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Identification of Aberrantly Expressed Long Non-Coding RNAs and Nearby Targeted Genes in Male Osteoporosis

Purpose To investigate different expression profiles of long non-coding RNAs (lncRNAs) and mRNAs between male osteoporosis and normal control by high throughput RNA sequencing. Methods We obtained the different expression profiles of long non-coding RNAs (lncRNAs) and mRNAs between male osteoporosis and normal control by high throughput RNA sequencing. Compared to normal control, we identified the differentially expressed genes (DEGs), differentially expressed lncRNAs (DElncRNAs) and the nearby targeted DEGs of DElncRNAs in male osteoporosis. Functional annotation was used to further study the functions of DEGs in male osteoporosis. The DElncRNAs–DEGs interaction network was constructed. One DElncRNA-nearby targeted DEG interaction pair of LINC02009-CCR2 was validated in vitro. Results Totally, 3296 DEGs, 204 DElncRNAs and 168 DElncRNAs-nearby targeted DEGs pairs were obtained. The most significantly up-regulated and down-regulated DElncRNAs in male osteoporosis were Loc105372801 and KCNQ1OT1, respectively. Osteoclast differentiation and chemokine signaling pathway were significantly enriched pathways in male osteoporosis. Based on the DElncRNAs–DEGs interaction network in male osteoporosis, we obtained several interaction pairs including SNHG5-SYNCRIP-HBA1-HBB, HCG27-HLA-C, LINC02009-CCR2, and LOC101926887-IFIT1-IFIT2/IFIT3/IFIT5. The expression of LINC02009 and CCR2 was down-regulated in keeping with the RNA sequencing data. Conclusion Identified DElncRNAs–DEGs interaction pairs may be involved in the development of male osteoporosis, which make a contribution to underlying the mechanism of male osteoporosis. Among which, the validated DElncRNAs-nearby targeted DEGs interaction pair of LINC02009-CCR2 may be important regulators in the development of male osteoporosis.


Introduction
Primary osteoporosis is characterized by decreased bone mass, low bone mineral density (BMD) and loss of microarchitectural integrity with consequent increases the risk of fracture. 1 Primary osteoporosis mainly occurs in postmenopausal women and senile men. Like postmenopausal osteoporosis, male osteoporosis is becoming a serious health problem worldwide with aging. 2 The loss of BMD is approximately 1% per year in senile men and 20% of senile man aged over 50 years suffer an osteoporosis-related fracture. 3 One third of men with osteoporosis suffer a hip fracture, a major risk factor for osteoporotic fracture-related death. 4 In the last decades, there has been considerable effort to uncover the mechanisms underlying osteoporosis, especially the postmenopausal osteoporosis. Bone loss of osteoporosis was induced by remodeling imbalance between bone resorption and bone formation. 4 Calcium, 5 parathyroid hormone, 6 estrogen 7 and vitamin D 8 are wellrecognized regulators in osteoporosis. Multiple cytokines such as interleukin-1 (IL-1), IL-6, macrophage colonystimulating factor (M-CSF), tumor necrosis factor-α (TNF-α) and receptor activator of nuclear factor-kB ligand (RANKL) were found to be involved in osteoporosis. 9,10 Immune system plays a crucial role in osteoporosis as well. 4,8 However, the molecular mechanisms of osteoporosis in men are not fully known.
Recently, accumulated evidence has indicated that long non-coding RNAs (lncRNAs) are involved in various biological progress by regulating the gene expression at transcriptional, epigenetic, or post-transcriptional levels. [11][12][13] It is reported that lncRNAs can alter the modification and location of transcription factors, act as precursor for siRNAs or miRNAs, regulate the alternative splicing of pre-mRNAs through SR complex, regulate histone modification by interacting with modification factors and bind to chromatin modification complexes to regulate chromatin remodeling and structure. 14 Previous studies have reported that several lncRNAs such as DNACR and MEG3 are associated with postmenopausal osteoporosis. 12,13,15 In this study, we investigated different expression profiles of lncRNAs and mRNAs between male osteoporosis and normal control by high throughput RNA sequencing. Differentially expressed lncRNAs (DElncRNAs) and differentially expressed genes (DEGs) in male osteoporosis were further identified. Since lncRNAs can regulate the expression of their nearby gene by cis-regulatory effects, 11 the nearby targeted DEGs of DElncRNAs in male osteoporosis were identified as well. To further study the functions of DEGs in male osteoporosis, functional annotation was carried out.

Patients and Samples
We collected full blood samples from three men with osteoporosis and two healthy men. Detailed full blood collection was as follows: (1) the full blood was collected with BD tube (2.5 mL per tube) at room temperature (18-25°C); (2) the full blood was slowly reversed and mixed 8 to 10 times to mix well with the anticoagulant in the BD tube; (3) the full blood was placed vertically at room temperature for 12-24 h to fully contacted with the RNA stabilizer in the BD tube; (4) the full blood was transferred to the refrigerator at −20°C for 24 h, then transferred to the refrigerator at −80°C for long-term preservation.
The inclusion criteria of male osteoporosis were as follows: (1) according to world health organization (WHO) standards, patients whose T-score of the femoral neck or lumbar vertebrae or total hip is ≤-2.5 were diagnosed as osteoporosis; (2) patients had a new vertebral compression fracture with no trauma history; (3) patients had no prior history of drug use that might affect bone metabolisms such as glucocorticoids, thyroid hormone, parathyroid hormone, fluoride, calcitonin, thiazides, barbiturates, antiepileptic drugs, vitamin D and calcium. The exclusion criteria of male osteoporosis were as follows: (1) patients had diseases that affect bone metabolic such as kidney disease, liver disease, thyroid disease, diabetes, hyperprolactinemia, rheumatoid arthritis, ankylosing spondylitis, chronic abdominal bay absorption adverse symptoms, malignant tumor, blood disease and abnormal reproductive system and related medical history; (2) patients had traumatic fractures. In addition, the inclusion criteria of normal controls (men with degenerative osteoarthritis of the lumbar spine) were as follows: (1) individuals were matched by gender, ageand frequency of the case group; (2) T-scores of the femoral neck and lumbar vertebrae (L 1-4 ) and total hip are all ≥-1. The exclusion criteria of normal controls were as follows: (1) People with a previous history of orthopedic system disease. The detailed information of participants is shown in Table 1. All the participants submitted the signed informed consent. The protocol for this study has been approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University (2017-P2-084-01).

RNA Sequencing
We isolated total RNA from these five blood samples through the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Nanodrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to test the concentration and purity of total RNA. 2% agarose gel was used to check the integrity of RNA. We also obtained the RNA integrity number (RIN) value by an Agilent 2100 Bioanalyzer. The threshold for cDNA library construction was as follows: (1) the total amount of RNA was more than 5μg; (2) the concentration of RNA was more than 200ng/mL; (3) the value of OD260/280 was 1.8-2.2. We removed ribosomal RNA by Ribo-Zero Magnetic kit (EpiCentre, Madison, WI, USA). RNA was fragmented into about 200 base pairs randomly. By using Truseq RNA sample Prep Kit (Illumina, Inc., San Diego, CA, USA), cDNA library was constructed. Firstly, the first cDNA strand was synthesized with RNA fragments primed with random hexamer primers. Secondly, we synthesized the second cDNA strand with dUTP instead of dTTP. Thirdly, end repair, 3ʹend adenylation and adapter ligation of double-stranded cDNAs were performed with End Repair Mix. Lastly, the second cDNA was digested by UNG enzyme (Illumina, Inc., San Diego, CA, USA). After the construction of the cDNA library, we amplified the library by performing polymerase chain reaction (PCR) for 15 cycles. Then, the amplified cDNA library was purified and quantified by Certified Low Range Ultra Agarose (Bio-Rad) and Picogreen (Molecular probes) on TBS380 (Turner Biosystems), respectively. The bridge PCR on cBot was performed by Truseq PE Cluster Kit v3-cBot-HS. In the end, sequencing of each library was performed by Illumina HiSeq 2000.

Data Processing
The FASTQ sequence data derived from RNA-seq results were obtained through Base Calling. After removed the low-quality reads by cutadapt (http://cufflinks.cbcb.umd. edu/), we obtained the high-quality clean data. Fasta and gff files of human reference genome (Ensemble GRCh38. p7) were downloaded from NCBI. TopHat (http://tophat. cbcb.umd.edu/) and Ensemble gene annotation were used to align clean reads to Ensemble GRCh38.p7. Cuffquant (http://cufflinks.cbcb.umd.edu/) was used to determine the expression of both mRNAs and lncRNAs and outputted the normalized data. The DEGs and DElncRNAs in male osteoporosis compared to normal control were obtained by using an R package, DEGseq. P-value <0.05 was the threshold of DEGs and DElncRNAs in male osteoporosis. The heat-maps of top 50 up-and down-regulated DEGs and DElncRNAs expression profile were performed with cluster in R language.

Protein-Protein Interaction (PPI) Network of DEGs
Based on Biological General Repository for Interaction Datasets (BioGRID) (http://thebiogrid.org/), we explored the proteins interacted with proteins encoded by the top 10 up-regulated and down-regulated DEGs in male osteoporosis. After removing these proteins that were not belonging to DEGs, we constructed the PPI network of top 10 up-and down-regulated DEGs in male osteoporosis.

Nearby Targeted DEGs of DElncRNAs
In order to identify the target DEGs of DElncRNAs, the DEGs transcribed within a 100 kbp window upstream or down-stream of DElncRNAs were researched and considered as nearby cis targets of DElncRNAs. The network of DElncRNA-near targeted DEGs was constructed by using Cytoscape.

Functional Annotation of All DEGs and Nearby Targeted DEGs of DElncRNAs
To further study the biological functions of identified all of DEGs and nearby targeted DEGs of DElncRNAs, we performed the Gene Ontology (GO) function and Kyoto

DElncRNAs-Nearby Targeted DEGs Interaction Network
Firstly, we constructed the DElncRNAs-nearby targeted DEGs interaction network by using all of the DElncRNAs and DEGs in male osteoporosis compared to normal control. Then, this DElncRNAs-nearby targeted DEGs interaction network and the male osteoporosisspecific PPI network were merged with the top 10 upand down-regulated DEGs.

In vitro Validation
One DElncRNA-nearby targeted DEGs interaction pair of LINC02009-CCR2 was selected randomly for validation. A total of 36 full blood samples (16 cases) from male osteoporosis patients and healthy individuals (20 normal controls) were used for validation of CCR2 and LINC02009. The clinical information of these individuals was shown in the supplementary Table 1. We performed QRT-PCR in an ABI 7500 real-time PCR system. Relative gene expression was analyzed using 2 −ΔΔCt method. The human GAPDH gene was used as an endogenous control for mRNA expression in the analysis. All the participants submitted the signed informed consent. The protocol for this study has been approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University (2017-P2-084-01).

PPI Network of DEGs in Male Osteoporosis
The PPI network consisted of 101 nodes and 105 edges ( Figure 2). Based on this network, HLA-C (degree=18), KRT1 (degree=17) and IFIT1 (degree=15) were three hub proteins in male osteoporosis.

Nearby Targeted DEGs of DElncRNAs
After searching the DEGs transcribed within a 100 kbp window upstream or down-stream of DElncRNAs, we

DElncRNAs-Nearby Targeted DEGs Interaction Network
After merged the DElncRNAs-nearby targeted DEGs interaction network and the PPI network (involved top 10 up-and down-regulated DEGs), we obtained key DElncRNA-nearby targeted DEGs (top 10 up-and downregulated DEGs) interaction network (Figure 7).

QRT-PCR Confirmation of DEGs
To manifest the expression of high throughput RNA sequencing for male osteoporosis, we compared the results of qRT-PCR and RNA sequencing of DElncRNA-nearby targeted DEGs interaction pair LINC02009-CCR2. In Figure 8, expressions of LINC02009 and CCR2 were all down-regulated and consistent with our RNA sequencing results.

Discussion
Accumulated evidence has emphasized the importance of lncRNAs in various cancers and other diseases, such as osteoporosis. [11][12][13] Herein, we obtained the lncRNA and mRNA expression profiles between male osteoporosis and normal control by high throughput RNA sequencing. Based on functional annotation, we identified several DElncRNA-DEGs interaction pairs, which may be associated with the process of male osteoporosis. Compared to normal control, HBB (hemoglobin subunit beta) was the most significantly up-regulated gene, while HBA1 was a significantly down-regulated gene in male osteoporosis. HBB and HBA1 encode the β and α 1 subunit of hemoglobin, respectively. As a major component of hemoglobin, iron is an important factor of bone metabolism. Iron can disorder the process of bone formation, make toxic effects on osteoblasts and reduce the recruitment of osteoblastic-lineage cells. 16 It is pointed out that hemoglobin level is related to BMD and bone mass negatively and independently. 17 Moreover, higher bone loss was found in men with lower hemoglobin level than women with lower hemoglobin level. 17 Moreover, we found an interaction of "SNHG5-SYNCRIP-HBA1-HBB" based on the DElncRNA-nearby targeted DEGs interaction network. SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) was a down-regulated gene in male osteoporosis compared to normal control. SYNCRIP has been reported to be involved in bone morphogenesis protein signaling pathway. 18 These three DEGs (HBA1, HBB and SYNCRIP) may be involved in the pathogenesis of male osteoporosis by their interactions. Small nucleolar RNA host gene 5 (SNHG5) is a member of the non-protein -coding multiple snoRNA host gene family which was significantly down-regulated in male osteoporosis in our study. SNHG5 has been reported to be associated with various cancers. 19,20 SNHG5 is involved in gastric cancer  19 However, no association between SNHG5 and male osteoporosis has been reported. Our study suggested that down-regulated SNHG5 may play an important role in male osteoporosis through regulating the expression of SYNCRIP, HBB and HBA1.
Based on the male osteoporosis-specific PPI network, major histocompatibility complex, class I, C (HLA-C) was a hub gene, which was up-regulated in male osteoporosis compared to normal control. HLA-C is a member of the HLA class I heavy chain paralogues. HLA class I molecule consists of a heavy chain and a light chain which plays a key role in the immune system. Accumulated evidence has demonstrated the relationship between the immune system and bone. 21 T cells play a crucial role in the formation and activity of osteoclast and osteoblast in multiple skeletal diseases, such as osteoporosis. 22 The production of T-lymphocyte, T-helper and T-cytotoxic cells is decreased with aging. 23 Additionally, other osteoporosis-related factors including estrogen, calcium, parathyroid hormone, and vitamin D play a regulatory in immune system. 23 The role of immune system in the pathogenesis of osteoporosis is mainly induced by decreased estrogen and secondary hyperparathyroidism. 23 In this study, HLA-C was a targeted nearby cis-DEG of HCG27 (HLA complex group 27). In this study, HCG27 was a significantly upregulated lncRNA in male osteoporosis. Our finding emphasized the importance of HCG27 in the process of male osteoporosis.
Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) was another hub gene based on PPI network which was up-regulated in male osteoporosis. Depending on the DElncRNAs-nearby targeted DEGs interaction network, IFIT1 could be interacted with three other up-regulated DEGs including IFIT2, IFIT3 and IFIT5. These four genes were interferon-induced proteins that contain tetratricopeptide repeats. 24 As an innate immune bottleneck, IFIT1 is expressed during the response of osteoblasts to immune cytokine interferon-β. 25 IFIT2 is a regulator of innate immunity as well. Both IFIT1 and IFIT2 play a role in the fracture healing process in osteoporosis, 26 28 Loss of imprinting of KCNQ1OT1 locus by epigenetic disruption is related to colorectal carcinogenesis. [29][30][31] A novel tetranucleotide repeat polymorphism (rs35622507) within KCNQ1OT1 confers risk for hepatocellular carcinoma. 30 KCNQ1OT1 involves in breast cancer through the epigenetic suppression of the cyclin-dependent kinase inhibitor 1C. 31 The precise function of KCNQ1OT1 in male osteoporosis needs further research. In our study, chemokine signaling pathway was a significantly enriched pathway in male osteoporosis. As one of the DEGs enriched in chemokine signaling pathway, C-C motif chemokine receptor 2 (CCR2) is a receptor for ligands like monocyte chemoattractant protein-1 (MCP-1). CCR2 is served as a regulator of bone loss, which is expressed on preosteoclasts and osteoclasts. 32 The MCP-1/CCR2 axis has been demonstrated to be involved in fusion and maturation of osteoclast and bone resorption. 33,34 In addition, gene variants of MCP-1 and CCR2 are reported to be risk factors for osteoporosis. 35 In young adult mice, knockout of the

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Ccr2 results in delay in fracture healing. 36,37 In the present study, CCR2 and LINC02009 were all significantly down-regulated in male osteoporosis, which was validated by qRT-PCR. CCR2 was a nearby targeted DEG of a down-regulated DElncRNA, LINC02009.
Our result suggested that LINC02009 may be associated with the process of osteoporosis by regulating the expression of CCR2 and other nearby targeted DEGs including lactotransferrin (LTF) and leucine-rich repeat containing 2 (LRRC2).
Osteoclast differentiation was another significantly enriched pathway for DEGs in male osteoporosis which was demonstrated to be crucial pathway of osteoporosis. These DEGs enriched in osteoclast differentiation may be important regulators in male osteoporosis as well.
Taken together, identified several DElncRNA-nearby targeted DEGs interaction pairs such as SNHG5-SYNCRIP-HBA1-HBB, HCG27-HLA-C, LINC02009-CCR2, LOC10 1926887-IFIT1-IFIT2, LOC101926887-IFIT1-IFIT3 and LOC101926887-IFIT1-IFIT5 may play an important role in  the pathogenesis of male osteoporosis. The validated DElncRNA-nearby targeted DEGs interaction pair of LINC02009-CCR2 may be important regulators in the process of male osteoporosis as well. Our findings may contribute to underlying the function of lncRNAs and mechanism of male osteoporosis and provide new clues for novel diagnostic and therapeutic strategies for male osteoporosis. However, there are limitations to our study. Firstly, the sample size in the RNA sequencing is small, larger numbers of blood samples or bone samples are further needed for further validation of identified lncRNAs and genes. Secondly, we did not validate the expression of identified other DElncRNAs and DEGs in male osteoporosis or any cell lines. Thirdly, we did not perform the deeper mechanism study of male osteoporosis, some in vivo or in vitro experiments are further needed.