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. 2022 May;13(5):11933-11944.
doi: 10.1080/21655979.2022.2067286.

Bone mesenchymal stem cells (BMSCs)-derived exosomal microRNA-21-5p regulates Kruppel-like factor 3 (KLF3) to promote osteoblast proliferation in vitro

Murong You  1 , Zisheng Ai  2 , Jihuan Zeng  1 , Yang Fu  1 , Liang Zhang  1 , Xin Wu  1
Affiliations

Bone mesenchymal stem cells (BMSCs)-derived exosomal microRNA-21-5p regulates Kruppel-like factor 3 (KLF3) to promote osteoblast proliferation in vitro

Murong You et al. Bioengineered. 2022 May.

Abstract

Bone mesenchymal stem cells (BMSCs)-derived exosomes (Exos) play important roles in osteoporosis, while the regulation of microRNA (miR)-21-5p remains unclear. The BMSCs-derived exosomes were isolated from femoral bone marrow of trauma patients, which were then used to stimulate human osteoblasts (hFOB1.19 cells). The miR-21-5p mimic or inhibitor was transfected into BMSCs to overexpress or knockdown miR-21-5p. The functions of miR-21-5p in osteoporosis were assessed by cell counting kit-8 (CCK-8) assay, alkaline phosphatase (ALP) staining and alizarin red staining assays. We found that BMSCs-derived exosomes could enhance proliferation, osteoblastic differentiation and ALP activity of hFOB1.19 cells. BMSCs-derived exosomes with upregulated miR-21-5p could further enhance these protective impacts compared with that in BMSCs-derived exosomes, while BMSCs-derived exosomes with downregulated miR-21-5p reduced these cell phenotypes. MiR-21-5p could directly bind to the 3'-untranslated region (UTR) of Kruppel-like factor 3 (KLF3), and knockdown of KLF3 obviously attenuated these inhibitory effects of BMSCs-derived exosomes with downregulated miR-21-5p on osteoblastic differentiation and ALP activity of hFOB1.19 cells. In summary, BMSCs-derived exosomal miR-21-5p improved osteoporosis through regulating KLF3, providing a potential therapeutic strategy for osteoporosis.

Keywords: Bone mesenchymal stem cells (BMSCs); Kruppel-like factor 3 (KLF3); exosomes; miR-21-5p; osteoporosis.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

None
Graphical abstract
Figure 1.
Figure 1.
The identification of BMSCs-derived exosomes. (a) The morphology of BMSCs by an inverted microscope. (b) Flow cytometry analysis of the BMSCs surface markers. (c) Exosomes were isolated from BMSCs under transmission electron microscopy (TEM) identification. (d) Western blot analysis of the exosome surface markers.
Figure 2.
Figure 2.
BMSCs-derived exosomes (Exos) enhanced the activities of hFOB1.19 cells. (a) hFOB1.19 cell proliferation determined by Cell counting kit-8 (CCK-8) assay after treated with BMSCs-derived exosomes at different time point. (b) Osteoblastic differentiation in hFOB1.19 cells evaluated by Alizarin Red staining after treated with BMSCs-derived exosomes. (c) Alkaline phosphatase (ALP) staining of hFOB1.19 cells after treated with BMSCs-derived exosomes. (d) hFOB1.19 cells cycle detected by flow cytometry after treated with BMSCs-derived exosomes. * p < 0.05, ** p < 0.01, *** p < 0.001, and NS indicates no significant difference.
Figure 3.
Figure 3.
BMSCs-derived exosomes (Exos) enhanced the activities of hFOB1.19 cells via exosomal miR-21-5p. (a) Relative level of miR-21-5p in BMSCs-derived exosomes after transfection. (b) hFOB1.19 cell proliferation determined by Cell counting kit-8 (CCK-8) assay after treated with BMSCs-derived exosomes with changed miR-21-5p. (c) Alkaline phosphatase (ALP) staining of hFOB1.19 cells after treated with BMSCs-derived exosomes with changed miR-21-5p. (d) Osteoblastic differentiation of hFOB1.19 cells evaluated by Alizarin Red staining after treated with BMSCs-derived exosomes with upregulated or downregulated miR-21-5p. ** p < 0.01, *** p < 0.001.
Figure 4.
Figure 4.
Kruppel-like factor 3 (KLF3) was targeted by miR-21-5p. (a) Relative level of miR-21-5p in hFOB1.19 cells after miR-21-5p mimics transfection. (b) The putative interaction predicted by Targetscan. (c) The impacts of miR-21-5p mimics on WT/MUT KLF3 in hFOB1.19 cells by luciferase reporter assay. (d and e) Relative level of KLF3 in hFOB1.19 cells after transfection of miR mimics or inhibitor determined by qRT-PCR (d) and western blot (e). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5.
Figure 5.
KLF3 mediated the impacts of miR-21-5p on the activities of hFOB1.19 cells. (a and b) Relative mRNA (a) and protein (b) expression level of KLF3 in hFOB1.19 cells after treating with BMSCs-Exo sh-KLF3. (c and d) Alkaline phosphatase (ALP) staining (c) and osteoblastic differentiation (d) of hFOB1.19 cells after co-treated with BMSCs-derived exosomes (Exos) carrying miR-21-5p inhibitor and sh-KLF3. * p < 0.05, ** p < 0.01, *** p < 0.001.
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