MIR205 host gene (MIR205HG) drives osteosarcoma metastasis via regulating the microRNA 2114-3p (miR-2114-3p)/twist family bHLH transcription factor 2 (TWIST2) axis

Abstract

Osteosarcoma (OS) is an aggressive malignant tumor with a high rate of lung metastasis and a lack of therapeutic targets. Although the anomalous expression of long non-coding RNA (lncRNA) has been extensively documented in human cancer, its contribution to OS metastasis remains poorly understood. In this study, we found that MIR205 host gene (MIR205HG) was significantly elevated in human OS tissues, especially in metastatic OS tissues. Stable knockdown of MIR205HG inhibited OS cell invasion and lung metastatic foci formation, but did not affect cell viability. The vast majority of MIR205HG was situated in the cytosol, and served as a competing endogenous RNA (ceRNA) that directly bound to microRNA 2114-3p (miR-2114-3p), resulting in increased twist family bHLH transcription factor 2 (TWIST2) level. Pre-clinically, high MIR205HG was linked with dismal overall and relapse-free survival. Functionally, the attenuated cell invasion caused by MIR205HG knockdown was effectively rescued by miR-2114-3p silencing or TWIST2 overexpression. Overall, our findings suggest that the previously uncharacterized regulatory axis of MIR205HG/miR-2114-3p/TWIST2 plays a critical role in promoting OS metastasis, which implies a potential therapeutic target in OS patients with metastasis.

Keywords: Competing endogenous RNA; MIR205HG; metastasis; osteosarcoma.

Graphical abstract

Figure 1.

High MIR205HG is identified in human OS tissues. (a). qRT-PCR analysis of MIR205HG level in normal and OS tissues; OS-NM: non-metastatic OS tissues; OS-M: metastatic OS tissues. (b, c). The survival curve of OS patients based on median MIR205HG level. (d–f). qRT-PCR analysis and FISH assay testing the subcellular localization of MIR205HG in OS cells. **P< 0.01, ***P< 0.001

Figure 2.

MIR205HG knockdown represses cell invasion and metastasis. (a). qRT-PCR analysis of MIR205HG levels in OS cells after infection with lentiviral vector. (b, c). CCK-8 assay testing cell viability after MIR205HG silencing. (d, e). Transwell assay testing cell invasion after MIR205HG silencing. (f, g). Lung metastasis assay testing the effect of MIR205HG on OS cell metastasis in vivo. **P< 0.01

Figure 3.

MIR205HG sponges miR-2114-3p. (a). RIP assay in OS cells with anti-Ago2 antibody, followed by qRT-PCR analysis of MIR205HG and GAPDH enrichment. (b,c). RNA pull-down assay using biotin-labeled MIR205HG/miR-2114-3p probe, followed by qRT-PCR analysis of relative enrichment. (d). Luciferase reporter assay detecting the binding of miR-2114-3p on MIR205HG. (e, f). qRT-PCR analysis of miR-2114-3p/MIR205HG levels in MIR205HG-silenced/miR-2114-3p-overexpressed OS cells. (g). qRT-PCR analysis of miR-2114-3p in normal and OS tissues. H. The correlation between MIR205HG and miR-2114-3p in OS tissues. **P< 0.01, ***P< 0.001

Figure 4.

MIR205HG elevates TWIST2 via miR-2114-3p. (a). Luciferase reporter assay detecting the binding of miR-2114-3p on TWIST2 3`-UTR. (b). RNA pull-down assay using biotin-labeled miR-2114-3p probe, followed by qRT-PCR analysis of relative enrichment. (c). qRT-PCR analysis of TWIST2 mRNA level in miR-2114-3p-overexpressed OS cells. (d). IF and IHC assays testing TWIST2 protein level in miR-2114-3p-overexpressed OS cells and metastatic lung tissues. (e). Western blot assay testing TWIST2 protein level in stable MIR205HG-silenced OS cells transfected with miR-2114-3p inhibitors. (f). qRT-PCR analysis of TWIST2 mRNA level in normal and OS tissues. (g). The correlation between MIR205HG and TWIST2 in OS tissues. (h). Transwell assay testing cell invasion in stable MIR205HG-silenced OS cells transfected with miR-2114-3p inhibitors or TWIST2-overexpressed vector. *P< 0.05, **P< 0.01, ***P< 0.001

Figure 5.

The schematic diagram of the action of MIR205HG in OS. In normal cells, miR-2114-3p binds to TWIST2 mRNA 3`-UTR, resulting in decreased TWIST2 level; In OS cells, MIR205HG sponges miR-2114-3p, antagonizing the inhibitory effect of miR-2114-3p on TWIST2, thereby increasing TWIST2 expression and subsequently promoting OS metastasis