To elucidate the effects of miR-616 on the malignant biological processes of osteosarcoma and to preliminarily explore its potential mechanisms.

In situ hybridization (ISH) was employed to analyze miR-616 expression in 11 paraffin-embedded osteosarcoma specimens collected in our department during January 2018 to December 2019. Quantitative real-time PCR (qRT-PCR) was used to compare the mRNA expression level of miR-616 in the osteoblast cell line hFOB1. 19 and osteosarcoma cell lines 143B and HOS. Stable cell lines with miR-616 knockdown or overexpression were established via lentiviral transfection in 143B and HOS cells. Cell proliferation and apoptosis were detected by flow cytometry, while cell invasion and migration were assessed using Transwell and colony formation assays, respectively. To evaluate the effect of miR-616 on tumor growth in vivo, 10 female nude mice (4 weeks old, weighing 18~20 g) were randomized into a control group and a miR-616 overexpression group. After the xenograft tumor model was constructed, the growth of subcutaneous tumors was monitored. Finally, next-generation sequencing and a dual-luciferase reporter assay were performed to identify the target genes of miR-616.

ISH results showed that miR-616 expression was up-regulated in osteosarcoma tissues than adjacent tissues, and primarily localized in the cytoplasm. qRT-PCR confirmed that miR-616 level was significantly higher in 143B and HOS cells than hFOB1. 19 cells (P<0.05). In vitro experiments revealed that miR-616 overexpression enhanced the proliferation, migration and invasion, while suppressing apoptosis in 143B and HOS cells (P<0.01). Conversely, miR-616 knockdown weakened these malignant phenotypes (P<0.05), with miR-616-3p showing a stronger effect on apoptosis than miR-616-5p. Animal experiments demonstrated that the tumor weight in the miR-616 overexpression group was significantly greater than that of the control group (98.00±17.22 vs 33.60±8.08 mg, P<0.01). Furthermore, KLF2 was identified and confirmed as a direct target of miR-616.

MiR-616 promotes malignant biological behaviors in osteosarcoma, and its expression level indicates that it may serve as a potential therapeutic target.

To screen a key molecule, leucine-rich glioma inactivated protein 4 (LGI4), which participates in endothelial cell-mediated osteosarcoma metastasis, and to further analyze its function and molecular mechanism in osteosarcoma metastasis.

A co-culture model of human umbilical vein endothelial cells (HUVECs)-osteosarcoma cells was established, and the genes with significant fold change were screened by whole transcriptome sequencing in combination with relevant literature. After, the candidate target gene, LGI4 was verified with real-time quantitative PCR (RT-qPCR) and Western blotting, the expression of LGI4 at mRNA and protein levels was also determined. Osteosarcoma cell line 143B and MTF LGI4 were transfected with the overexpression plasmid of LGI4 to construct the cells with stable overexpression of LGI4. Then the experiments included 5 groups of osteosarcoma cells, including negative control group (normally cultured), co-culture group (co-cultured with HUVECs), co-culture medium group (treated with the HUVECs medium), vector control group (transfected with control plasmid) and LGI4 group (overexpression of LGI4). Transwell assay and wound healing test were used to detect the migration and invasion abilities of osteosarcoma cells. A tumor metastasis model in nude mice was used to verify the effect of LGI4 overexpressed osteosarcoma cells on distant metastasis in vivo. Bioinformatics analysis was applied to analyze the potential downstream target signaling pathways of LGI4, and recovery experiments were performed with related inhibitors to clarify the role of target signaling pathways in the regulation of osteosarcoma metastasis by LGI4.

The expression levels of LGI4 were markedly decreased in 143B and MTF cells after co-culture with HUVECs (P<0.05). Compared with the negative control group, the cell migration and invasion abilities of the co-culture group and co-medium group were significantly enhanced, whereas the cell migration and invasion rates of the LGI4 overexpression group were significantly lower than that of the vector control group (P<0.05). The nude mice metastasis model revealed that the metastasis ability of osteosarcoma cells was suppressed with LGI4 overexpression. Further analysis revealed that NOTCH4 signaling pathway was the downstream target signaling pathway of LGI4, and functional recovery experiments confirmed that LGI4 resulted in osteosarcoma metastasis by inhibiting NOTCH4 signaling pathway (P<0.05).

Endothelial cells activate NOTCH4 signaling pathway and thus promote tumor metastasis by inhibiting the expression of LGI4 in osteosarcoma cells.