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. 2017 Jul 18;24(1):46.
doi: 10.1186/s12929-017-0353-9.

LncRNA SNHG15 contributes to proliferation, invasion and autophagy in osteosarcoma cells by sponging miR-141

Ke Liu  1 , Yi Hou  1 , Yunke Liu  1 , Jia Zheng  2
Affiliations

LncRNA SNHG15 contributes to proliferation, invasion and autophagy in osteosarcoma cells by sponging miR-141

Ke Liu et al. J Biomed Sci. .

Abstract

Background: LncRNA small nucleolar RNA host gene 15 (SNHG15) was reported to play an oncogenic role in tumors. However, the role of SNHG15 and its molecular mechanism in osteosarcoma (OS) cells are largely unknown.

Methods: qRT-PCR was performed to evaluate the expression levels of SNHG15 and miR-141 in OS tissues and cells. Cell transfection with different siRNAs, miRNAs or pcDNAs into U2OS and MG63 cells were carried out by Lipofectamine 2000. The effects of SNHG15 and miR-141 on OS cell proliferation, invasion and the levels of autophagy-related proteins were analyzed by MTT assay, Transwell invasion/migration assay and western blot, respectively. Luciferase reporter assay was used to confirm whether SNHG15 could directly interact with miR-141.

Results: We found that up-regulation of SNHG15 was inversely correlated with miR-141 expression in OS tissues. SNHG15 knockdown and miR-141 overexpression significantly suppressed cell proliferation, invasion, migration and autophagy while SNHG15 overexpression and miR-141 repression exhibited the opposite effects on OS cells. Besides, SNHG15 could directly interact with miR-141 and regulate its expression. Furthermore, miR-141 suppressing significantly overturned the inhibition on proliferation, invasion, migration and autophagy mediated by SNHG15 knockdown while miR-141 overexpression remarkably attenuated SNHG15 overexpression-induced proliferation, invasion, migration and autophagy in OS cells.

Conclusion: Our data showed that SNHG15 contributes to proliferation, invasion, migration and autophagy in OS by negatively regulating miR-141, providing a new potential target and prognostic biomarker for the treatment of OS.

Keywords: Osteosarcoma; Sponge; lncRNA SNHG15; miR-141.

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Conflict of interest statement

Authors’ information

Ke Liu, the first author. Jia Zheng, the corresponding author.

Ethics approval and consent to participate

Not applicable.

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All the authors have consented for publication.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Expression levels of SNHG15 and miR-141 in OS tissues. qRT-PCR was performed to evaluate the expression levels of SNHG15 (a) and miR-141 (b) in 35 paired OS tissues and the adjacent normal tissues. GAPDH was used as the endogenous control. (c) Correlation between SNHG15 and miR-141 expression. *P < 0.05 vs. control group
Fig. 2
Fig. 2
Effects of SNHG15 on OS cell proliferation, invasion, migration and autophagy. a qRT-PCR was performed to determine the expressions of SNHG15 in five OS cell lines (143B, U2OS, HOS, MG63 and SaOS2) and osteoblastic cell line HFOB1.19. GAPDH was used as the endogenous control. b The expression of SNHG15 in U2OS cells transfected with si-SNHG15–1, si-SNHG15–2, si-SNHG15–3 or si-control was evaluated by qRT-PCR. GAPDH was used as the endogenous control. c The expression of SNHG15 in MG63 cells transfected with pcDNA-SNHG15 or vector was assessed by qRT-PCR. GAPDH was used as the endogenous control. MTT assay was carried out to detect cell proliferation at 24 h, 48 h, 72 h, and 96 h in U2OS cells (d) transfected with si-SNHG15 or si-control and MG63 cells (e) transfected with pcDNA-SNHG15 or vector. Transwell invasion assay in U2OS cells (f) transfected with si-SNHG15 or si-control and MG63 cells (g) transfected with pcDNA-SNHG15 or vector were performed to detect cell invasiveness. Transwell migration assay in U2OS cells (h) transfected with si-SNHG15 or si-control and MG63 cells (i) transfected with pcDNA-SNHG15 or vector were performed to detect cell migration. Western blot was used to evaluate the levels of Atg5, LC3-I, LC3-II and p62 in U2OS cells (j) transfected with si-SNHG15 or si-control and MG63 cells (k) transfected with pcDNA-SNHG15 or vector. β-actin was used as the internal control. *P < 0.05 vs. control group
Fig. 3
Fig. 3
Effect of miR-141 on OS cell proliferation, invasion and autophagy. a The expressions of miR-141 in OS cell lines (U2OS and MG63) and osteoblastic cell line HFOB1.19 were analyzed by qRT-PCR. GAPDH was used as the endogenous control. Cell proliferation in U2OS cells (b) transfected with miR-141 or miR-control and MG63 cells (c) transfected with anti-miR-141 or anti-miR-control at 24 h, 48 h, 72 h and 92 h was examined by MTT assay. The number of invasive cells in U2OS cells (d) transfected with miR-141 or miR-control and MG63 cells (e) transfected with anti-miR-141 or anti-miR-control were determined by Transwell invasion assay. The levels of Atg5, LC3-I, LC3-II and p62 in U2OS cells (f) transfected with miR-141 or miR-control and MG63 cells (g) transfected with anti-miR-141 or anti-miR-control were detected by western blot. β-actin was used as the internal control. *P < 0.05 vs. control group
Fig. 4
Fig. 4
The regulatory relationship between SNHG15 and miR-141. a The bioinformatics predicted binding sites of miR-141 on SNHG15. Luciferase reporter assay was performed in U2OS (b) and MG63 (c) cells cotransfected with pGL3-SNHG15-WT or pGL3-SNHG15-MUT and miR-141 or miR-control. d qRT-PCR was carried out to determine the expressions of miR-141 in U2OS cells transfected with si-SNHG15, si-control, si-SNHG15 + anti-miR-141, or si-SNHG15 + anti-miR-control. GAPDH was chosen as the internal control. e qRT-PCR was carried out to examine the expression of miR-141 in MG63 transfected with pcDNA-SNHG15, vector, pcDNA-SNHG15 + miR-141, or pcDNA-SNHG15 + miR-control. GAPDH was chosen as the internal control. *P < 0.05 vs. control group
Fig. 5
Fig. 5
SNHG15 contributed to proliferation, invasion, migration and autophagy by sponging miR-141 in OS cells. U2OS cells were transfected with si-SNHG15, si-control, si-SNHG15 + anti-miR-141, or si-SNHG15 + anti-miR-control. MG63 cells were transfected with pcDNA-SNHG15, vector, pcDNA-SNHG15 + miR-141, or pcDNA-SNHG15 + miR-control. Transfected U2OS and MG63 cells were maintained for 48 h. MTT assay was performed to detect cell proliferation at 24 h, 48 h and 72 h in transfected U2OS (a) and MG63 (b) cells. Transwell invasion assay was used to assess cell invasiveness in transfected U2OS (c) and MG63 (d) cells. Transwell migration assay was used to assess cell migration in transfected U2OS (e) and MG63 (f) cells. Western blot analysis was carried out to determine the levels of Atg5, LC3-I, LC3-II and p62 in transfected U2OS (g) and MG63 (h) cells. β-actin was used to normalize the levels of Atg5, LC3-I and LC3-II. *P < 0.05 vs. control group
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