A mouse MLO-Y4 osteocyte cell line (provided by JENNIO Biological Technology, Guangzhou, China) was cultured in dishes with α-MEM medium (α-MEM, Invitrogen), containing 10% FBS and 1% penicillin. Then the cells were transferred to mechanical loading dishes that were reformed from cell culture dishes (Nalge Nunc International).
Mouse MC3T3-E1 osteoblastic cells (JENNIO Biological Technology, Guangzhou) were cultured with the same medium as mentioned above.
Application of mechanical strain
At confluence, the medium was renewed with FBS-free medium, then the MLO-Y4 cells were stimulated with mechanical tensile strain of 2500 με at 0.5 Hz for 8 h by a four-point bending device, as previously described .
Enzyme-linked immunosorbent assay (ELISA)
Following mechanical tensile strain, the expression levels of IGF-1 and PGE2 in the collected culture supernatant were detected using an IGF-1 ELISA kit (Boster Bioengineering Co., Ltd., Wuhan China) and PGE2 EIA kit (Cayman Chemical, Michigan USA), according to the manufacturers’ instructions. An ELISA reader (Thermo Scientific Multiskan FC ELISA Reader, Rockford, IL, USA) was used to measure the absorbances at 450 nm and 420 nm respectively, with the results presented as the content of changes, compared to the unstrained control.
Following mechanical tensile strain, cell lysates were prepared in RIPA lysis buffer (Beijing Solarbio Science & Technology, Co. Ltd., Beijing, China). Protein in cell lysates was quantified using the BCA method. Equal amounts of proteins were separated by electrophoresis on a polyacrylamide gel containing 0.15% SDS, then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk and incubation with primary antibodies overnight at 4 °C, the membranes were incubated with horseradish peroxidase conjugated secondary antibody. The immunoreactive bands were visualized using an ECL detection kit (7 sea biotech Co. Ltd., Shanghai, China). β-actin in cell lysates was used as a loading control. Data were normalized against those of corresponding optical density of β-actin.
Detection of NOS activity
After mechanical tensile strain of MLO-Y4 for 8 h, cells were collected and bathed gently in an ultrasonic processor (UP 400S, Hielscher, Germany) for 2 min. NOS activity was measured using a colorimetric method based on NOS ability to catalyze L-Arg and molecular oxygen to generate NO, and generated NO produces colored compounds with nucleophiles. The optical densities at 530 nm wavelength were obtained using an ELISA reader (Thermo Scientific Multiskan FC ELISA Reader) and activities of NOS were calculated according to the calibration formula provided in the instructions.
Microarray and RT-qPCR validation of miR
The RiboArray miDETECT mouse array (Ribobio Co., Guangzhou, China) and RT-qPCR were used to detect and validate the miR expression levels in MLO-Y4 cells. The miR expression levels of the mechanically strained group were compared with the unstrained group.
Briefly, total RNA extraction and miR enrichment procedures were performed using the Trizol method and an mirVana miR Isolation kit (Ambion Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Target labeling, hybridization, imaging and data processing were performed, according to the manufacturer’s instructions using a RiboArray miDETECT mouse array (Ribobio Corporation) which contained all mouse miRNAs of Sanger miRBase 19. Data were acquired using Agilent Feature Extraction software version 10.7 (Agilent, Palo Alto, CA, USA) [16, 17]. Further data analyses were performed using GeneSpring GX 10.0 software (Agilent, Palo Alto, CA, USA). Following microarray detection, expression levels of 40 miRs with significant differences were validated by RT-qPCR at Ribobio Co., Ltd. in Guangzhou. The miDETECT A Track Uni-Reverse Primers and miDETECT A Track miRNA Forward Primers (specific primers) for RT-qPCR of these miRs were provided by Ribobio Corporation (Ribobio Co., Ltd. Guangzhou, China). Poly(A) tailing, reverse transcription and qPCR were performed successively using the miDETECT A Track miRNA qRT-PCR Starter Kit (Ribobio Co., Ltd). The reactions were incubated in a 96-well optical plate at 95 °C for 20 s, followed by 40 cycles of 10 s at 95 °C, 20 s at 60 °C, and 10 s at 70 °C. Expression analysis was performed in triplicate for each sample. U6 was used as the normalization control. The miR expression levels were quantified using a CFX 96 system (Bio-Rad Laboratories, Hercules, CA).
miRs transfection and preparation of osteocytes’ conditioned medium
MLO-Y4 osteocyte cells, at 70% confluence, were transfected by miR-29b-3p mimic, miR-29b-3p inhibitor and miR control (Ribobio Co., at a final concentration of 50 nM) respectively, using the riboFect CP Transfection Kit (Ribobio Co.) according to the manufacturer’s method.
The cells were carefully washed in serum-free medium to remove proteins from the bovine serum supplement and then incubated in fresh serum-free medium for 24 h (stimulated with mechanical tensile strain or not). After centrifugation at 3000 g for 12 min, the conditioned medium was collected and prepared for the next experiment.
Detection of osteoblastic differentiation
MC3T3-E1 osteoblastic cells at confluence were carefully washed in serum-free medium, then the cells were cultured in osteocytes’ conditioned medium for 24 h. Cells were harvested, washed with a phosphate buffer solution (PBS), and lysed with a lysis buffer (Beijing Solarbio Science & Technology, Co. Ltd). The ALP activity of the lysates was measured with the ALP Activity Assay Kit (Nanjing Jiancheng Biotechnology Co. Ltd., China) at 25 °C according to the provider’s protocol. The bone morphogenetic protein 2 (BMP-2) of the osteoblastic cells was assayed with an ELISA kit (Elabscience Biotechnology Co., Ltd., Wuhan, China), according to the manufacturer’s instructions mentioned above.
cDNA was synthesized using the TIANScript RT kit (Tiangen Biotech Co., Ltd., Beijing, China), then the Runx 2 mRNA was detected using SYBR Green I PCR Mix (Beijing CoWin Biotech Co., Ltd., Beijing, China) according to the manufacturer’s method. The PCR amplification reaction included a denaturation step at 94 °C for 3 min followed by 40 cycles of 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Relative expression was normalized to mRNA levels of GAPDH using the 2-ΔΔCq method.
miRWalk2.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/), MicroRNA.
org (www.microrna.org/) and TargetScan (www.targetscan.org/) were applied to predict target genes for these differently expressed miRs. The same target genes for one corresponding miR presented in three online databases were considered as potential targets.
All data are presented as the mean ± standard deviation from three separate experiments (n = 5 or 6). Data were tested for normal distribution using the Shapiro-Wilk test and differences between groups were analyzed using one-way analysis of variance and determined by the least significant difference test. Statistical analysis was performed using SPSS software (version 18; SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant difference.