypm gene were involved in neuropathic pain of DRGs after spared sciatic nerve injury, thus contributing to peripheral nerve regeneration via putative mechanisms.
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P. Mao , C.R. Li , S.Z. Zhang , Y. Zhang , B.T. Liu  and B.F. Fan 
 Department of Pain Medicine, China-Japan Friendship Hospital, Beijing, China
 State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing, China
Correspondence: B.F. Fan: <email@example.com>
Received September 10, 2017 | Accepted June 11, 2018
Caption: Figure 1. Genetic clustering of differential expression genes (threshold: fold-change >2). Red color represents high fold-change, green color represents low fold-change. Names were omitted due to many lines overlapping. DRG: dorsal root ganglia.
Caption: Figure 2. Co-expression network of gene rno-Cntnap2 (ENSRNOG00000006617).
Caption: Figure 3. Co-expression network of novel long non-coding RNA (IncRNAs) with target mRNAs. The network comprises nodes and edges. Central round nodes are IncRNAs, green rectangle nodes are mRNAs. Purple edges indicate positive correlation and blue edges indicate negative correlation.
Caption: Figure 4. QPCR relative mRNA quantification. Upper panel shows rno-IncRNA AC111653.1 levels. Lower panel shows rno-Hypm mRNA levels in each group. Negative ctrl: pcDNA3.1 vector transfection; overexpress: pcDNA3.1-lnc-AC111653.1 transfection; siRNA interfere: siRNA-lnc-AC111653.1 transfection; normal ctrl: normal cells without treatment.
Caption: Figure 5. Western blot images. Cells were transfected with pcDNA3.1-lnc-AC111653.1 for overexpression and with siRNA-Inc-AC111653.1 for RNA interference. pcDNA3.1 vector and scrambled RNA served as negative controls. *P<0.05 compared to the other groups (ANOVA).
Table 1. Primer sequences. Gene name Sequence (5' [right arrow] 3') mo-Cntnap2-201_F gcacctaccacaccaacga mo-Cntnap2-201_R tttgctctcgtcaatggtctct mo-Fam171b_5267F aggagttctgctttgctctgg mo-Fam 171b_5460R tccacacacaaccaagggta mo-Hebp2_415F agatccgacactacggacca mo-Hebp2_662R cctgggtggatcatgttgct mo-Gde1_20F acgggtctggccgattatgt mo-Gde1_586R gcactctgtaactgcttccct mo-GAPDH_1096F gcccagcaaggatactgaga mo-GAPDH_1252R ggtattcgagagaagggaggg mo-AC111653.1_201F agctacagtcatggaaacaccc mo-AC111653.1_201R agatagcctcagctttgctcact mo-Hypm_71F cgacatgatg gtttgatggt mo-Hypm_251R ccatgcttga ttaccttacc Table 2. Mapping results of transcriptome to referenced genome. Sample Total Reads (M) Total Mapped (M) Sham 1 34,697 27,586 (79.50%) Sham 2 31,648 25,293 (79.92%) Sham 3 40,603 30,892 (76.08%) DRG1 24,133 14,275 (59.15%) DRG2 25,319 17,235 (68.07%) DRG3 41,835 30,454 (72.80%) Sample Multiple Mapped (M) Uniquely Mapped (M) Sham 1 7,890 (22.74%) 19,696 (56.76%) Sham 2 7,034 (22.23%) 18,258 (57.69%) Sham 3 9,391 (23.13%) 21,501 (52.95%) DRG1 6,434 (26.66%) 7,840 (32.49%) DRG2 7,139 (28.20%) 10,096 (39.88%) DRG3 10,614 (25.37%) 19,841 (47.43%) DRG: dorsal root ganglia. Table 3. Number and distribution of known gene expression. Sample Genes Min. 1st Qu. Median Mean Sham 17856 0 0.83 4.75 151.65 DRG 17296 0 1.15 5.15 400.95 Sample Genes 3rd Qu. Max. Sd. Sum. Sham 17856 14.99 482618 5987.02 2707906 DRG 17296 14.28 1828940 19976.57 6934761 DRG: dorsal root ganglia. Table 4. Number and percentage of known gene expression. Sample 0-0.5 >0.5-1 > 1 -5 Sham 3634 (20.35%) 1184 (6.63%) 4322 (24%) DRG 2859 (16.53%) 1175 (6.79%) 4504 (26%) Sample >5-10 >10-50 > 50 Sham 2764 (15.48%) 4488 (25.13%) 1464 (0.08%) DRG 2996 (17.32%) 4416 (25.53%) 1346 (0.08%) DRG: dorsal root ganglia. Table 5. Known long non-coding RNA genes and their node degrees in Cytoscape co-expression network. nodes_label nodes_degree ENSRNOG00000002734 47 ENSRNOG00000003025 47 ENSRNOG00000005811 1 ENSRNOG00000006617 43 ENSRNOG00000009373 1 ENSRNOG00000011160 45 ENSRNOG00000017974 4 ENSRNOG00000019648 122 ENSRNOG00000024799 55 ENSRNOG00000031706 4 ENSRNOG00000033581 88 ENSRNOG00000043199 13 ENSRNOG00000043866 5 ENSRNOG00000046171 21 ENSRNOG00000046774 3 ENSRNOG00000047117 1 ENSRNOG00000048929 31 ENSRNOG00000049537 12 ENSRNOG00000051356 2 ENSRNOG00000051492 13 ENSRNOG00000051664 29 ENSRNOG00000051722 3 ENSRNOG00000051924 3 ENSRNOG00000052027 3 ENSRNOG00000052373 1 ENSRNOG00000052439 3 ENSRNOG00000052563 37 ENSRNOG00000052573 2 ENSRNOG00000053160 2 ENSRNOG00000053367 6 ENSRNOG00000053827 1 ENSRNOG00000054418 2 ENSRNOG00000054489 1 ENSRNOG00000054529 2 ENSRNOG00000054533 5 ENSRNOG00000054867 3 ENSRNOG00000054897 3 ENSRNOG00000054935 1 ENSRNOG00000054984 1 ENSRNOG00000055021 2 ENSRNOG00000055067 1 ENSRNOG00000055278 2 ENSRNOG00000055939 42 ENSRNOG00000056040 7 ENSRNOG00000056054 5 ENSRNOG00000056490 1 ENSRNOG00000056599 4 ENSRNOG00000056608 2 ENSRNOG00000056656 1 ENSRNOG00000056824 3 ENSRNOG00000057161 12 ENSRNOG00000057278 13 ENSRNOG00000057291 1 ENSRNOG00000057463 1 ENSRNOG00000057991 1 ENSRNOG00000058263 1 ENSRNOG00000058571 12 ENSRNOG00000058935 2 ENSRNOG00000058944 3 ENSRNOG00000059449 3 ENSRNOG00000059660 1 ENSRNOG00000060090 6 ENSRNOG00000060430 2 ENSRNOG00000060483 1 ENSRNOG00000060700 1 ENSRNOG00000060863 64 ENSRNOG00000061151 2 ENSRNOG00000061536 3 ENSRNOG00000061622 1 Table 6. Gene Ontology (GO) terms of rno-Cntnap2 long non-coding RNA gene. Category Term Class GO:0071205 protein localization biological_process to juxtaparanode region of axon GO:0044224 juxtaparanode region cellular_component of axon GO:0030673 axolemma cellular_component GO:0008076 voltage-gated potassium cellular_component channel complex GO:0019899 enzyme binding molecular_function GO:0043204 perikaryon cellular_component GO:0031175 neuron projection biological_process development GO:0005769 early endosome cellular_component GO:0007420 brain development biological_process GO:0030424 axon cellular_component GO:0030425 dendrite cellular_component GO:0043025 neuronal cell body cellular_component GO:0009986 cell surface cellular_component Category Gene_id GO:0071205 ENSRNOG00000006617 GO:0044224 ENSRNOG00000006617 GO:0030673 ENSRNOG00000006617 GO:0008076 ENSRNOG00000006617 GO:0019899 ENSRNOG00000006617 GO:0043204 ENSRNOG00000006617 GO:0031175 ENSRNOG00000006617 GO:0005769 ENSRNOG00000006617 GO:0007420 ENSRNOG00000006617 GO:0030424 ENSRNOG00000006617 GO:0030425 ENSRNOG00000006617 GO:0043025 ENSRNOG00000006617 GO:0009986 ENSRNOG00000006617 Table 7. Transcripts of 26 novel long non-coding RNA genes, of which 4 (#-in bold) are involved in co-expression network with target mRNA genes. Transcript Length bp gene_id TCONS_00000233 509 ENSRNOG00000058258 TCONS_00000914 1884 ENSRNOG00000058637 TCONS_00001056# 586# ENSRNOG00000056324# TCONS_00002131 879 ENSRNOG00000055411 TCONS_00002609 194 ENSRNOG00000060612 TCONS_00003909 1655 ENSRNOG00000054067 TCONS_00009651# 593# ENSRNOG00000059555# TCONS_00011039 350 ENSRNOG00000061204 TCONS_00012047 330 ENSRNOG00000035462 TCONS_00012411 134 ENSRNOG00000052738 TCONS_00014951# 153# ENSRNOG00000059564# TCONS_00015936 235 ENSRNOG00000035501 TCONS_00016823# 828# ENSRNOG00000057337# TCONS_00016834 220 ENSRNOG00000036434 TCONS_00017920 149 ENSRNOG00000058995 TCONS_00019224 1441 ENSRNOG00000000809 TCONS_00021430 262 ENSRNOG00000053319 TCONS_00021456 187 ENSRNOG00000047611 TCONS_00023970 2730 ENSRNOG00000056448 TCONS_00028110 255 ENSRNOG00000036492 TCONS_00028111 217 ENSRNOG00000040358 TCONS_00028926 281 ENSRNOG00000047126 TCONS_00033369 1000 ENSRNOG00000057258 TCONS_00034793 312 ENSRNOG00000032609 TCONS_00035961 1447 ENSRNOG00000051245 TCONS_00036296 341 ENSRNOG00000035579 Table 8. Real time qPCR quantification of interest/GAPDH (interest DRG/Sham) gene expression levels. Sham DRG1 DRG2 rno-Cntnap2-20 0.0530 (1.0000) 0.0074 (0.1411) 0.0069 (0.1310) IncRNA rno-Fam171b 0.0265 (1.0000) 0.0053 (0.1993) 0.0012 (0.0446) rno-Hebp2 0.0417 (1.0000) 0.0112 (0.2673) 0.0145 (0.3475) rno-Gde1 0.0128 (1.0000) 0.0097 (0.7526) 0.0042 (0.3231) rno-AC111653.1 0 0.08652 0.04228 IncRNA rno-Hypm 0 0.29365 0.1263 DRG3 Sequencing (Sham/DRG) rno-Cntnap2-20 0.0039 (0.0736) 20.3371/3.9422 IncRNA rno-Fam171b 0.0026 (0.0981) 19.7562/6.0137 rno-Hebp2 0.0082 (0.1966) 97.8422/22.9642 rno-Gde1 0.0065 (0.5078) 93.1178/36.7535 rno-AC111653.1 0.03684 0/0.52789 IncRNA rno-Hypm 0.1046 0/2.6653 DRG: dorsal root ganglia. The numbers outside parenthesis indicate the gene expression ratio of interest to GAPDH and those inside indicate the gene expression ratio of interest DRG groups to the Sham group.
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|Title Annotation:||Research Article|
|Author:||Mao, P.; Li, C.R.; Zhang, S.Z.; Zhang, Y.; Liu, B.T.; Fan, B.F.|
|Publication:||Brazilian Journal of Medical and Biological Research|
|Date:||Oct 1, 2018|
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