Mechanical strain affects some microRNA profiles in pre-oeteoblasts.

Abstract MicroRNAs (miRNAs) are important regulators of cell proliferation, differentiation and function. Mechanical strain is an essential factor for osteoblast proliferation and differentiation. A previous study revealed that a physiological mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz applied once a day for 1 h over 3 consecutive days promoted osteoblast differentiation. However, the mechanoresponsive miRNAs of these osteoblasts were not identified. In this study, we applied the same mechanical tensile strain to in vitro cultivated mouse MC3T3-E1 pre-osteoblasts and identified the mechanoresponsive miRNAs. Using miRNA microarray and qRT-PCR assays, the expression patterns of miRNAs were evaluated and 5 of them were found to be significantly different between the mechanical loading group and the control group: miR-3077-5p, 3090-5p and 3103-5p were significantly upregulated and miR-466i-3p and 466h-3p were downregulated. Bioinformatics analysis revealed possible target genes for these differentially expressed miRNAs. Some target genes correlated with osteoblast differentiation. These findings indicated that the mechanical strain changed the expression levels of these miRNAs. This might be a potential regulator of osteoblast differentiation and responses to mechanical strain.


INTRODUCTION
Mechanical strain plays a pivotal role in the growth and maintenance of bone. Mechanical loading can activate anabolic genes and induce bone remodeling, while a lack of loading leads to bone loss [1][2][3][4][5][6]. In bone tissue, osteoblasts act as receptors, transforming mechanical stimuli into biochemical signals to guide bone formation. Previous studies have revealed that mechanical loading is an important regulator for osteoblast differentiation, proliferation and apoptosis [7,8]. However, there is no detailed knowledge on the miRNAs and regulating mechanisms involved in the osteoblast response to mechanical stimuli. MiRNAs are a class of short, non-coding, single-strand small RNAs, with a length of ~22 nt. They govern gene expression by targeting to special regions (known as 3' untranslated regions) resulting in translational repression or mRNA degradation [9,10]. It was previously found that microRNA plays a pivotal role in cell growth and apoptosis [11,12]. Researchers have discovered many miRNAs that regulate cell differentiation, especially osteoblast differentiation [9,13]. Some mechanosensitive or mechanoresponsive miRNAs were recently identified. Some of these play significant roles in osteoblast proliferation and differentiation. For example, miRNA-542-3p suppresses osteoblast proliferation and promotes apoptosis, while miRNA-27 promotes osteoblastogenesis [14,15]. It has been reported that mechanical tensile strain of 2500 με at 0.5 Hz for 1 h per day over 3 consecutive days promoted osteoblast differentiation [16,17]. However, the mechanoresponsive miRNAs of osteoblasts subjected to such mechanical tensile strain were not identified. In this study, some novel mechanosensitive miRNAs applied to MC3T3-E1 cells were selected.

Cell culture
A mouse pre-osteoblast cell line, MC3T3-E1 (provided by Institute of Basic Medicine of Peking Union Medical College, Beijing, 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).

Application of mechanical strain
At confluence, the medium was renewed and the cells were stimulated with mechanical tensile strain of 2500 με at 0.5 Hz for 1 h per day over 3 days. The mechanical strain was generated using a 4-point bending device, as previously described [16].

MiRNA microarray
MiRNA microarrays were performed at Phalanx Biotech. MiRNAs were enriched from total RNA extracted using a mirVana miRNA Isolation Kit and labeled with a mirVana Array Labeling Kit (Ambion). The labeled miRNAs were used for hybridization on each miRNA microarray, to determine the differential expression of miRNAs. This procedure was repeated twice. Target labeling, hybridization, imaging and data processing were performed at Phalan Biotech according to the protocols described by the manufacturers using the Mouse & Rat miRNA OneArray Microarray and Sanger miRBase 19. Data were acquired using Agilent Feature Extraction software version 10.7. Further data analyses were performed using GeneSpring GX 10.0.

Gene-specific quantitative RT-PCR
Total RNA was extracted with Trizol reagent according to the manufacturer's instructions. After treatment with DNase, the total RNA was reverse transcribed to cDNA with gene-specific RT primers using the TaqMan MicroRNA Reverse Transcription Kit (ABI) according to the manufacturer's instructions. Seven gene-specific stem-loop RT primers were designed according to the miRNA sequences listed in the Sanger miRBase (microRNA sequences, targets and gene nomenclature) with U6 as the reference gene (shown in Table 1). Each reaction mixture of RT primer contained 400 ng RNA sample per 15 μl. Random primers were used for U6. The reactions were incubated in a 96-well plate for 30 min at 16ºC, 30 min at 42ºC and 5 min at 85ºC, and then held at 4ºC. The expressions of the miRNA precursors were determined via qRT-PCR performed on a BIO-RAD CFX Connect Real-Time PCR detection system. 50 ng per 10 μl reaction volume and 500 nM of forward and reverse primers were used. Cycling conditions were 95ºC for 2 min, followed by 45 cycles of 95ºC for 5 sec and 55ºC for 20 sec, using Kappa Probe Fast qPCR Master Mix (Kapa Biosystem) and Roche Universal Probe Library Probe 21 according to the manufacturers' protocols.

Statistical analysis
To identify differentially expressed miRNAs in the strained group and control group, Student's t-test was employed using SPSS 19.0. Experiments were repeated in triplicate. Statistical significance was measured using Student's t-test, with p < 0.05 considered statistically significant. Table 1. RT-Primers and F-Primers used for qRT-PCR of miRNAs.

RESULTS
Five miRNAs were responsive to mechanical strain applied to MC3T3-E1 cells.

MicroRNA
Oligonucleotide sequence ( 2. Hierarchical clustering analysis of miRNA from MC3T3-E1 cells subjected to a mechanical tensile strain of 2500 με. Each row represents one miRNA. Five miRNAs were expressed at a higher level in the mechanically strained group than in the control (unstrained) group, and eleven miRNAs were expressed at a lower level. Fig. 3. The differential expression levels of miRNAs were verified using qRT-PCR. Mechanical strain of 2500 με significantly upregulated the expression level of mmu-miR-3077-5p, 3090-5p and 3103-5p, and downregulated the expression level of mmu-miR-466h-3p and 466i-3p in the mechanically strained group compared with the unstrained group. n = 5, *p < 0.05, **p < 0.01 compared with the unstrained group.

Putative target gene prediction
MicroRNASeq, DIANA LAB and miRDB were applied to predict target genes for these differentially expressed miRNAs. The results indicated that several target genes correlated with osteoblast differentiation (Table 2).

DISCUSSION
Skeleton and bone are responsive to dynamic mechanical loading, the presence of which prevents bone loss and promotes bone formation, while its absence results in a decline in bone mass [18,19]. The mechanical strain loaded on bone tissue normally stimulates a response via bone cells [20]. The osteoblasts are bone-forming cells that reside in the surface of the bone so they can be responsive to mechanical strain in vivo [21]. It has been reported that osteoblasts are responsive to mechanical strain [22][23][24]. Differentially expressed miRNAs in mechanically stimulated tissue or cells are considered mechanoresponsive (also called mechanosensitive) miRNAs. In endothelial cells, smooth muscle cells and chondrocytes, the identified mechanoresponsive miRNAs play important roles in cell proliferation, differentiation and apoptosis [25][26][27].
In this study, the results indicated that a mechanical tensile strain of 2500 με at 0.5 Hz for 1 h per day over 3 consecutive days changed the expression level of five miRNAs of MC3T3-E1 pre-osteoblasts: miR-3077-5p, 3090-5p, 3103-5p, 466h-3p and 466i-3p. Bioinformatics analysis revealed the possible target genes of these miRNAs. In all of the putative target genes, we found 12 genes correlating with osteoblast differentiation, including BMP-2, Creb1 and Runx 2 [17,35,36]. These target genes play important roles in osteoblast differentiation and bone formation. These results suggest that the proper mechanical strain changed the expression levels of these miRNAs, and this might be a potential regulator of osteoblast differentiation and response to mechanical strain. Further study is needed to verify these target genes and investigate the detailed mechanisms of these mechanically responsive miRNAs in osteoblast proliferation, differentiation and maturation. Mechanical strain could promote osteoblast differentiation [16,17]. In our previous study, a mechanical strain of 2500 με at 0.5 Hz for 8 hours also promoted osteoblast differentiation and four miRNAs (miR-218, 191*, 3070a and 33) were responsive to the mechanical strain [42]. However, the mechanoresponsive miRNAs of our previous study were different from the mechanoresponsive miRNAs found in this study. These results suggest that mechanical strains of 2500 με at 0.5 Hz with different loading methods resulted in different mechanoresponsive miRNAs in MC3T3-E1 pre-osteoblasts. In summary, five novel miRNAs that were mechanoresponsive and probably involved in osteoblast differentiation were discovered in this study, and the five mechanoresponsive miRNAs were different from those identified with mechanical strain with different loading method in a previous study.