| Objective: Osteocytes are the most abundant mechanically sensitive cells in bone tissue,accounting for more than 90%.Besides,previous studies have shown that some miRNAs differentially expressed in osteocytes are not differentially expressed in osteoblasts stimulated by the same mechanical stimulation,and the conditioned medium of osteocytes under appropriate mechanical stimulation could promote the differentiation of osteoblasts.In addition,mechanical stimulation can promote osteocytes to produce secretion factors and exosomes.In this study,the same cyclic tensile strain was applied to osteocytes MLO-Y4 and osteoblasts MC3T3-E1 cultured in vitro to screen out Micro RNAs(miRNAs)and mRNAs that are only significantly differentially expressed in osteocytes,to further screen out mRNAs,miRNAs and miRNAs’ target genes related to osteogenic differentiation or bone metabolism,and analyze these mechanical response RNAs of osteocytes by bioinformatics,which laid a foundation for the follow-up study of these RNAs being transported to osteoblasts through exosomes to regulate osteogenic differentiation.Methods: 1.Osteocytes MLO-Y4 and osteoblasts MC3T3-E1 cultured in vitro were subjected to the same cyclic tensile strain(2500,0.5Hz,1h/d,lasting for 3 days),and the control groups were set up respectively.2.To detect the mechanical and biological responses of osteocytes and osteoblasts,so as to carry out follow-up experiments.3.Exosomes were extracted from the osteocytes and osteoblasts experimental group conditioned medium by using ultracentrifugation,and the extracted exosomes were analyzed using an electron microscope,particle size,and nano-flow fluorescence.4.The RNA of cells was extracted and high-throughput sequencing was carried out,and the miRNAs and mRNAs with significant differential expression in osteocytes and osteoblasts were screened,and the miRNAs and mRNAs with differential expression only in osteocytes and not in osteoblasts were further screened out.5.Bioinformatics predicts the target genes of miRNAs that are differentially expressed only in osteocytes,and further screens out the target genes related to osteogenic differentiation or bone metabolism by combining literature research;analyzing the relationship between differentially expressed mRNAs and osteogenic differentiation;and screening out the mRNAs whose function are related to osteogenic differentiation or bone metabolism;GO and KEGG functions were analyzed for mRNAs differentially expressed only in osteocytes.The function of the screened miRNAs’ target genes are analyzed by software such as Targetscan,and the signal transduction pathways related to osteogenic differentiation or bone metabolism were selected by combining the previous research work and related literature materials.Results: 1.Compared with the control group,the expressions of BMP-2,ALP,RUNX-2,and COL-1 in the osteoblasts experimental group increased.Compared with the control group,the expressions of NO,NOS,IGF-1,and PGE-2 in the osteocytes experimental group were up-regulated.2.Exosomes are identified both in osteocytes and osteoblasts,and the average particle size and concentration of exosomes in the experimental group are higher than those in the control group.Compared with the control group,the exosomes concentration in the osteocytes experimental group increased more than the ratio of the osteoblasts experimental group to the control group.3.The results of differential RNAs screening showed that there were 22 miRNAs differentially expressed only in osteocytes but not in osteoblasts.It was predicted that the target genes of 13 miRNAs,such as Arfgef1,Negr1,and Kcnmb2,were related to osteogenic differentiation or bone metabolism.There are 30 mRNAs differentially expressed only in osteocytes but not in osteoblasts.Combined with mRNA function analysis,10 mRNAs related to osteogenic differentiation or bone metabolism,such as Arhgef1,Egr2,and Trmp,were screened out.4.Through GO database analysis and KEGG analysis,these signal pathways related to the regulation of osteogenic differentiation or bone metabolism by mRNAs of osteocytes mechanical response are mainly enriched in six signal pathways such as C-type lectin receptor.The differentially expressed miRNAs of target gene function analysis are mainly enriched in 6 signal pathways,such as pluripotent stem cells.Conclusion: Under the cyclic tensile strain of 2500 με,0.5 Hz,and 1 h/d for 3days,some target genes of miRNAs(Arfgef1,Negr1,Kcnmb2,etc.)that are only differentially expressed in osteocytes or the proteins translated by these target genes,as well as some mRNAs(Arhgef1,Adamts,Egr2,etc.)expressed proteins are probably to be factors that regulate osteogenic differentiation.After further literature investigation,it was found that some of these RNAs(miR-205-5p,miR-2137,miR-32-5p,miR-374b-5p,Arhgef1,Adamts,Mapk9,Mras,etc.)were present in the exosomes.This work will lay the foundation for subsequent verification that some of these RNAs may be transported by exosomes to osteoblasts to regulate osteogenic differentiation. |
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