miRNAs in bone tissue correlate to bone mineral density and circulating miRNAs are gender independent in osteoporotic patients

We previously demonstrated the aberrant expression of nine specific miRNAs in serum from osteoporotic patients. In the present study, we further evaluated the expression of these miRNAs in bone tissue, osteoblasts, and osteoclasts from 28 patients. We hypothesize that miRNA expression in serum from osteoporotic patients may be gender-independent. A further hypothesis is that the miRNA expression in bone could be correlated with BMD values. Moreover, intracellular expression of these osteoporosis-related miRNAs may indicate the role of these molecules during osteoporosis. Our results indeed show that miRNA expression in serum was gender-independent except for miR125b-5p. A correlation with BMD was confirmed for miR-21-5p, miR-24-3p, miR-93-5p, miR-100-5p and miR125b-5p with linear correlation coefficients r > 0.9. Intracellular studies revealed a simultaneous up-regulation of miR-21-5p, miR-93-5p, miR-100-5p and miR125b-5p in osteoblasts and in osteoclasts. miR-148a-3p up-regulation in cells was specific for osteoporotic osteoclasts. Altogether, miR-21-5p, miR-93-5p, miR-100-5p, and miR-125b-5p showed significant upregulation in serum, tissue and bone cells of osteoporotic patients. All except miR-125b-5p showed gender independent expression and good correlation to BMD values. Our results suggest that these miRNAs may be important for an earlier diagnosis of osteoporosis.


Primary human osteoblast isolation
Cancellous bone from femoral heads was used for osteoblast isolations. Bone samples were mechanically shredded to small bone fragments by using a Luer forceps and transferred into a cell culture flask. Culture medium (D-MEM with 10% FCS, 1% P/S and 50µg L-ascorbate-2phosphate) was added and the flask was incubated for 7 days, with no further medium changes, at 37°C and 5% CO 2 in a humidified environment . Thereafter, the cell culture medium was changed to osteoblast expansion medium (Low glucose D-MEM, 5% FCS, 1% penicillin/streptomycin, 10 mM β-glycerol-phosphate disodium salt hydrate, 1.56 mM CaCl 2 , 3 0.025 M HEPES, 100 nM dexamethasone and 0.2 mM L-ascorbic acid 2-phosphate) 49 . Cells were expanded until reaching passage 3 to ensure homogeneity of the culture and adequate purity. Cells in passage 3 were used for the miRNA expression measurements.

Characterization of isolated osteoblasts: Alkaline phosphatase activity and alizarin red as indication for calcium deposition
Alkaline phosphatase (ALP) activity was evaluated at days 3, 7 and 14 days after isolation.
Thus, p-nitrophenyl phosphate (pNPP) was used as phosphatase substrate. In brief, isolated osteoblasts were washed twice with D-PBS and subsequently incubated with ALP substrate solution (i.e. 3.5 mM p-nitrophenyl phosphate in the presence of 50 mM glycine, 100 mM Tris-base and 2 mM magnesium chloride) for 30 minutes at 37°C. Next, 100 µl of the solution were transferred to a 96 well plate and the absorbance was measured at 405 nm using an Omega FLUOStar plate reader (BMG Labtech GmbH). The ALP activity was reported as means of p-nitrophenol content produced and calculations were performed based on a standard curve. Triplicates were evaluated in all cases.
Alizarin red staining was similarly performed at days 3, 7 and 14 days after osteoblast isolation to evaluate the presence of calcium deposits. In brief, ethanol-fixed cells were incubated with Alizarin red solution (5 mg/mL in D-PBS) for 10 minutes at room temperature. Stained cells were washed extensively with water and subsequently photographed using a microscope (BZ-9000, Keyence). Calcium deposits are stained as red spots. Thereafter, the alizarin red dye was extracted with 100 mM cetylpyridinium chloride by incubating the stained cells for 3 hours at room temperature. Absorbance was measured at 570 nm and calculations were performed based on a standard curve. Experiments were performed in triplicates.

Primary human osteoclast generation
4 Peripheral blood mononuclear cells (PBMCs) were isolated from collected blood by using Lymphocyte Separation Medium (LSM, density 1077 kg/m 3 , Biowest, Nuaillé, France). In brief, 30 ml of the collected patient's blood was carefully layered on top of 20 ml LSM.
Subsequently, the tubes were centrifuged during 20 minutes at 1000g without brakes. PBMCs were then collected from the interphase layer. Washed PBMCs were resuspended in α-MEM supplemented with 10% FCS and 1% penicillin/streptomycin (α-MEM complete) and plated at 3 x 10 6 cells/cm 2 . 24 hours later, the cell culture medium was changed to α-MEM complete additionally containing 25 ng/ml M-CSF. After 6 days of M-CSF stimulation, 10 ng/ml RANKL were added and the cells were cultured under these conditions for the next 24 hours. At that time, the cell culture medium was replaced with α-MEM complete additionally containing solely 20 ng/ml RANKL. Osteoclasts were then harvested either at 21 days or 28 days after culture for further analysis.

Characterization of functional osteoclasts generated from PBMCs: TRAP staining and activity
In order to assess osteoclast differentiation and function, tartrate-resistant acid phosphatase (TRAP) activity was determined. TRAP staining was performed to visualize the presence of osteoclasts. Thus, confirming successful differentiation from PBMCs. TRAP activity was examined to determine the level of osteoclast resorption activity.
The TRAP staining was determined at 21 and 28 days after initiation of osteoclast differentiation. In brief, cells were fixed with 4% formaldehyde for 5 minutes and subsequently allowed to air-dry at room temperature. Next, cells were covered with a TRAP staining solution (i.e. 40 mM sodium acetate, 10 mM sodium tartrate, 0.01% naphthol AS-MX phosphate, 0.06% fast red violet LB salt, and 1% N,N-dimethylformamide) and incubated for 20 minutes at 37°C. After a washing step with Dulbecco's phosphate buffered 5 saline (D-PBS), photographs were taken using the fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). Experiments were performed in triplicates.
The TRAP activity of stimulated PBMCs was determined at 6, 21 and 28 days after initiation of osteoclast differentiation. For this analysis, 50 µl of cell culture supernatant were mixed with 150 µl 5 mM 4-nitrophenyl phosphate disodium solution (pNPP) in a 96 well plate. After incubation for 1 hour at 37°C, the reaction was stopped by addition of 50 µl 3 M NaOH and the absorbance was determined at 405 nm using an Omega FLUOStar plate reader (BMG Labtech GmbH, Jena, Germany). Experiments were performed in triplicates. TRAP activity was calculated based on a standard curve.

miRNA extraction from serum samples using miRNeasy Serum/Plasma Kit
Serum samples were thawed on ice. Next, 1000 µl QIAzol lysis reagent was added to 200 µl serum sample and vortexed to ensure complete mixing. The mixture was incubated at room temperature for 5 minutes and 3.5 µl miRNeasy Spike-In control (1.6 x 10 8 copies/µl C. elegans miR-39-3p miRNA mimic, Qiagen) was subsequently added. RNA extraction was performed by adding 200 µl chloroform to the mixture. After a brief vortex, the mixture was incubated at room temperature for 2 minutes and subsequently centrifuged at 12000g, 4 o C for 15 minutes. The upper aqueous phase was thoroughly mixed with 100% ethanol and the mixture was pipetted into an RNeasy MinElute spin column (Qiagen). After a short centrifugation (15 seconds) at 8000g, 700 µl buffer RWT and 500 µl buffer RPE were added to the spin columns always followed by a short centrifugation step (8000g). Finally, the spin columns were washed with 80% ethanol and the RNA was eluted by adding 15 µl RNase-free water and centrifuging for 1 minute at full speed. Obtained samples were stored at -80 o C until further cDNA transcription. 6

Evaluation of the purity and integrity of isolated miRNA from serum, tissue and cells
To guarantee a correct quantification of circulating miRNAs levels as well as miRNAs in tissue and cellular material, several quality control steps were applied namely, hemolysis test to serum samples, use of RNA spike-in control and RNA integrity checks.
• Hemolysis test to serum samples: Free hemoglobin was determined spectrophotometrically using a 3-points measurement at 415, 380 and 450 nm (Harboe method). Samples showing hemolysis were not included in further miRNA extraction steps.
• RNA integrity checks: Purity of isolated miRNA was determined spectrophotometrically (BioPhotometer plus UV, Eppendorf AG) by reading the absorbance at 230, 260 and 280 nm. The ratio A 260 /A 280 was considered as indication of the presence of contaminants. Values in the range of 1.8-2.0 were considered as pure RNA. In addition, the ratio A 260 /A 230 was used as secondary measurement of RNA purity. In this case, a range of 2.0-2.2 was considered as pure RNA. Samples in which obtained A 260 /A 280 and A 260 /A 230 values were out of the mentioned range were excluded from the evaluation.
In addition, total RNA-integrity was assessed running aliquots of the isolated miRNA samples on a denaturing 1.5% agarose gel containing 7 µl ethidium bromide (EtBr).
Of each sample, 0.3 µg miRNA was brought into solution with 15 µl RNase-free water and 5 µl loading buffer. The samples were placed in the gel. Subsequently, 90 volts were applied for 40 minutes. The gels were evaluated using INTAS Gel iX20 Doc System (Intas Science Imaging Instruments GmbH, Göttingen, Germany).

cDNA synthesis using the miScript II RT Kit (Qiagen)
7 A reverse transcription master mix was prepared by mixing together 4 µl 5x miScript HiSpec Buffer, 2 µl 10x miScript Nucleics Mix (contains dNTPs, rATP, oligo-dT primers, and miRNA reverse transcription internal control), 2 µl miScript Reverse Transcriptase Mix and adequate amount of RNase-free water. By using miScript HiSpec Buffer, only mature miRNAs are converted into cDNA. RNA samples (i.e. isolated miRNA from serum, tissue and cells) were thawed on ice and 20 ng RNA were used as template in the reverse transcription reaction. The RNA template was added to the reverse transcription master mix maintaining the final reaction volume in 20 µl for all the reactions. The mixture was gently mixed and incubated at 37°C for 60 minutes followed by an incubation step at 95°C for 5 minutes in an Eppendorf mastercycler Nexus. Undiluted cDNA was stored at -20 o C until further use in the qPCR reactions.  Fig. S1. TRAP staining (A, B) and activity (C) of generated osteoclasts after respective periods of M-CSF and RANKL stimulation. Black arrows in (A) indicate multinucleated cells. In addition, higher magnification images provided in (B) illustrate the multinucleated character of isolated osteoclasts. Significant differences are indicated in (C) by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Normality of the data was tested by D'Agostino Pearson test. Statistical analysis was performed by means of a multiple t-test corrected for multiple comparisons using Holm-Sidak. Scale bar = 100 µm.