Effects And Mechanisms Of Human Umbilical Cord-derived Mesenchymal Stem Cells On Osteosarcoma Cells In Vitro

Objective:To investigate the effects of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs) on the proliferation and migration of osteosarcoma cells(Saos-2)and the underlying molecular mechanism in vitro.Methods:hUC-MSCs were isolated and cultured by tissue explants adherent technique,the cell surface markers CD19, CD29, CD90 and CD105 on hUC-MSCs were identified by flow cytometry, osteogenic and adipogenic differentiation of cells were detected by alizarin red staining and oil red O Staining respectively. Collecting and preparing the conditioned medium of hUC-MSCs(hUC-MSCs-CM) with different concentrations. The effects of hUC-MSCs-CM and recombinant human interleukin 6(rh IL-6) on the proliferation of Saos-2 cells were detected by cytometry and cell counting kit 8(CCK8) assay. The IL-6 secretion volume of hUC-MSCs and Saos-2cells was assayed by enzyme-linked immunosorbent assays(ELISA). The effects of IL-6, hUC-MSCs-CM and AG490 on the protein levels of STAT3 and p-STAT3 in extracts from Saos-2 cells were determined by western-blot assay. RT-PCR was used to assess the transcription level of PCNA, Cyclin D1, Survivin and STAT3 gene, and the proliferation of Saos-2 cells were measured by cytometry and CCK-8 assay. The potential of hUC-MSCs target Saos-2 cells and migration potential of Saos-2 cells were measured by wound-healing assay and Transwell assay.Results:Fibroblast-like adherent cells were successfully isolated and cultured from human umbilical cord. The cells were positive(>97%) for CD29, CD90 and CD105,and negative(0.1%) for CD19, capable of differentiating into osteogenic, adipogenic.Cytometry and CCK-8 assay demonstrated that hUC-MSCs-CM and rh IL-6 promoted the proliferation Saos-2 cells, and the effects of promoting among different concentrations had significant differences, showed a concentration-dependence manner. The IL-6 concentration in hUC-MSCs-CM was 1804.8 ± 152.2 ng/L(5×105hUC-MSCs/m L *24 h), while the IL-6 concentration secreted by the same number of Saos-2 cells was only 170.1± 22.5 ng/L. The activation of STAT3 in Saos-2 cells was inhibited by AG490, STAT3 was activated by conditioned medium from hUC-MSCs and rh IL-6 and that the activation could be attenuated by a neutralization antibody against IL-6. There was no significant difference of both the m RNA and protein expression levels of STAT3 in Saos-2 cells among different groups(P>0.05). Both hUC-MSCs-CM and rh IL-6 up-regulated the m RNA expression of proliferation related genes PCNA, Cyclin D1 and Survivin, and the inhibition of STAT3 in Saos-2cells by IL-6 neutralizing antibody or AG490 down-regulated the tanscriptional levels of this proliferation related genes. The changes in proliferative capability of Saos-2cells in different groups were consistent with the changes of genes expression. The wound-healing assay and Transwell results confirmed that hUC-MSCs could migrate to Saos-2 cells, the migration ability of Saos-2 cells was significantly enhanced by rh IL-6 and hUC-MSCs-CM, the migration of cells was reduced when the neutralizing IL-6 antibody was added to hUC-MSCs-CM, and lower than that of control group.The numbers of the Saos-2 cells crossed polycarbonate membrane in group CM+AG490 were significantly less than that of control group and group CM.Conclusion:(1) hUC-MSCs could migrate to osteosarcoma cells and promote the proliferation and migration of osteosarcoma cells through secreting IL-6 in vitro.(2)STAT3 activation by IL-6 from hUC-MSCs might up-regulates the m RNA expression of PCNA, Cyclin D1 and Survivin, and promotes the proliferation osteosarcoma cells.(3) STAT3 activation by IL-6 from human umbilical cord-derived mesenchymal stem cells enhance the migration of osteosarcoma cells.