Inhibition of LCMR1 and ATG12 by demethylation-activated miR-570-3p is involved in the anti-metastasis effects of metformin on human osteosarcoma

Xing Bao,Libo Zhao,Feng Li,Hanfeng Guan

doi:10.1038/s41419-018-0620-z

Abstract

Epidemiological studies have demonstrated that metformin could mitigate the progression of several tumors. Although it has been proved that metformin could cause demethylation of DNA and lead to up-regulation of some encoding genes and non-coding RNAs, there is little data about the effects of metformin on metastasis, and the interaction between metastasis and autophagy in human osteosarcoma cells. Here, we found miR-570-3p was significantly down-regulated in human metastatic osteosarcoma tissues but not in non-metastatic osteosarcoma tissues. Metformin attenuates the metastasis and autophagy in osteosarcoma. Interestingly, this autophagy favors osteosarcoma cells invasion. Moreover, reduction of metformin-induced inhibition of autophagy could reverse the invasion suppression in osteosarcoma. Mechanistically, metformin increases miR-570-3p by the demethylation of DNA, and the upregulation of miR-570-3p repressed the translation of its target, LCMR1 and ATG12. Our results, for the first time, presents evidence that the miR-570-3p-mediated suppression of LCMR1 and ATG12 is involved in the metformin-induced inhibition of metastasis in osteosarcoma cells.

Highlights

Osteosarcoma (OS) is the most common primary malignant bone tumor, which is characterised by an awfully high aggressiveness with rapid development of distant metastasis[1,2,3]
We explored the molecular mechanisms by which miR570-3p affects metformin-mediated metastasis and autophagy suppression
Our western blot assay suggested that LCMR1 and autophagy-related gene 12 (ATG12) expression was notably increased in metastatic osteosarcoma compared with the primary non-metastatic ones (Fig. 6g)

Summary

Result

Metformin did not markedly affect the vitality of MG63, U2OS or 143B cells at concentrations of 2, 5 or 10 mM (Fig. 1a). In metformin-treated 143B and U2OS cells, there was These data suggest that miR-570-3p has a significant role in the metformin-induced inhibition of invasion and autophagy in human osteosarcoma. The results demonstrated that treatment with metformin in 143B and U2OS cells significantly decreased DNA methylation levels at the CpG islands of miR-570-3p promoter regions (Fig. 5b). Taken together, these data indicated that metformininduced demethylation of miR-570-3p promoter may, at least in part, conduce to upregulation of miR-570-3p in osteosarcoma cells. Our western blot assay suggested that LCMR1 and ATG12 expression was notably increased in metastatic osteosarcoma compared with the primary non-metastatic ones (Fig. 6g). Compared to the anti-miR-570-3p group, fewer lung metastatic nodules were found in the anti-con group (Fig. 7e).The xenograft tumors were removed and evaluated by qRT-PCR, immunohistochemistry and WB (Fig. 7a–c)

Discussion

Findings

Materials and methods