http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(7): 553–556 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.935957

RESEARCH ARTICLE

Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients Hongqiu Li1#, Zhe Wang2, Qin Fu1, and Jing Zhang1 Department of Orthopaedics and 2Department of Blood Transfusion, Shengjing Hospital of China Medical University, Shenyang, China

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Abstract

Keywords

In our study, we detect the levels of three micro-RNAs (miRNAs; miR-21, miR-133a and miR146a) in the plasma of 120 Chinese postmenopausal women who were divided into three groups (normal, osteopenia and osteoporosis) according to the T-scores. Downregulation of miR-21, as well as upregulation of miR-133a, was validated in the plasma of osteoporosis and osteopenia patients versus the normal group. The difference in expression regarding the miR146a level in plasma among the three groups was not significant (p40.01). The circulating miRNA expression levels and bone mineral density (BMD) were examined during a multiple correlation analysis as a dependent variable after adjusting for age, weight and height. We have demonstrated that specific miRNAs species are significantly changed in the plasma of osteoporosis and osteopenia patients and correlated with the BMD. Our study suggested a potential use of miR-21 and miR-133a as sensitive and plasma biomarkers for postmenopausal osteoporosis.

Bone mineral density, miRNA, osteoporosis, postmenopausal

Introduction Postmenopausal osteoporosis is the most common bone disease and features bone loss and susceptibility to fragility fractures that are associated with a low bone mineral density (BMD) (Cummings & Melton, 2002; Sambrook & Cooper, 2004). It is related to unbalanced bone resorption by osteoclasts and bone formation by osteoblasts (Khosla et al., 2011). A series of related expression profiling studies have indicated that deregulation of micro-RNA (miRNA) expression could affect both bone resorption and bone formation (Mizoguchi et al., 2010; Sugatani & Hruska, 2009; Taipaleenma¨ki et al., 2012) as well as osteoporosis (van Wijnen et al., 2013). miRNAs are small, non-protein-encoding RNAs that posttranscriptionally regulate gene expression through the suppression of specific target mRNAs (Bartel, 2004). Cultured mammalian cells have also been reported to export miRNAs into the extracellular environment, and significant amounts of miRNA have been found in blood plasma (Wang et al., 2010). In addition, in Thorarinn Blondal et al.’s (2013) review, 119 miRNAs were found in the serum and plasma, including miR21, miR-133a and miR-146a. The role of plasma miRNAs as novel non-invasive biomarkers in rheumatoid arthritis (Murata et al., 2013) and osteosarcoma (Ouyang et al., 2013) has been verified. #Hongqiu Li is responsible for statistical design/analysis. E-mail: [email protected] Address for correspondence: Qin Fu, Department of Orthopaedics, Shengjing hospital of China Medical University, No. 36 Sanhao Street, Shenyang, China. E-mail: [email protected]

History Received 14 May 2014 Revised 8 June 2014 Accepted 14 June 2014 Published online 18 September 2014

Several reviews (van Wijnen et al., 2013; Xia et al., 2011; Singh et al., 2013) have previously illustrated the importance of miRNAs (miR-21, miR-218, miR-2861, miR-146a, miR146b, miR-34c, miR-133a, miR-146a and miR-155) in osteoporosis. In these miRNAs, only miR-21, miR-133a and miR-146a could be detected in the plasma, according to Thorarinn Blondal et al.’s review. As such, we detected the levels of these three miRNAs in the plasma of postmenopausal women. Seeliger et al. (2014) also identified that circulating miRNAs are associated with osteoporotic fractures. However, the associations between plasma miRNAs and BMD have not been studied well in osteoporosis. In order to fill the gap, we examined the levels of miR-21, miR-133a and miR-146a in the plasma of postmenopausal women and investigated whether these plasma miRNAs levels correlate with sensitivity to BMD.

Materials and methods One-hundred twenty Chinese postmenopausal women from Shengjing Hospital, China Medical University, were enrolled between January 2012 and January 2013 and were divided into three groups according to the T-scores (total hip BMD). A T-score of  –1.0 was classified as normal; a T-score between4–2.5 and5–1.0 was classified as osteopenia; and a T-score of –2.5 was classified as osteoporosis. We obtained the demographic characteristics such as age, ethnicity, menstrual status, medication history and disease history through a questionnaire. The patients’ fasting blood samples were obtained, and all extracted plasma samples were stored in liquid nitrogen. For the sake of minimizing the freeze–thaw effect on circulating

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miRNAs, we only used plasma samples that had not been previously thawed. The BMD (g/cm2) was measured for the lumbar spine (L1-4) and total hip (femoral neck, trochanter and intertrochanteric region) by using daily checked dual energy X-ray absorptiometry scanners. As reflected by the coefficient of variation, the measurement precision was 0.9% and 1.4% for spine and hip BMDs, respectively. The postmenopausal status was defined as the date of last menses, followed by at least 12 months of no menses. This study was approved by the Human Research Ethics Committee, Shengjing Hospital, and written informed consent was obtained from all participants before study commencement.

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RNA isolation The total RNA was extracted from the plasma using a commercial, column-based system, according to the manufacturer’s instructions (Qiagen miRNeasy, Mini Kit, Shanghai, China). To allow for the normalization of sampleto-sample variation, miR-16 (RNA oligonucleotides synthesized by Qiagen, Shanghai, China) was added (as a mixture of 25 fmol for a total volume of 51 L) to each denatured sample (i.e. after combining the plasma sample with a denaturing solution), as previously described. The RNA was stored at 80  C. cDNA synthesis and real-time PCR Eight microliters of RNA eluate was reverse transcribed in 40-mL reactions using the miRCURY LNAÔ Universal RT cDNA Synthesis Kit (Exiqon). The cDNA was diluted 50 times and assayed in 10-mL PCR reactions according to the protocols of the miRCURY LNAÔ Universal RT microRNA PCR System (Exiqon A/S); each microRNA was assayed once using qPCR with the plasma Focus microRNA PCR panel. A no template control (with water) was purified with the samples and was profiled as the samples were. The amplification was performed in a Light CyclerÕ Real-time PCR System (Roche) in well plates. The amplification curves were analyzed using the Roche LC software for the determination of Cp (by the second derivative method), as well as for a melting curve analysis. The levels of miRNAs were all normalized to miR-16 and calculated by using the following equation: 2–DDCT. The RQ of each miRNA for each sample was determined by using 2–DDCT, in which DCT ¼ (average of the triplicate CT Target miRNA – average of the triplicate CTmiR-16) and DDCT ¼ (DCT – average DCT from all samples).

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statistical analyses were performed with the SPSS 13.0 software. All tests were two tailed, and a p of 50.01 was considered a significant difference.

Results Human subjects and characteristics The detailed characteristics of the study subjects are summarized in Table 1. There were insignificant differences observed in the age at menopause, weight and height among the groups regarding osteopenia, osteoporosis and the controls. The mean age of the osteoporosis group was 57.5 ± 11.3 years, while it was 56.7 ± 10.7 years for the osteopenia group and 56.5 ± 10.5 years for the normal group; both the hip and spine BMDs were significantly different among the groups (p50.01). qRT-PCR for miRNAs The differential expression results for miR-21, miR-133a and miR-146a using qRT-PCR are shown in Figure 1. Downregulation of miR-21 (*p50.01), as well as upregulation of miR133a (**p50.01), was validated in the plasma of osteoporosis and osteopenia patients versus the normal group. The difference in expression regarding the miR-146a level in plasma among the three groups was not significant (p40.01). Table 1. Characteristics of the study subjects.

Traits

Osteoporosis (n ¼ 40)

Age (years) Menopause (years) Height (cm) Weight (kg) HBMD (g/cm2) Hip T-score SBMD (g/cm2) Spine T-score

57.5 ± 11.3 51.5 ± 12.4 157.1 ± 15.3 61.4 ± 10.9 0.423 ± 0.053 –3.23 ± 0.64 0.536 ± 0.061 –3.75 ± 0.73

Osteopenia (n ¼ 40)

Normal (n ¼ 40)

p Value

56.7 ± 10.7 56.5 ± 10.5 NS 52.6 ± 11.8 52.3 ± 13.1 NS 156.1 ± 13.2 156.3 ± 11.4 NS 65.4 ± 11.5 64.6 ± 13.2 NS 0.865 ± 0.087 1.215 ± 0.24 50.01 –1.85 ± 0.75 0.81 ± 0.19 50.01 0.982 ± 0.047 1.347 ± 0.65 50.01 –2.03 ± 0.43 0.76 ± 0.13 50.01

ANOVA was used to determine whether any significant differences existed among the means of the three groups. The data are mean ± SD. HBMD, bone mineral density of the hips; SBMD, bone mineral density of the spine; and NS, not significant.

Statistical analysis All results for the continuous variables were expressed as M ± SD. For the normally distributed data, differences between the cases and controls characteristics were examined by using ANOVA. The circulating miRNAs expression levels were highly skewed; therefore, we used the non-parametric tests to examine differences among the groups. The circulating miRNA expression levels and BMDs were evaluated by using a multiple correlation analysis and used as a dependent variable after adjustment for age, weight and height. All

Figure 1. The levels (2–44CT) of miR-21, miR-133a and miR-146a were measured by using a qRT-PCR analysis in the plasma of the normal, osteopenia and osteoporosis groups (*p50.01; **p50.01).

Plasma miRNA levels in postmenopausal osteoporosis patients

DOI: 10.3109/1354750X.2014.935957

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Table 2. Associations between miRNAs and spine and hip BMDs. Osteoporosis –DDCT

miR (2

)

miR-21 a

miR-133a a

Osteopenia miR-146a

miR-21 a

miR-133a a

Normal miR-146a

miR-21 a

miR-133a a

miR-146a

SBMD

r p

0.466 0.000

–0.593 0.000

–0.056 0.542

0.457 0.000

–0.612 0.000

–0.062 0.571

0.457 0.000

–0.604 0.000

–0.049 0.563

HBMD

r p

0.464a 0.000

–0.619a 0.000

–0.040 0.666

0.482a 0.000

–0.621a 0.000

–0.037 0.678

0.475a 0.000

–0.588a 0.000

–0.038 0.654

a

Correlations significant 50.01.

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Correlations between the miRNA plasma levels and BMD The results of the multiple correlation analysis are listed in Table 2. After adjusting for age, weight and height, the plasma miR-21 levels had significantly positive correlations in the hip and spine BMDs in all groups, while the plasma miR-133a levels had significantly negative correlations. No significant correlations were found between the measured BMD values and miR-146a plasma levels, even after controlling for age, weight and height values in all groups.

Discussion The miRNA levels in the plasma of postmenopausal osteoporosis patients have not been studied well, and in this study, we examine the plasma miRNA levels of postmenopausal women and detect the underlying associations with BMD. We evaluated and analyzed the levels of three miRNAs (miR-21, miR-133a and miR-146a) in the plasma of 40 osteoporosis patients, 40 osteopenia patients and 40 healthy normal controls and found significantly lower plasma miR-21 levels and higher plasma miR-133a levels in osteopenia and osteoporosis than in normal patients. The different plasma miR-146a levels were insignificant in the osteoporosis and osteopenia patients. The plasma miR-21 and miR-133a levels correlated with the BMD; both could be used to differentiate osteoporosis and osteopenia patients from healthy normal controls with high accuracy. According to the research of Nan Yang et al. (2013), miR21 promoted MSC osteogenesis by repressing its target gene, Spry1. The study also verified the function of the miR-21– Spry1 axis through a phenomenon that promoted bone formation by blocking tumor necrosis factor a in OVX mice. The important roles of miR-21 have been demonstrated in various diseases such as osteosarcoma (Yuan et al., 2012), breast cancer (Zhao et al., 2013) and other cancers (Kishimoto et al., 2013; Zeng et al., 2013). Yang Wang et al. (2012) showed the association of miR-133a expression levels in the circulating monocytes and osteoclast precursors of postmenopausal osteoporosis patients. They suggested that miR-133a in circulating monocytes was a potential biomarker for postmenopausal osteoporosis. Our study replicated and extended these findings. There were distinct miR-21 and miR-133a plasma expressions in osteoporosis patients, as compared with normal women during the postmenopausal period: miR-21 was downregulated, and miR-133a had an increased expression. However, Seeliger et al. (2014) reported that miR-21 was increased, this may be both of studies’ sample size were small and sample source different. Although

the study by Lei SF et al. (2011) predicted that miR-146a could serve as potential binding sites for osteoporosis, we did not find differences in its plasma levels between the osteoporosis and normal patients. This may be because of the abnormal expression of miR-146a and the lack of its release in osteoporosis patients’ plasma. The BMD value is currently a gold standard tool, both for the diagnosis and evaluation of the response of the medical treatment in osteoporosis patients (Levine, 2011). Previous studies have discussed the associations between miRNAs and BMD. For example, one research study examined a panel of miRNAs in circulating monocytes from a small patient cohort and suggested that miR-133a was associated with a lower BMD (Wang et al., 2012). Shu-Feng Lei et al. (2011) identified three significant poly-miRNAs that were associated with the BMD in the 3’-UTR of the FGF2 gene; this finding was supported by three independent gene expression analyses that consistently demonstrated a depressed FGF2 gene expression in high-BMD subjects, as compared with lowBMD subjects, including miR-146a, which was not found in our study. We saw a significantly negative correlation between the plasma miR-133a level and BMD and a significantly positive correlation between the miR-21 level and BMD in the osteoporosis, osteopenia and normal groups. Thus, we have demonstrated that specific miRNAs species are significantly changed in the plasma of osteoporosis and osteopenia patients and correlated with the BMD. These findings suggested a potential use of miR-21 and miR-133a as sensitive and plasma biomarkers for postmenopausal osteoporosis. It is important to consider the limitations of this study. First, the sample size was small, so research with a larger sample size is necessary to reconfirm the results. Second, a plasma miRNA-microarray analysis might be an efficient way to obtain more information and could be used as a method in future studies. Third, we would conduct a bioinformatic sequence analysis for each significant miRNA to identify potential target genes and to explore the mechanisms by which circulating miRNAs are dysregulated in osteoporosis.

Acknowledgements The authors thank Shengjing Hospital for its assisting in the preparation of this manuscript.

Declaration of interest The authors declare no conflict of interest This study was funded by Shengjing Hospital (2012– 2013), Shenyang, China. This study was supported by the

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National Natural Science Foundation of China (Grant No 81370981).

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Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients.

In our study, we detect the levels of three micro-RNAs (miRNAs; miR-21, miR-133a and miR-146a) in the plasma of 120 Chinese postmenopausal women who w...
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