Expression of miR-616 in osteosarcoma and its role in proliferation, apoptosis, migration and invasion of tumor cells

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.