Identification of miR-194-5p as a potential biomarker for postmenopausal osteoporosis

Participant characteristics

This study was approved by the Ethics Committee of Harbin Medical University (Approval number: 2014-R-020). Each participant enrolled in this study was informed of the project and had signed a written consent. Twenty-three postmenopausal Chinese women with osteopenia (moderate decrease in BMD, spine T-score ≤ − 1.0 and > − 2.5) and 25 postmenopausal Chinese women with osteoporosis (marked decline in BMD, spine T-score ≤ − 2.5) were recruited and further divided into six subgroups according to their T-score measurements (Table 1). Dividing the patients into six subgroups allowed investigation of whether altered expression of a particular miRNA may be correlated with bone loss during pathological progression from osteopenia to osteoporosis. Both the spine and femoral neck T-score was measured for each participant. In addition, an independent collection of participants was recruited for the purpose of external validation. This group was comprised of 24 postmenopausal women with a normal BMD, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (Table S1). Participants were given a questionnaire to obtain their medication history, and a hospital examination verified that no serious complications, such as cancer, Type II diabetes, and/or cardiac disease were present. All the participants ranged in age from 59 to 70 and had been postmenopausal for at least 12 months (no menses 12 months after the date of the last menses). T-scores of the spine (L1-L4) and femoral neck were obtained using a dual energy X-ray absorptiometry scanner (Hologic Inc., Bedford, Massachusetts, USA).

Blood sample collection and total RNA extraction

Five milliliters of whole blood was obtained from each participant. Each whole blood sample was independently lysed using Red Blood Cell (RBC) Lysis Solution (Beyotime, Shanghai, China) and centrifuged for 10 min at 450× g. TRIzol reagent (Invitrogen, Shanghai, China) was used to extract RNA from the precipitate. RNA extraction was completed within 30 min after blood collection from each participant. Isolated RNA eluate was stored at −80 °C. After blood sample collection was complete, all RNA extraction samples from individual participants were thawed and pooled separately into six subgroups corresponding to the spine T-score values (Table 1). The pooled samples were then stored at −80 °C for future experiments. The same procedure was applied to the whole blood samples for external validation (Table S1); however, these RNA samples were not pooled.

Microarray scanning

An Agilent Human miRNA microarray (Release 19.0, 8 × 60 K) was used for global scanning of miRNA expression in pooled RNA samples. Sample labeling, microarray hybridization, and washing were performed based on the manufacturer’s standard protocols (Agilent Technologies Inc., Santa Clara, California, USA). Briefly, total RNA was dephosphorylated, denatured, and then labeled with Cyanine-3-CTP. After purification, labeled RNAs were hybridized onto the microarray. After washing, the arrays were scanned with an Agilent Scanner G2505C (Agilent Technologies Inc., Santa Clara, California, USA). Feature Extraction software (version 10.7.1.1; Agilent Technologies Inc., Santa Clara, California, USA) was used to analyze microarray images and obtain raw data. Next, GeneSpring software (version 12.5; Agilent Technologies Inc., Santa Clara, California, USA) was used to complete the basic analysis using raw data. The raw data was normalized with the quantile algorithm. If the probes with a positive normalized expression value were flagged as “Detected” in at least 100% of samples, they were chosen for further analysis. Differentially expressed miRNAs were then identified through fold change as well as the p value calculated using a Student’s t-test. The threshold set for significantly up- and down-regulated genes was a fold change >2.0 and a p value <0.05. The miRNA microarray assay was performed by Shanghai OEBiotech Technology Co, Ltd. (Shanghai, China). Microarray scanning data have been submitted to the Gene Expression Omnibus with an accession number GSE64433.

qRT-PCR analysis

To avoid false positives during microarray detection, a qRT-PCR assay was used to validate further the significantly dysregulated miRNAs identified in the pooled RNA extraction samples. RNA eluate (0.4 µL) was reverse transcribed into cDNA in a 10-µL reaction volume, using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Briefly, 1.0 µL 10 × RT Buffer, 0.4 µL 25 × dNTP Mix (100 mM), 1.0 µL 100 mM RT miRNA primers, 0.5 µL MultiScribe™ Reverse Transcriptase, and 0.5 µg RNA were mixed, and the reaction volume was brought up to 10 µL by using DEPC H₂O. Transcription parameters were as follows: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min. The cDNA was diluted 1:100 and assayed in 10 µL PCR reactions. miRNA primer sequences and the U6 internal control are listed in Table S2. For real-time PCR, 10 µL 2 × SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), 7 µL DEPC H₂O, 1 µg cDNA, and 1 µL (each) F and R primers were mixed and run on a Bio-Rad CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Singapore). Samples were briefly heated (10 min, 95 °C) and amplified as follows: 95 °C for 15 s and 60 °C for 1 min for 40 cycles. MiRNA cycle threshold (CT) values were normalized to U6 and calculated using the equation 2^−ΔΔCT, as previously described (Wang et al., 2012). The same procedure was applied to the RNA samples from the external validation set (Table S1).

Additionally, the efficiency and specificity of the qRT-PCR protocol was evaluated for the miR-194-5p primers designed for this study. A synthetic miR-194-5p was generated according to its nucleotide sequence in miRBase (Kozomara & Griffiths-Jones, 2014). Synthetic miR-194-5p was dissolved using DEPC H₂O at a concentration of 10 µg/µL. The solution was then further diluted 4 × 10³, 4 × 10⁴, 4 × 10⁵, and 4 × 10⁶ times. After this, 1 µL of each solution at different concentrations was used as a template to be co-amplified with the cDNA reverse-transcribed from the RNA obtained from blood samples and be subjected to real-time PCR assay.

Pathway analysis

The HuGE Navigator Gene Prospector online tool was used to search for literature-reported osteoporosis-related genes (Yu et al., 2008). After uploading the official symbols of osteoporosis-related genes onto the DAVID website, the online functional annotation tool was applied to identify osteoporosis-related pathways by selecting ‘Homo sapiens’ as the species background and ‘KEGG_pathway’ as the only annotation for functional analysis (Huang da, Sherman & Lempicki, 2009). Over-representation of osteoporosis-related genes on a KEGG pathway was considered statistically significant only if the Bonferroni-adjusted p < 0.05 (Bland & Altman, 1995).

An integrative retrieval from two miRNA-target interaction (MTI) databases, including miRSel (Naeem et al., 2010) and miRTarBase (Hsu et al., 2014), was applied to search for experimentally validated target genes of six miRNAs, including miR-130b-3p, -133a, -151a-3p, -151b, -194-5p, and -590-5p in humans. After uploading the official symbols of the target genes of each miRNA onto the DAVID website, the online functional annotation tool was used to reveal whether these target genes were involved in osteoporosis-related KEGG pathways.

Statistical analysis

All data are expressed as the mean ± SD. Statistical analysis was performed using Student’s t-test or a Pearson correlation test. GraphPad Prism v6.0 was used to conduct statistical analysis. Differences were considered as statistically significant when p < 0.05.

Microarray scanning identified six miRNAs with increased peripheral blood expression in participants with osteoporosis

To search for potential miRNA biomarkers for postmenopausal osteoporosis, a comprehensive miRNA expression analysis was performed on pooled RNA samples isolated from postmenopausal Chinese women with osteopenia or osteoporosis (Table 1). We identified 331 unique miRNAs with detectable expression in every sample tested (Table S3). Among them, six miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, -590-5p, and -660-5p) were found to have significantly increased peripheral expression in the blood of participants with osteoporosis compared those with osteopenia (p < 0.05, Fig. 1). MiR-194-5p was the most highly upregulated (with a more than5-fold change), followed by miR-151a-3p, -151b, and -590-5p (with a more than 3-fold change), and miR-130b-3p and -660-5p (with only a 2-fold increase in expression) (Table S3).

qRT-PCR assay validated significant upregulation of five miRNAs in the blood of patients with osteoporosis

Due to the high false positive rate of the microarray method, we validated the circulating blood expression levels of the six significant microarray-identified miRNAs by using qRT-PCR. Except for miR-660-5p, all of the miRNAs tested showed remarkably higher expression in the peripheral blood of patients with osteoporosis compared to that observed in the blood of patients with osteopenia (p < 0.05, Fig. 2). Furthermore, we investigated whether a significant linear correlation existed between miRNA expression and spine T-score. Our results showed that the expression of four miRNAs, including miR-130b-3p, -151a-3p, -151b, -194-5p, in circulating blood were significantly and negatively correlated with a decline in BMD (p < 0.05, Fig. S1). Of note, the expression levels of miR-151b and -194-5p were also significantly and negatively correlated with femoral neck T-scores (p < 0.05, Fig. S1). This finding suggested that these two miRNAs might be more suitable for further consideration as potential biomarkers for postmenopausal osteoporosis.

miR-194-5p is implicated in multiple osteoporosis-related pathways

To explore a potential functional association between the miRNAs identified in this study and osteoporosis, the online bioinformatics tool DAVID was used to investigate whether the identified miRNAs regulate osteoporosis-related pathways by targeting the mRNA genes in each pathway. There were 985 genes reported in the literature to be functionally associated with osteoporosis. Analysis of their official gene symbols with the DAVID website revealed 19 KEGG pathways with over-represented osteoporosis-related genes (Table 2). These pathways were defined as osteoporosis-related due to this significantly enrichment with osteoporosis-related genes. Experimental evidence of MTIs were retrieved from the miRSel and miRTarBase databases for the five PCR-validated miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, -590-5p) identified in this study, as well as miR-133a,which was previously suggested as a feasible biomarker for postmenopausal osteoporosis (Wang et al., 2012; Li et al., 2014). We then investigated involvement of each miRNAs in regulating osteoporosis-related pathways. MiR-151b was found to lack experimentally-validated MTIs; therefore, it was excluded from further analysis. The remaining four miRNAs and miR-133a, the known osteoporosis biomarker, underwent subsequent pathway analysis. As expected, miR-133a was implicated in six osteoporosis-related pathways such as cytokine-cytokine receptor interaction (Table 2). This finding was consistent with the previous study, in which the authors demonstrated that miR-133a targeted multiple pathway-associated genes, such as chemokine (C-X-C motif) ligand 11 (CXCL11) and chemokine (C-C motif) receptor 3 (CXCR3) (Wang et al., 2012). In our analysis, we determined that miR-194-5p was functionally associated with eight osteoporosis-related pathways. This result implies that by targeting many genes, miR-194-5p may have effects on the TGF-beta and Wnt signaling pathways, which were shown to play critical roles in the pathology of postmenopausal osteoporosis (Krishnan, Bryant & Macdougald, 2006; Nistala et al., 2010).

External validation confirmed increased expression of miR-194-5p in postmenopausal osteoporosis

External validation of miR-194-5p expression in the blood circulation of postmenopausal women with osteoporosis was performed by qRT-PCR analysis. To do this, an independent collection of participants was recruited, which was comprised of 24 postmenopausal women with normal BMD, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (Table S1). A significant increase in peripheral expression of miR-194-5p was found in postmenopausal women with osteopenia or osteoporosis, compared to postmenopausal women with normal BMD (p < 0.001, Fig. 3A). A remarkable negative correlation was also found between miR-194-5p expression and T-scores (p < 0.05, Figs. 3B and 3C). Moreover, the qRT-PCR method established in this study efficiently detected miR-194-5p expression in peripheral blood with high specificity (Fig. S2), thus increasing our confidence in the reliability of the external validation results.