miRNA-296-5p functions as a potential tumor suppressor in human osteosarcoma by targeting SND1

Abstract

Background: The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p.

Methods: We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis.

Results: We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003).

Conclusion: Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.

Figure 1

Levels of mature miR-296-5p expression in osteosarcoma cell lines and tissues. (A) Expression levels of miR-296-5p were measured by qRT-PCR in osteoblasts. (B) Expression levels of miR-296-5p were measured by qRT-PCR in five osteosarcoma cell lines. qRT-PCR: quantitative reverse transcription polymerase chain reaction; OS: osteosarcoma; hFOB 1.19: a normal osteoblast cell line; ^∗P < 0.05, compared to hFOB 1.19; ^†P < 0.01, compare to Control.

Figure 2

Effects of miR-296-5p on the proliferation, migration, and invasion of osteosarcoma cells in vitro. (A) HOS and Saos-2 cells were transfected with either miR-296-5p mimics or the negative control (NC). Twenty-four hours later, cells were assessed by qRT-PCR. (B and C) MTT assay. (D and E) Wound healing assay (bright field; original magnification 10×) and transwell migration and invasion assays (crystal violet staining; original magnification 10×) of HOS and Saos-2 cells transfected with miR-296-5p mimics or NC. ^∗P < 0.01. qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Figure 3

Levels of SND1 expression in osteosarcoma cell lines and tissues. (A and B) Expression levels of SND1 were measured by western blotting and IHC in normal and osteosarcoma tissues (original magnification, 40×). (C and D) The expression level of miR-296-5p was measured by qRT-PCR in osteoblasts and four osteosarcoma cell lines and humanosteoblasts (HOB) cells. CTRL: control; IHC: immunohistochemistry; OS: osteosarcoma; qRT-PCR: quantitative reverse transcription polymerase chain reaction. ^∗P < 0.05, ^†P < 0.01, compared to hFOB 1.19.

Figure 4

Effects of Staphylococcal Nuclease Domain-Containing Protein 1 (SND1) on the proliferation, migration, and invasion of osteosarcoma cells in vitro. (A) HOS and Saos-2 cells were transfected with SND1 shRNA or NC. Twenty-four hours later, cells were assessed by qRT-PCR. (B) MTT assay. (C and D) Wound healing assay (bright field; original magnification 10×) and transwell migration and invasion assays (crystal violet staining; original magnification 10×) of HOS and Saos-2 cells transfected with SND1 shRNA or NC. NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; shRNA, short hairpin RNA. ^∗P < 0.01.

Figure 5

SND1 is a direct target of miR-296-5p. (A) miR-296-5p and its putative binding sequences in the 3′-UTR of SND1. Mutations were generated in the complementary site that binds to the seed region of miR-296-5p. (B) The qRT-PCR analysis of miR-296-5p and SND1 expression in 12 human osteosarcoma tissues. (C) Spearman correlation analyses between relative miR-296-5p expression and relative SND1 mRNA expression in 12 osteosarcoma tissues. (D) Luciferase reporter assay in HOS and Saos-2 cells revealed that miR-296-5p overexpression suppressed the activity of the firefly luciferase carrying the wild-type (wt) 3′-UTR of SND1. ^∗P < 0.01. OS: osteosarcoma; qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Figure 6

SND1 overexpression rescues the phenotype of osteosarcoma cells with miR-296-5p overexpression. (A and B) Western blot and qRT-PCR analyses of SND1-flag expression in HOS cells transfected with miR-296-5p mimics, SND-flag, or NC. (C) MTT assay. (D and E) Wound healing assay (bright field; original magnification, 10×) and transwell assay (crystal violet staining; original magnification 10×) of HOS and Saos-2 cells transfected with the SND1-flag, miR-296-5p mimics, or NC. NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction. ^∗P < 0.05, ^†P < 0.01. ns: no significance.