IJMM International Journal of Molecular Medicine 1107-3756 1791-244X D.A. Spandidos 10.3892/ijmm.2018.4038 ijmm-43-03-1179 Articles Identification of the key genes and long non-coding RNAs in ankylosing spondylitis using RNA sequencing XuZhengkuan ZhouXiaopeng LiHao ChenQixin ChenGang Department of Orthopedics, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China Correspondence to: Dr Gang Chen, Department of Orthopedics, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jie Fang Road, Hangzhou, Zhejiang 310009, P.R. China, E-mail: chengang1976@zju.edu.cn 03 2019 20 12 2018 43 3 1179 1192 12 04 2018 07 12 2018 Copyright: © Xu et al. 2018 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Ankylosing spondylitis (AS) is an insidious and debilitating form of arthritis that involves the axial skeleton, and its etiology and pathogenesis remain unclear. In the present study, three patients with AS and three normal controls from our hospital were enrolled. RNA sequencing and bioinformatics analysis were performed in order to identify the differentially expressed (DE) mRNAs (DEmRNAs) and DE long non-coding RNAs (DElncRNAs) between the patients with AS and normal controls. Construction of an AS-specific protein-protein interaction network, a weighted DElncRNA-DEmRNA co-expression network and functional annotation of the DEmRNAs co-expressed with DElncRNAs was performed. Nearby cis-targeted DEmRNAs or DElncRNAs were identified by searching for DEmRNAs that were transcribed within 100-kb up- or downstream of DElncRNAs. Based on the Gene Expression Omnibus datasets GSE25101 and GSE73754, the expression of selected DEmRNAs and DElncRNAs were verified using published RNA sequencing data from blood samples, and receiver operating characteristic analysis of selected DEmRNAs was performed. Compared with the normal controls, 1,072 DEmRNAs and 372 DElncRNAs in the patients with AS were identified. Caspase recruitment domain family member 11 and DNA methyltransferase 1 have great diagnostic value for AS. MSTRG.8559 and LINC00987 were also identified as two hub DElncRNAs. The T-cell receptor signaling pathway was a significantly enriched pathway of the DEmRNAs co-expressed with DElncRNAs in patients with AS. In conclusion, the present study identified the key DEmRNAs and DElncRNAs in AS, which provides novel information for understanding the pathogenesis of AS and developing potential biomarkers for AS.

 ankylosing spondylitis RNA sequencing lncRNAs Introduction

Ankylosing spondylitis (AS) is the prototypic and debilitating type of spondyloarthritis, which is an autoimmune disorder (1). AS often affects the axial joints, including the sacroiliac joint and spine, and induces new bone formation and ultimately ankylosis (2,3).

AS is associated with the interplay of genetic risks and environmental triggers, and its etiology and pathogenesis remain largely unknown. A number of studies have reported a strong association between the major histocompatibility complex class I allele human leukocyte antigen B27 (HLA-B27) and AS (4-6). However, the mechanism by which HLA-B27 causes a predisposition to AS remains unclear. T cells and a number of immune pathways, including the interleukin (IL)-17/IL-23 pathway and control of nuclear factor-κB (NF-κB) activation, have been reported to be closely associated with AS (5,6). Currently, it is difficult to diagnose AS during the early stages due to the lack of accurate diagnostic biomarkers.

Long non-coding RNA (lncRNA) is a type of non-protein-coding transcript with a length of >200 nucleotides; they are emerging as key regulators in various biological processes (7). Recent studies have indicated that lncRNAs, including lncRNA-AK001085, lnc-zinc finger protein 354A (ZNF354A)-1, lnc-Lin-54 DREAM MuvB core complex component (LIN54)-1, lnc-Facioscapulohumeral muscular dystrophy region gene 2 family member C (FRG2C)-3 and lnc-ubiquitin specific peptidase 50 (USP50)-2, serve roles in AS (8,9). Some researchers have also revealed that mRNAs, such as programmed cell death 1 (PDCD1) (1), caspase recruitment domain-containing protein 11 (CARD11) (10), phospholipase Cγ1 (PLCG1) (11,12) and DNA methyltransferase 1 (DNMT1) (13), may be involved in the pathogenesis of AS.

In the present study, the key differentially expressed (DE) mRNAs (DEmRNAs) and DElncRNAs in AS were identified using RNA sequencing and bioinformatics analysis. DElncRNA-DEmRNA co-expression network construction, identification of nearby target DEmRNAs of DElncRNAs and functional annotation of DEmRNAs were performed in order to understand the biological functions of the key DEmRNAs and DElncRNAs in AS.

 Materials and methods Patients and samples

Three patients with AS and three normal controls were enrolled in the present study from 2nd Affiliated Hospital, School of Medicine, Zhejiang University (Zhejiang, China). The patients with AS were aged 37, 36 and 40 years old, and all were HLA-B27+ and male. These patients were diagnosed with AS based on kyphotic deformity, bilateral damage of the sacroiliac joint observed in the computed tomography results, and spinal fusion and sacroiliac arthrodesis observed in the X-ray results. All of the patients had not received treatment with non-steroidal anti-inflammatory drugs or biologics and they had not exhibited complications. The normal controls were aged 35, 36 and 37 years old and were male. None of the six participants had any other type of autoimmune disease. Blood samples were obtained from all six participants. All of the participants submitted written informed consent and the present study was approved by the Ethics Committee of 2nd Affiliated Hospital, School of Medicine, Zhejiang University.

 Library preparation and high-throughput sequencing

Total RNA was isolated from the blood samples with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. A NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Inc.) was used to check the concentration and purity of the RNA. The integrity of the RNA was confirmed using 2% agarose gel and the RNA integrity number (RIN) was obtained using an Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Inc., Santa Clara, CA, USA). The thresholds of total RNA for cDNA library construction were as follows: i) Amount of RNA, >5 μg; ii) concentration of RNA; ≥200 ng/ml; iii) 1.8<optical density (OD)260/280<2.2; iv) 2.0<OD260/280; and v) RIN value, >8.0.

Firstly, the ribosomal RNA was removed from the total RNA using a Ribo-Zero Magnetic kit (Epicentre; Illumina, Inc., San Diego, CA, USA). In addition, 'Soap' software (version 1.03; soap.genomics.org.cn/SOAPdenovo-Trans.html#intro2) was used to compare the reads of the rRNA, and then to write a perl script to remove it from the FastQ file. Subsequently, the cDNA library for RNA sequencing was contrasted using the TruSeq RNA Sample Prep kit (Illumina, Inc.). Briefly, the retrieved RNA was fragmented by adding First Strand Master Mix and then first-strand cDNA was generated using the First Strand Master mix and Super Script II reverse transcription (Invitrogen; Thermo Fisher Scientific, Inc.) with the following reaction conditions: 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. Following purification of the product with Agencourt RNA Clean XP beads (Beckman Coulter, Inc., Brea, CA, USA), Second Strand Master mix and dATP, dGTP, dCTP and dUTP mix (Beckman Coulter, Inc.) were added to synthesize the second-strand cDNA (16°C for 1 h). Subsequently, the purified cDNA was combined with End Repair Mix (Thermo Fisher Scientific, Inc.) and incubated at 30°C for 30 min. Following purification with beads (Qiagen, Inc., Valencia, CA, USA), A-Tailing mix (Qiagen, Inc.) was added into the reaction system, which was incubated at 37°C for 30 min. Adenylate 3' Ends DNA, Index Adapter and Ligation mix (Qiagen, Inc.) were combined and incubated at 30°C for 10 min. Subsequently, the Uracil-N-Glycosylase enzyme was added into the purified ligation product and incubated at 37°C for 10 min. A total of 15 rounds of polymerase chain reaction (PCR) amplification were conducted with PCR Primer Cocktail (Illumina, Inc.) and PCR Master Mix (Illumina, Inc.) to enrich the cDNA fragments. The PCR products were then purified with AMPure XP beads (Qiagen, Inc.). The qualified libraries were amplified on cBot (Illumina, Inc.) to generate a cluster on the flow cell (TruSeq PE Cluster kit v3-cBot-HS; Illumina, Inc.). The amplified flow cell was sequenced on an Illumina HiSeq X Ten platform (Illumina, Inc.).

 Quality control of raw sequence

Quality control of the raw reads derived from the RNA sequencing was performed to obtain clean reads of high quality. Briefly, quality control involved trimming low-quality reads, including adaptor sequences, sequences with a quality score <20 and sequences with an N-base rate of raw reads >10%, using SeqPrep (version 1.2; github.com/jstjohn/SeqPrep) and Sickle (version V3.4.0; github.com/najoshi/sickle).

 Clean reads mapping

Sequences were aligned to the human reference genome GRCh38.p7 (Ensembl v84; www.ensembl.org/index.html) using hierarchical indexing for the spliced alignment of the transcripts programme HISAT2 (version 2.1.0; ccb.jhu.edu/software/hisat2/index.shtml). Then, StringTie (version v1.3.4; ccb.jhu.edu/software/stringtie/) was used to assemble and quantify the transcripts in each sample. Ultimately, differential gene expression analysis was performed with Ballgown (version 3.7; www.bioconductor.org/packages/release/bioc/html/ballgown.html) in R environment.

 Identification of DEmRNAs and DElncRNAs

Using Ballgown, the DEmRNAs and DElncRNAs between the patients with AS and normal controls were identified with P<0.05. The false discovering rate (FDR)-adjusted P-value of the test statistic was used. Hierarchical clustering of the DElncRNAs and DEmRNAs expression profile was performed using hcluster in R language (version 3.3.3; stat.ethz.ch/R-manual/R-devel/library/stats/html/hclust.html).

 AS-specific protein-protein interaction (PPI) network construction

PPI networks of the top 30 up- and down-regulated DEmRNAs, respectively, were constructed using BioGRID (version 3.5; thebiogrid.org).

 AS-specific weighted DEmRNA-DElncRNA co-expression network analyses

Weighted Gene Co-expression Network Analysis (WGCNA_1.64-1; horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/) (14) is an R package for weighted correlation network analysis, which is also known as weighted gene co-expression network analysis. Using WGCNA, AS-specific weighted DEmRNA-DElncRNA co-expression network analysis was performed. The pairwise Pearson's correlation coefficients (PCCs) between the DElncRNAs and DEmRNAs in patients with AS were calculated. DElncRNA-DEmRNA pairs with |PCC value ≥0.90| and P<0.001 were used to construct an AS-specific weighted DEmRNA-DElncRNA co-expression network, which was deciphered using the online-based software GeneCodis3 (genecodis.cnb.csic.es/analysis).

 Functional analyses of DEmRNAs co-expressed with DElncRNAs in AS

The DEmRNAs of these DElncRNA-DEmRNA pairs with |PCC value ≥0.90| and P<0.001 were used to conduct Gene Ontology (GO; www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; www.genome.jp/kegg/) molecular pathway enrichment analysis using GeneCoDis3.

 DEmRNAs close to DElncRNAs with cis-regulatory effects

Previous studies have reported that lncRNAs can regulate genes that are transcribed near to them, consistent with activity in cis-regulatory elements (15,16). Therefore, in the present study DEmRNAs that were close cis targets of DElncRNAs were identified by searching for DEmRNAs that were transcribed within a 100-kb window up- or downstream of DElncRNAs between the patients with AS and the normal controls (17).

 Validation of the expression of DEmRNAs and DElncRNAs using the GSE73754 and GSE25101 datasets

The mRNA expression profile of 52 patients with AS and 20 normal controls (Canadian cohort) in the GSE73754 dataset (GPL10558 Illumina HumanHT-12 V4.0 expression beadchip), which was downloaded from the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/gds). The mRNA and lncRNA expression profile of 16 patients with AS and 16 normal controls (Australian cohort) in the GSE25101 dataset (GPL6947 Illumina HumanHT-12 V3.0 expression beadchip) was also downloaded from the GEO database. The expression levels of the selected DEmRNAs and DElncRNAs between the patients with AS and normal controls in the present study were validated using the GSE73754 and GSE25101 datasets, and the difference in the expression levels was visualized using box plots.

 Receiver operating characteristic (ROC) analyses

To assess the diagnostic value of the DEmRNAs in AS, ROC analyses were conducted using pROC package (version 1.13.0) in R language (cran.r-project.org/web/packages/pROC/index.html). The area under the curve (AUC) under the binomial exact confidence interval was calculated. ROC curves were then generated.

 Statistical analysis

Values are displayed as the mean ± standard deviation. Student's t-test was used to assess differences among the groups, and P≤0.05 was considered to indicate a statistically significant difference. Co-expression associations between the lncRNAs and the protein-coding genes were estimated using pairwise PCC analysis using R language (version 3.3.3; stat. ethz.ch/R-manual/R-devel/library/stats/html/hclust.html).

 Results Identification of DEmRNAs and DElncRNAs between patients with AS and normal controls

Compared with the normal controls, 1,072 DEmRNAs (320 upregulated and 752 down-regulated) and 372 DElncRNAs (230 upregulated and 142 downregulated) in patients with AS were identified. The top 10 up- and downregulated DEmRNAs and DElncRNAs between the patients with AS and normal controls are shown in Tables I and II, respectively.

MANSC domain containing 1 (MANSC1) and DNMT1 were the most significantly up- and downregulated DEmRNAs between the patients with AS and normal controls, respectively (data not shown). NOTCH1 associated lncRNA in T-cell acute lymphoblastic leukemia 1 (NALT1) and RP11-837J7.4 were the most significantly up- and downregulated DElncRNAs between the patients with AS and normal controls, respectively (data not shown).

 AS-specific PPI network construction

The AS-specific PPI network consisted of 159 nodes and 164 edges. The top 10 mRNAs that had the highest degrees were Myc proto-oncogene basic helix-loop-helix transcription factor (MYC; degree=65), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1; degree= 40), spectrin-a non-erythrocytic 1 (SPTAN1; degree=18), TATA-box binding protein associated factor 10 (TAF10; degree=9), ETS proto-oncogene 1 transcription factor (degree=7), NCK adaptor protein 1 (degree=6), eukaryotic translation initiation factor 4B (degree=5), KIAA1033 (degree=5), extended synaptotagmin 1 (degree=5) and tumor protein 53 (degree=5); of these, MYC, HNRNPA1, SPTAN1 and TAF10 were hub proteins of the AS-specific PPI network (Fig. 1).

 AS-specific weighted DEmRNA-DElncRNA co-expression network analysis

A total of 3,505 lncRNA-mRNA pairs were identified, which included 302 DElncRNAs and 602 DEmRNAs with |PCC value ≥0.90| and P<0.001. Based on these lncRNA-mRNA pairs, the negatively and positively co-expressed DEmRNA-DElncRNA interaction networks were constructed, respectively. The top 100 co-expressed DEmRNA-DElncRNA interaction network is presented in Table III. Based on the positively co-expressed DEmRNA-DElncRNA interaction network, MSTRG.8559 (degree=226) and long intergenic non-protein coding RNA 987 (LINC00987; degree=209) were the top 2 DElncRNAs that were co-expressed with the greatest number of DEmRNAs (Fig. 2). The selected co-expression DEmRNA-DElncRNA interaction network is presented in Fig. 2.

 Functional analysis of DEmRNAs co-expressing DElncRNAs between patients with AS and normal controls

Based on the GO enrichment analysis (Fig. 3) of the 602 DEmRNAs that were co-expressed with DElncRNAs in patients with AS and normal controls, the significantly enriched GO terms were as follows: 'DNA repair' (FDR=3.94×10−4; Fig. 3A), 'carbohydrate metabolic process' (FDR=5.66×10−4; Fig. 3A), 'nucleus' (FDR=3.34×10−30; Fig. 3B) and 'protein binding' (FDR=7.54×10−31; Fig. 3C). 'T-cell receptor signaling pathway' (FDR=2.29×10−3; Fig. 4) and 'cell cycle' (FDR=1.52×10−3; Fig. 4) were the significantly enriched KEGG pathways.

 DEmRNAs close to DElncRNAs with cis-regulatory effects

A total of 84 DElncRNAs and nearby cis target DEmRNA pairs, which included 73 DElncRNAs and 82 DEmRNAs, were obtained (Table IV).

 Validation of the expression of DEmRNAs and DElncRNAs using the GSE73754 and GSE25101 datasets

A total of 4 DEmRNAs (DNMT1, PDCD1, CARD11 and PLCG1) were selected to perform expression validation using the GSE73754 dataset (Fig. 5). The expression of all four DElncRNAs was downregulated in patients with AS when compared with the normal controls, which was generally consistent with the RNA sequencing data (DNMT1, P<0.05; CARD11, P<0.05; PDCD1, P>0.05; PLCG1, P>0.05). Three lncRNAs (cat eye syndrome chromosome region candidate 7, hydatidiform mole associated and imprinted and KIAA0125) and seven DEmRNAs [MANSC1, adenosine triphosphatase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), myeloid zinc finger 1 (MZF1), PDCD1, DNMT1, CARD11 and PLCG1] were selected to perform expression validation using the GSE25101 dataset (Fig. 6). The expression of KIAA0125 (P>0.05), MANSC1 (P>0.05), ATP2A2 (P>0.05), MZF1 (P>0.05), PDCD1 (P>0.05), DNMT1 (P<0.0001), CARD11 (P<0.05) and PLCG1 (P>0.05) was generally consistent with the RNA sequencing data.

 ROC curve analysis

ROC curve analyses and the AUC were used to assess the discriminatory ability of the four DEmRNAs (DNMT1, PDCD1, CARD11 and PLCG1) among the 52 patients with AS and 20 normal controls of the GSE73754 dataset. The AUCs of PDCD1 and PLCG1 were <0.7 (data not shown). The AUCs of CARD11 and DNMT1 were 0.782 and 0.737, respectively (Fig. 7). For AS diagnosis, the sensitivity (proportion of true positive) and 1-specificity (proportion of false positive) of CARD11 were 70.0 and 80.8%, respectively, and for DNMT1 were 75.0 and 69.2%, respectively (Fig. 7).

 Discussion

AS is a type of autoimmune disorder that is associated with HLB-27 and T-cells; however, its etiology and pathogenesis remain unclear (18). The delays in the diagnosis of AS and the insufficient responses to the currently available therapeutics supports the requirement for a greater understanding of its pathogenesis.

A previous microarray study identified four lncRNAs, lnc-ZNF354A-1, lnc-LIN54-1, lnc-FRG2C-3 and lnc-USP50-2, that are involved in the abnormal osteogenic differentiation of mesenchymal stem cells (MSCs) in patients with AS. The expression of these four lncRNAs was positively correlated with that of bone morphogenetic protein 2 and Noggin in MSCs from healthy donors (8). A recent study reported that lncRNA-AK001085 was downregulated in patients with AS and served as a potential diagnostic indicator, thus, lncRNA-AK001085 was considered to be a potential suppressor of AS (9). Due to the lack of research, the regulatory mechanism of the majority of lncRNAs in AS remains unknown. In the present study, the key DEmRNAs and DElncRNAs associated with AS were identified and their functions in AS were investigated using RNA sequencing and bioinformatics analysis.

LINC00342 was demonstrated to be upregulated in patients with non-small cell lung cancer and its expression was positively correlated with lymph node metastasis and the Tumor-Node-Metastasis stage (19). Coiled-coil domain containing 26 (CCDC26) is also a tumor-associated lncRNA that regulates the growth of glioma, pancreatic cancer and myeloid leukemia cells (20-22). In the present study, LINC00342 and CCDC26 were downregulated in patients with AS, which suggests that they may serve roles in AS. Further studies are required in order to identify their precise function in AS.

Furthermore, the results of the present study indicated that many novel DElncRNAs may be involved in AS. In order to investigate their functions in AS, a weighted DElncRNA-DEmRNA co-expression network was constructed and functional annotation of the DElncRNAs co-expressed with DElncRNAs was performed. A total of 3,505 DElncRNA-DEmRNA co-expression pairs, which included 302 DElncRNAs and 602 DEmRNAs, were obtained. Based on the DEmRNAs co-expressed with DElncRNAs, 'T-cell receptor signaling pathway' was a significantly enriched pathway. T-cells have been demonstrated by previous studies to serve important roles in the pathology of AS (23-25). HLA-B27-reactive cluster of differentiation-4+ T-cells have been reported to be involved in the pathogenesis of spondyloarthropathies (26). The number of peripheral T-helper (Th)-2 and Th17 lymphocytes has been demonstrated to be increased in AS, which is suggestive of their potential roles in AS (27,28). Therefore, the results of the present study support those of previous studies as well as the importance of the T-cell receptor signaling pathway in AS. Furthermore, the DEmRNAs that were enriched in the T-cell receptor signaling pathway, including NF-κB inhibitor β, CARD11, P21 (Ras-related C3 botulinum toxin substrate 1) activated kinase 6, protein kinase Cθ, PLCG1, PDCD1, FYN proto-oncogene Src family tyrosine kinase, protein kinase B2 and Vav guanine nucleotide exchange factor 3, may be involved with AS by regulating the T-cell receptor signaling pathway.

Among these DEmRNAs, PDCD1 is a known AS-associated gene. PDCD1 is a member of the immunoglobulin superfamily that is expressed on the surface of peripheral T-cells, and regulates T-cell responses and the maintenance of peripheral tolerance (29,30). A previous study demonstrated that the expression levels of PDCD1 on activated T-cells were decreased in patients with AS (1). Downregulation of PDCD1 may be involved in AS by stimulating the activity of T-cells and elevating the production of cytokines, which promotes spinal inflammation and destruction in patients with AS (1). Downregulated PDCD1 was also identified in patients with AS in the present study, which provides evidence to support the results of the previous study.

CARD11 is a shared member of the CARD and CARD-containing membrane-associated guanylate kinase protein 1 families that has been reported to serve a vital role in regulation of inflammation and the immune response (31). Although to the best of our knowledge, there have been no studies that have reported on the association between CARD11 and AS, downregulated CARD11 has been implicated in another type of autoimmune disease, rheumatoid arthritis, via NF-κB activation, reduced Th17 responses and the decreased production of proinflammatory cytokines, including IL-1β, IL-6 and IL-17 (10,32,33). Joint inflammation and destruction were demonstrated to be attenuated by CARD11 small interfering RNA treatment in mice (10). Considering the crucial roles of NF-κB activation, Th17 cells and proinflammatory cytokines in AS, it was hypothesized that CARD11 may also be a key regulator of AS.

Similar to CARD11, PLCG1 has been reported to be associated with other types of autoimmune disease, including multiple sclerosis and lymphoproliferative syndrome (11,12). Furthermore, the interaction between PLCG1 and linker for activation of T-cells (LAT) was revealed to be involved with the activation and proliferation of T-cells (11,12). The production of IL-6 by T-cells is also regulated by PLCG1-LAT (11,12). Therefore, downregulated PLCG1 in the patients with AS in the present study may serve important roles in the progression of AS by regulating T-cells and the production of IL-6.

DNMT1 encodes an enzyme that establishes and regulates patterns of methylated cytosine residues (13). A previous study observed downregulated and hypermethylated DNMT1 in the peripheral blood mononuclear cells of patients with AS when compared with normal controls, which suggests that DNMT1 may be a potential biomarker of AS (13). Thus, the downregulation of DNMT1 observed in patients with AS in the present study is consistent with this previous study.

Based on the ROC analysis in the present study, CARD11 and DNMT1 may have great diagnostic value for AS, and therefore may be potential biomarkers.

LINC00987 was a downregulated lncRNA in the patients with AS in the present study, and was co-expressed with DNMT1, CARD11 and PLCG1. In addition, DNMT1 and PLCG1 were co-expressed with another downregulated lncRNA, MSTRG.8559, in the patients with AS. Furthermore, LINC00987 and MSTRG.8559 were two hub lncRNAs of the positively co-expressed DElncRNA-DEmRNA network, which regulates the majority of the DEmRNAs in AS. It was hypoth-esized that these two DElncRNAs may serve crucial roles by regulating the expression of DNMT1, the T-cell receptor signaling pathway and its associated genes. Further studies are required to further investigate the biological functions of LINC00987 and MSTRG.8559, particularly those in AS.

RP11-837J7.4 and NALT1 were the most significantly down- and upregulated, respectively, DElncRNAs in patients with AS in the present study; however, their biological functions remain known. Further studies are required in order to identify whether these two DElncRNAs could serve as diagnostic biomarkers for AS.

Previous studies have revealed the prevalence of lncRNA-mediated cis regulation on nearby transcription (34-36). The 84 AS-specific DElncRNA and nearby cis target DEmRNA pairs obtained in the present study provide novel information for understanding the biological functions of lncRNAs in AS.

In conclusion, the present study obtained lncRNA and mRNA expression profiles from patients with AS and normal controls using RNA sequencing. A number of key genes, including PDCD1, DNMT1, CARD11 and PLCG1, that are involved in AS were identified. In addition, the results indicated that numerous novel DElncRNAs may be involved in AS. Furthermore, the functions of DElncRNAs in AS were investigated using functional annotation of DEmRNAs co-expressed with DElncRNAs and through the identification of nearby target DEmRNAs of DElncRNAs. These results may support further studies on the potential roles of lncRNAs in AS. However, the sample size for RNA-seq was small, which is a limitation of the present study, therefore, studies with larger sample sizes are required in order to confirm this conclusion.

 Funding

No funding was received.

 Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Authors' contributions

ZX and GC conceived and designed the experiments. ZX, XZ and HL performed the experiments. ZX, XZ and QC analyzed the data. HL and QC were significant contributors in the manuscript. All authors revised the manuscript and have agreed to the publication of this manuscript.

 Ethics approval and consent to participate

All of the participants submitted written informed consent and the present study was approved by the Ethics Committee of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University (Zhejiang, China).

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 Abbreviations AS

ankylosing spondylitis

 DEmRNAs

differentially expressed mRNAs

 DElncRNAs

differentially expressed long non-coding RNAs

 GO

Gene Ontology

 KEGG

Kyoto Encyclopedia of Genes and Genomes

 PPI

protein-protein interaction

 Acknowledgments

The authors would like to thank Beijing Yangshen Bioinformatic Technology (Beijing, China) for their assistance during high-throughput sequencing and data analysis.

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AS-specific PPI network. Blue and red shapes represent proteins encoded by down- and upregulated DEmRNAs, respectively, when comparing patients with AS and normal controls. Among these, blue shapes with green outlines represent proteins encoded by the top 10 downregulated DEmRNAs; red shapes with red outlines represent proteins encoded by the top 10 upregulated DEmRNAs. AS, ankylosing spondylitis; DEmRNA, differentially expressed mRNA.

Selected DElncRNA-DEmRNA co-expression network. Ovals and rhombuses represent DEmRNAs and DElncRNAs, respectively, comparing AS patients and normal controls. Shapes with bold black outlines indicate the top 10 DEmRNAs/DElncRNAs when comparing AS and normal controls. AS, ankylosing spondylitis; DEmRNA, differentially expressed mRNA; DElncRNA, differentially expressed long non-coding RNA.

Top 15 most significantly enriched GO terms of DEmRNAs co-expressed with DElncRNAs. The x-axis presents the number of DEmRNAs enriched in the GO terms and the y-axis presents the GO terms. (A) Biological process; (B) Cellular component; (C) Molecular function. GO, Gene Ontology; DEmRNA, differentially expressed mRNA; DElncRNA, differentially expressed long non-coding RNA; FDR, false discovery rate.

Top 15 most significantly enriched KEGG pathways of DEmRNAs co-expressed with DElncRNAs. The x-axis presents the number of DEmRNAs enriched in KEGG pathways and the y-axis presents the KEGG pathways. KEGG, Kyoto Encyclopedia of Genes and Genomes; DEmRNA, differentially expressed mRNA; DElncRNA, differentially expressed long non-coding RNA; FDR, false discovery rate.

Validation the expression of selected DEmRNAs between patients with AS and normal controls in the GSE73754 dataset. The x-axis presents the AS and normal control groups and the y-axis presents the expression levels of (A) CARD11, (B) DNMT1, (C) PDCD1 and (D) PLCG1. Data are presented as the median and the 75th and 25th percentiles. *P<0.05 vs. control. AS, ankylosing spondylitis; DEmRNA, differentially expressed mRNA; CARD11, caspase recruitment domain-containing protein 11; DNMT1, DNA methyltransferase 1; PDCD1, programmed cell death 1; PLCG1, phospholipase Cγ1.

Validation of the expression of selected DEmRNAs and DElncRNAs between patients with AS and the normal controls in the GSE25101 dataset. The x-axis presents the AS and normal control groups and the y-axis presents the expression levels. (A) The selected DEmRNAs and (B) The selected DElncRNAs. AS, ankylosing spondylitis; DEmRNA, differentially expressed mRNA; DElncRNA, differentially expressed long non-coding RNA; CARD11, caspase recruitment domain-containing protein 11; DNMT1, DNA methyltransferase 1; PDCD1, programmed cell death 1; PLCG1, phospholipase Cγ1; MANSC1, MANSC domain containing 1; ATP2A2, adenosine triphosphatase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2; MZF1, myeloid zinc finger 1; CECR7, cat eye syndrome chromosome region candidate 7; HYMAI, hydatidiform mole associated and imprinted.

ROC curves of CARD11 and DNMT1 comparing patients with ankylosing spondylitis and normal controls in the GSE73754 dataset. ROC curves displayed the diagnostic ability of differentially expressed long non-coding RNA with sensitivity (the proportion of true positive) and 1-Specificity (the proportion of false positive). The x-axis presents 1-specificity and y-axis presents sensitivity. (A) CARD11 and (B) DNMT1. ROC, receiver operating characteristic; CARD11, caspase recruitment domain-containing protein 11; DNMT1, DNA methyltransferase 1; AUC, area under the curve.

Top 10 up- and downregulated DEmRNAs between ankylosing spondylitis and normal controls.

DEmRNAs log2.Fold change P-value SD_fpkm_C SD_fpkm_T Regulation
IGHG1 −2.021979095 0.047255959 8.80361236 0.515381703 Down
IGHG4 −1.859252007 0.025210597 3.368882506 0.227307135 Down
TRAJ18 −1.643179443 0.035866398 10.07677638 4.09159482 Down
TRAJ11 −1.524711275 0.014505975 8.654159887 4.620202967 Down
TRAJ5 −1.49467071 0.000440647 6.737567008 2.426348758 Down
TRAJ44 −1.46271809 0.001143618 4.42364856 1.848651282 Down
ETS1 −1.335519855 0.031765089 32.5763664 9.312805914 Down
LAIR2 −1.302776176 0.044790098 0.834353066 1.050550336 Down
ESYT1 −1.259200819 0.002673524 15.14634661 6.658023234 Down
NCR3 −1.247053856 0.033379705 5.641796936 2.719067002 Down
CLC 1.194865831 0.043003696 21.60725157 83.0330712 Up
S100A12 0.98788469 0.00518168 40.98384277 110.0364144 Up
CMPK2 0.904925259 0.01474919 0.622597776 2.021786682 Up
ZFP36 0.842014426 0.012071156 6.481954564 7.910723637 Up
MME 0.746984037 0.031312145 9.013396028 20.24882244 Up
LIN7A 0.711395439 0.049387957 3.456739121 4.043733587 Up
CYP4F3 0.707298362 0.025967428 4.648357447 8.950444777 Up
CXCL8 0.651965182 0.048148598 6.882829746 11.04384805 Up
CLEC4D 0.636486225 0.047074356 2.579585737 3.692774365 Up
FAM118A 0.634156346 0.010715671 0.992441488 2.574184644 Up

DEmRNAs, differentially expressed mRNAs; fpkm, fragments per kilobase million; C, control group; T, case group.

Top 10 up- and downregulated DElncRNAs between the patients with ankylosing spondylitis and the normal controls.

DElncRNAs log2.Fold change P-value SD_fpkm_C SD_fpkm_T Regulation
MSTRG.9221 −2.900321700 0.018550654 4.278074312 0.276811741 Down
MSTRG.1368 −2.619486415 0.003067593 1.673345961 0 Down
AL928768.3 −2.320417178 0.018613919 1.432426837 0.766958784 Down
WDR86-AS1 −1.749482818 0.045879379 5.820719606 0.430167793 Down
GS1-393G12.12 −1.595574045 0.025413087 2.892968281 0.573066828 Down
RP11-75C10.7 −1.578454562 0.028918409 1.681378986 0.27755832 Down
MSTRG.22984 −1.515487489 0.028151217 6.307372767 6.856887292 Down
RP1-29C18.8 −1.491219357 0.007182308 1.813751882 0.780203396 Down
RP11-20I20.4 −1.489870084 0.045895116 1.882568915 0.702213888 Down
CTD-2531D15.5 −1.452569255 0.044459184 1.198212257 0 Down
AC010084.1 1.812545185 0.005654266 0 0.808113485 Up
PSMD5-AS1 1.788651473 0.011076928 8.927660428 20.42027077 Up
RP11-535M15.1 1.639437785 0.00820557 0 1.477784502 Up
LLPH-AS1 1.498657545 0.005471274 0.230318651 2.298519226 Up
RP4-811H24.9 1.452389476 0.004968106 0.053490925 0.499762058 Up
MSTRG.30231 1.342076952 0.040559246 2.874551067 6.364192991 Up
TMEM92-AS1 1.339293051 0.014889801 0 1.352052251 Up
WI2-87327B8.2 1.289651543 0.017494757 1.241932483 1.714464098 Up
AC099668.5 1.25464443 0.023121561 0 1.059053016 Up
LINC01151 1.231390756 0.00790838 0.384761558 0.385097694 Up

DElncRNA, differentially expressed long non-coding RNA; fpkm, fragments per kilobase million; C, control group; T, case group.

The top 100 co-expressed differentially expressed mRNA-differentially expressed long non-coding RNA interaction network.

mRNAsig lncRNAsig corsig pvalsig
OR5B2 RP11-524O1.4 0.999999784 7.02×10−14
ADAMTS14 RP5-906A24.2 0.999998855 1.97×10−12
ADAMTS14 KCNMB2-AS1 0.99999619 2.18×10−11
DMRTC2 RP11-756H6.1 0.9999959 2.52×10−11
PRY RP11-355N15.3 0.999995538 2.99×10−11
CAMSAP3 LINC01337 0.999993952 5.49×10−11
GPR62 LINC01337 0.999993931 5.52×10−11
ACTN3 AC004471.9 0.999985033 3.36×10−10
COL21A1 LINC01337 0.999984742 3.49×10−10
OR5B2 RP11-91I20.2 0.9999843 3.70×10−10
SFTA2 AC004471.9 0.999975676 8.88×10−10
ADAMTS14 TSPEAR-AS1 0.999948189 4.03×10−9
DMRTC2 TSPEAR-AS1 0.999936548 6.04×10−9
FOXI1 RP5-906A24.2 0.999888814 1.85×10−8
FOXI1 AC022431.3 0.999855298 3.14×10−8
ERICH6 RP1-72A23.4 0.999853976 3.20×10−8
DMRTC2 KCNMB2-AS1 0.99982545 4.57×10−8
FOXI1 KCNMB2-AS1 0.999815958 5.08×10−8
OR2C3 RP11-355N15.3 0.999806865 5.59×10−8
FOXI1 RP11-320N21.1 0.999759427 8.68×10−8
PAK6 PSMD5-AS1 0.999743004 9.91×10−8
DMRTC2 RP5-906A24.2 0.999736476 1.04×10−7
CAMSAP3 RP11-254I22.2 0.999726506 1.12×10−7
GPR62 RP11-254I22.2 0.99972637 1.12×10−7
SFTA2 RP11-320N21.1 0.99970509 1.30×10−7
ADAMTS14 RP11-756H6.1 0.999704563 1.31×10−7
ERICH6 RP11-254I22.2 0.999685951 1.48×10−7
COL21A1 RP11-254I22.2 0.999676565 1.57×10−7
OR2C3 RP11-524O1.4 0.999662773 1.71×10−7
FOXI1 TSPEAR-AS1 0.999646107 1.88×10−7
SFTA2 AC022431.3 0.999573412 2.73×10−7
PRY RP11-203E8.1 0.999549564 3.04×10−7
PRY RP11-104N10.1 0.999534887 3.24×10−7
OR2C3 RP11-91I20.2 0.999522111 3.43×10−7
PRY RP4-736L20.3 0.999509557 3.61×10−7
FCRL4 CTD-2534I21.8 0.999489932 3.90×10−7
ADAMTS14 AC022431.3 0.999441016 4.69×10−7
PRY AC008781.7 0.999421699 5.02×10−7
ACTN3 RP11-320N21.1 0.999325423 6.82×10−7
FZD9 AC008781.7 0.999315145 7.03×10−7
RP11-505K9.4 RP11-756H6.1 0.999273631 7.91×10−7
ADAMTS14 RP11-320N21.1 0.999264308 8.12×10−7
FZD9 RP4-736L20.3 0.999211985 9.31×10−7
FZD9 RP11-104N10.1 0.99917912 1.01×10−6
FOXI1 RP11-756H6.1 0.999170485 1.03×10−6
FZD9 RP11-203E8.1 0.99915935 1.06×10−6
PDZD4 RP11-159N11.4 0.999141006 1.11×10−6
ACTN3 AC022431.3 0.999132502 1.13×10−6
RP11-219A15.1 RP11-457M11.7 0.999106184 1.20×10−6
CAMSAP3 RP1-72A23.4 0.999004264 1.49×10−6
GPR62 RP1-72A23.4 0.999004022 1.49×10−6
OR5B2 RP11-355N15.3 0.998989141 1.53×10−6
ERICH6 LINC01337 0.998988895 1.53×10−6
CXXC5 LINC01588 0.998981114 1.56×10−6
FZD9 RP1-72A23.4 0.998951532 1.65×10−6
COL21A1 RP1-72A23.4 0.99891675 1.76×10−6
NDUFC2-KCTD14 RP11-45A17.2 -0.998877807 1.89×10−6
MAP4 MSTRG.8559 0.998862021 1.94×10−6
PRY RP11-524O1.4 0.998818651 2.09×10−6
SRGN CTD-2562G15.3 0.998661182 2.69×10−6
RP11-505K9.4 TSPEAR-AS1 0.998635484 2.79×10−6
OR2C3 RP11-203E8.1 0.998613936 2.88×10−6
OR2C3 RP11-104N10.1 0.998588286 2.99×10−6
FOXI1 AC004471.9 0.998585363 3.00×10−6
PRY RP11-91I20.2 0.998566098 3.08×10−6
ZNF804A TCEAL3-AS1 0.99856452 3.09×10−6
FAM111A MSTRG.6714 0.998552192 3.14×10−6
OR2C3 RP4-736L20.3 0.998544416 3.18×10−6
DMRTC2 AC022431.3 0.998494339 3.40×10−6
OR2C3 AC008781.7 0.99839571 3.86×10−6
HHAT AC012074.2 0.998323875 4.21×10−6
IL18BP MSTRG.8559 0.998273402 4.47×10−6
RP5-862P8.2 RP11-1H8.5 0.998269298 4.49×10−6
SMAD6 RP5-1092A3.5 0.998257122 4.55×10−6
CFAP20 MSTRG.8559 0.998252424 4.58×10−6
RP11-505K9.4 KCNMB2-AS1 0.998220553 4.75×10−6
DMRTC2 RP11-320N21.1 0.998212166 4.79×10−6
PCOLCE2 RP11-105C19.2 -0.998176846 4.98×10−6
SFTA2 RP5-906A24.2 0.998131862 5.23×10−6
FZD9 RP11-254I22.2 0.998116114 5.32×10−6
ARL11 DNAJC3-AS1 0.998052713 5.68×10−6
MYOF LINC01151 -0.998035735 5.78×10−6
NT5C2 RP11-139H15.6 0.998022562 5.86×10−6
MME RP11-295I5.4 0.998001494 5.99×10−6
RP11-505K9.4 RP5-906A24.2 0.99795661 6.26×10−6
IL18BP MYCBP2-AS2 0.997935664 6.39×10−6
HSPA1L LINC01588 0.997924492 6.46×10−6
SAMHD1 MYCBP2-AS2 0.997902735 6.59×10−6
SFTA2 KCNMB2-AS1 0.997861652 6.85×10−6
CSNK2B RP11-214K3.23 0.997833473 7.04×10−6
NOLC1 MSTRG.8559 0.997817608 7.14×10−6
TNXB RP11-441F2.2 0.997791211 7.31×10−6
RHOB LINC00671 0.997708778 7.87×10−6
DDB1 RP11-429J17.2 0.997652855 8.26×10−6
ORMDL3 MYCBP2-AS2 0.997626959 8.44×10−6
OR5B2 RP11-927P21.2 0.99758326 8.75×10−6
ADAMTS14 AC004471.9 0.997577319 8.80×10−6
TRIM73 RP11-565F19.2 0.997494288 9.41×10−6
PEG3 RP11-17E13.2 0.997467797 9.61×10−6

lncRNA, long non-coding RNA.

Nearby targeted DEmRNAs of DElncRNAs between ankylosing spondylitis and normal controls.

Chr DElncRNA
Nearby DEmRNA
Symbol Start -100 kb End +100 kb Symbol Start End
chr1 PIK3CD-AS1 9,552,610 9,754,586 CLSTN1 9,729,026 9824526
chr14 LINC01588 49,827,571 50,192,643 SOS2 50,117,120 50,231,558
chr22 AC004471.9 19,021,529 19,224,503 SLC25A1 19,175,575 19,178,830
chr2 AC007879.5 207,139,864 207,629,795 KLF7 207,074,137 207,167,267
ChrX INE1 47,104,921 47,305,865 UBA1 47,190,861 472,151,28
ChrX INE1 47,104,921 47,305,865 USP11 47,232,690 47,248,328
chr1 TAF1A-AS1 222,489,825 222,693,032 MIA3 222,618,086 222,668,012
chr7 AC007285.6 29,888,600 30,125,660 SCRN1 29,920,103 29,990,289
chr1 RP11-318C24.2 175,804,762 176,020,513 RFWD2 175,944,831 176,207,493
chr5 AC008781.7 141,518,414 141,726,481 DIAPH1 141,515,016 141,619,055
chr3 U73166.2 50,160,303 50,363,358 HYAL3 50,292,831 50,299,468
ChrX RP13-314C10.5 23,672,992 23,882,956 PRDX4 23,664,262 23,686,399
chr1 RP11-195C7.1 176,107,648 176,329,330 RFWD2 175,944,831 176,207,493
chr21 AP001056.1 44,075,489 44,276,453 PWP2 44,107,290 44,131,181
chr21 AP001056.1 44,075,489 44,276,453 TRAPPC10 44,012,319 44,106,552
chr1 RP11-7O11.3 43,844,370 44,046,551 ST3GAL3 43,705,824 43,931,165
chr3 RP11-379K17.4 169,839,353 170,066,734 GPR160 170,037,929 170,085,403
chr2 AC092620.2 138469090 138,674,458 SPOPL 138,501,801 138,573,547
chr3 LINC00969 195,558,062 195,839,964 MUC20 195,720,882 195,741,123
chr4 RP11-15B17.1 99,850,006 100,295,099 LAMTOR3 99,878,336 99,894,490
chr5 SCAMP1-AS1 78,242,365 78,460,507 AP3B1 78,000,525 78,294,755
chr15 RP11-30K9.6 58,668,072 58,870,974 ADAM10 58,588,807 58,749,978
chr8 BAALC-AS1 103,056,990 103,398,772 SLC25A32 103,39,8635 103,415,189
chr5 RP11-159F24.5 43,415,274 43,625,310 NNT 43,602,692 43,707,405
chr5 LINC01187 170,091,579 170,299,141 FOXI1 17,010,5897 170,109,725
chr8 LINC01151 122,570,385 122,794,106 ZHX2 122,781,394 122,974,512
chr11 RP11-727A23.4 83,080,144 83,284,520 PCF11 83,156,988 83,187,451
chr8 GS1-393G12.12 144,214,590 144,415,138 BOP1 144,262,102 144,291,370
chr12 RP11-561P12.5 8,448,361 8,667,613 CLEC4E 8,533,305 8,540,963
chr12 RP11-561P12.5 8,448,361 8,667,613 CLEC4D 8,509,475 8,522,366
chr12 RP11-996F15.2 29,180,418 29,417,848 ERGIC2 29,337,352 29,381,189
chr14 RP11-44N21.1 104,993,609 105,199,004 CDCA4 105,009,573 105,021,148
chr14 RP11-44N21.1 104,993,609 105,199,004 C14 or f79 104,985,775 105,010,482
chr12 RP11-753H16.3 54,253,661 54,597,688 HNRNPA1 54,280,193 54,287,088
chr14 RP11-193F5.1 60,779,714 61,082,585 SLC38A6 60,981,114 61,083,733
chr14 RP5-1021I20.1 73,687,360 73,903,270 PNMA1 73,711,783 73,714,372
chr3 AC099668.5 49,584,480 49,784,983 MST1 49,683,947 49,689,501
chr6 RP11-425D10.10 109,282,795 109,483,666 SMPD2 109,440,763 109,443,919
chr14 C TD-2547L24.4 91,158,299 91,359,003 CCDC88C 91,271,323 91,417,844
chr14 RP11-524O1.4 21,284,292 21,484,920 CHD8 21,385,194 21,456,126
chr14 RP11-524O1.4 21,284,292 21,484,920 SUPT16H 21,351,472 21,384,266
chr16 RP11-459F6.3 58,029,529 58,259,133 CFAP20 58,113,588 58,129,450
chr16 RP11-264B17.3 28,874,804 29,090,775 LAT 28,984,826 28,990,783
chr1 RP11-196G18.22 149,744,498 149,949,024 HIST2H2BF 149,782,689 149,812,373
chr16 LA16c-306E5.3 3,358,071 3,615,564 NLRC3 3,539,033 3,577,400
chr16 RP11-461A8.4 3,550,636 3,751,703 NLRC3 3,539,033 3,577,400
chr17 RP1-59D14.5 2,275,061 2,479,306 TSR1 2,322,503 2,337,507
chr17 RP1-59D14.5 2,275,061 2,479,306 METTL16 2,405,562 2,511,891
chr17 RP1-59D14.5 2,275,061 2,479,306 SMG6 2,059,839 2,303,771
chr17 RP11-927P21.2 64,799,766 65,000,716 LRRC37A3 64,854,312 64,919,480
chr17 RP11-1094M14.11 35,468,109 35,674,843 SLFN14 35,548,125 35,558,098
chr17 C TD-2534I21.8 44,847,912 45,048,939 EFTUD2 44,849,943 44,899,662
chr19 PTOV1-AS1 49,738,639 49,951,676 NUP62 49,906,825 49,929,763
chr20 RP4-591C20.9 63,761,212 63,964,293 UCKL1 63,939,829 63,956,415
chr19 AC007292.3 4,256,637 4,458,448 CCDC94 4,247,079 4,269,090
chr19 RAB11B-AS1 8,274,373 8,490,685 RAB11B 8,389,981 8,404,434
chr5 CTD-2033C11.1 65,824,629 66,025,135 ERBIN 65,926,475 66,082,549
chr4 RP11-572O17.1 1,612,821 1,813,622 FAM53A 1,617,915 1,684,302
chr4 RP11-572O17.1 1,612,821 1,813,622 LETM1 1,811,479 1,856,247
chr1 RP4-736L20.3 10,329,881 10,530,677 DFFA 10,456,522 10,472,526
chr10 RP11-574K11.29 73,603,735 73,813,581 USP54 73,497,538 73,625,953
chr1 RP11-156E8.1 244,869,350 245,071,088 EFCAB2 244,969,705 245,127,164
chr3 RP11-767C1.2 12,732,219 12,932,728 IQSEC1 12,897,220 13,073,117
chr1 RP11-11N7.4 244,764,738 244,965,272 HNRNPU 244,840,638 244,864,560
chr3 RP11-778D9.13 184,032,942 184,233,561 AP2M1 184,174,689 184,184,091
chr12 RP1-197B17.4 47,631,908 47,832,351 HDAC7 47,782,722 47,833,132
chr17 AC142472.6 45,046,730 45,248,470 NMT1 45,051,610 45,109,016
chr15 C TD-2562G15.3 75,352,964 75,553,947 SIN3A 75,369,379 75,455,842
chr17 RP11-333J10.3 36,898,598 37,100,034 AATF 36,948,875 37,056,871
chr15 C TD-2382E5.6 41,808,204 42,008,714 JMJD7 41,828,085 41,837,581
chr18 RP11-405M12.3 74,970,197 75,171,091 ZNF407 74,597,870 75,065,671
chr17 HEXDC-IT1 82,325,498 82,527,310 NARF 82,458,180 82,490,537
chr17 RP11-159D12.6 57,906,674 58,108,187 CUEDC1 57,861,243 57,955,323
chr15 RP11-1H8.5 34315,450 34,520,273 LPCAT4 34,358,618 34,367,278
chr15 RP11-1H8.5 34,315,450 34,520,273 SLC12A6 34,229,996 34,338,060
chr5 CTC-487M23.6 112,794,933 112,996,531 SRP19 112,861,222 112,869,788
chr5 CTC-487M23.6 112,794,933 112,996,531 DCP2 112,976,702 113,020,970
chr5 AC005593.2 131,697,415 131,897,929 FNIP1 131,641,714 131,797,063
chr17 RP13-638C3.5 82,548,849 82,750,657 FN3KRP 82,716,683 82,730,328
chr14 RP11-298I3.6 22,923,083 23,124,217 AJUBA 22,971,174 22,982,642
chr16 RP11-451N19.3 58,605,799 58,806,297 SLC38A7 58,665,109 58,685,104
chr16 RP11-451N19.3 58,605,799 58,806,297 GOT2 58,707,131 58,734,357
chr19 CTD-2233K9.1 46,846,535 47,049,156 ARHGAP35 46,918,676 47,005,077
chr6 XXbac-BPG283O16.9 30,182,349 30,386,054 RPP21 30,345,131 30,346,884
chr6 XXbac-BPG283O16.9 30,182,349 30,386,054 TRIM26 30,184,455 30,213,427

Chr, chromosome; DEmRNAs, differentially expressed mRNAs; DElncRNA, differentially expressed long non-coding RNA.