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Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells
Published on: March 8, 2019
An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using
Zhiwei Zhang1, Jiandan Wang, Xiaoying Lü
1State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, People's Republic of China.
Abstract:
This work combined transcriptomics, proteomics, and microRNA (miRNA) analyses to elucidate the mechanism of natural hydroxyapatite (NHA)-induced osteogenic differentiation of mesenchymal stem cells (MSCs). First, NHA powder was obtained from pig bones and fabricated into disc-shaped samples. Subsequently, the proliferation and osteogenic differentiation of MSCs cultured on NHA were investigated. Then, proteomics was employed to detect the protein expression profiles of MSCs cultured on NHA, and the effect of NHA on MSCs was analyzed through an integrated pathway analysis (including proteomics and previous transcriptomics data) in which specific NHA-induced differentiation pathways were analyzed. The pathway nodes with expression data at both the mRNA and protein levels (mRNA-protein pairs) were filtered in differentiation-related pathways. miRNAs corresponding to these target mRNA-protein pairs were predicted, screened and tested, and the regulatory effects of miRNAs on mRNA-protein pairs were analyzed. Finally, the NHA-induced osteogenic pathways were verified. The results of an MTT assay and alkaline phosphatase (ALP) staining showed that the cell proliferation rate decreased and the osteogenic performance improved in the presence of NHA. By integrating transcriptomics and proteomics, the genes and proteins involved in 89 pathways were shown to be differentially expressed. Among them, 5 differentiation-associated pathways, in which 9 miRNAs and 8 regulated-target mRNA-protein zby inhibiting the target mRNA-protein pair HSPA8 in the MAPK signaling pathway, and miR-26a and miR-26b might inhibit adipogenic differentiation by repressing the target mRNA-protein pair HMGA1 in the adipogenesis pathway. A verification experiment for the osteogenic pathway indicated that the ERK1/2 or JNK MAPK pathways might play an important role in NHA-induced osteogenic differentiation. In conclusion, NHA affected MSCs at both the transcriptional and translational levels, and MSC osteogenic differentiation eventually occurred through the MAPK and adipogenesis pathways, in which miRNAs and target mRNAs/proteins participated cooperatively.
