BackgroundThe extracellular matrix (ECM) provides a supportive microenvironment for cells, which is suitable as a tissue engineering scaffold. Mechanical stimulus plays a significant role in the fate of osteoblast, suggesting that it regulates ECM formation. Therefore, we investigated the influence of mechanical stimulus on ECM formation and bioactivity.MethodsMouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with mechanical tensile strain. After removing the cells, the ECMs coated on dishes were prepared. The ECM protein and calcium were assayed and MC3T3-E1 cells were re-seeded on the ECM-coated dishes to assess osteoinductive potential of the ECM.ResultsThe cyclic tensile strain increased collagen, bone morphogenetic protein 2 (BMP-2), BMP-4, and calcium levels in the ECM. Compared with the ECM produced by unstrained osteoblasts, those of mechanically stimulated osteoblasts promoted alkaline phosphatase activity, elevated BMP-2 and osteopontin levels and mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and increased secreted calcium of the re-seeded MC3T3-E1 cells.ConclusionMechanical strain promoted ECM production of osteoblasts in vitro, increased BMP-2/4 levels, and improved osteoinductive potential of the ECM. This study provided a novel method to enhance bioactivity of bone ECM in vitro via mechanical strain to osteoblasts.

BackgroundMechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation.MethodsMouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated.ResultsThe mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts’ miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation.ConclusionsIn osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes.

MicroRNA (miRNA) is an important regulator of cell differentiation and function. Mechanical strain is important in the growth and differentiation of osteoblasts. Therefore, mechanresponsive miRNA may be important in the response of osteoblasts to mechanical strain. The purpose of the present study was to select and identify the mechanoresponsive miRNAs of osteoblasts. Mouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with a mechanical tensile strain of 2,50 με at 0.5 Hz, and the activity of alkaline phosphatase (ALP), mRNA levels of ALP, osteocalcin (OCN), and collagen type I (Col I), and protein levels of bone morphogenetic proteins (BMPs) in the cell culture medium were assayed. Following miRNA microarray and reverse transcription-quantitative polymerase chain reaction analyses, differentially expressed miRNAs in the mechanically strained cells and unstrained cells were selected and identified. Using bioinformatics analysis, the target genes of the miRNAs were then predicted. The results revealed that the mechanical strain of 2,500 με increased the activity of ALP, the mRNA levels of ALP, OCN and Col I, and the protein levels of bone morphogenetic protein(BMP)-2 and BMP-4 Continuous mechanical stimulation for 8 h had the most marked stimulant effects. miR-218, miR-191*, miR-3070a and miR-33 were identified as differentially expressed miRNAs in the mechanically strained MC3T3-E1 cells. Certain target genes of these four miRNAs were involved in osteoblastic differentiation. These findings indicated that a mechanical strain of 2,500 με, particularly for a period of 8 h, promoted osteoblastic differentiation, and the four mechanoresponsive miRNAs identified may be a potential regulator of osteoblastic differentiation and their response to mechanical strain.

Long non-coding RNAs (lncRNAs) play important functional roles in many diverse biological processes. However, not all expressed lncRNAs are functional. Thus, it is necessary to manually collect all experimentally validated functional lncRNAs (EVlncRNA) with their sequences, structures, and functions annotated in a central database. The first release of such a database (EVLncRNAs) was made using the literature prior to 1 May 2016. Since then (till 15 May 2020), 19 245 articles related to lncRNAs have been published. In EVLncRNAs 2.0, these articles were manually examined for a major expansion of the data collected. Specifically, the number of annotated EVlncRNAs, associated diseases, lncRNA-disease associations, and interaction records were increased by 260%, 320%, 484% and 537%, respectively. Moreover, the database has added several new categories: 8 lncRNA structures, 33 exosomal lncRNAs, 188 circular RNAs, and 1079 drug-resistant, chemoresistant, and stress-resistant lncRNAs. All records have checked against known retraction and fake articles. This release also comes with a highly interactive visual interaction network that facilitates users to track the underlying relations among lncRNAs, miRNAs, proteins, genes and other functional elements. Furthermore, it provides links to four new bioinformatics tools with improved data browsing and searching functionality. EVLncRNAs 2.0 is freely available at https://www.sdklab-biophysics-dzu.net/EVLncRNAs2/.