LINC01128 regulates the development of osteosarcoma by sponging miR-299-3p to mediate MMP2 expression and activating Wnt/β-catenin signalling pathway

Abstract

Osteosarcoma (OS) is one of the most common metastatic bone cancers, which results in significant morbidity and mortality. The important role of long non-coding RNAs (lncRNAs) in the biological processes of OS has been demonstrated through several studies. In the current study, we evaluated the role of the lncRNA, LINC01128, in OS. We analysed the expression of LINC01128 in three OS gene expression omnibus (GEO) data sets GSE21257, GSE36001 and GSE42352. The expression of LINC01128 in OS tissues and matched non-tumour tissues obtained from 50 OS patients was detected using qRT-PCR. The association between LINC01128 expression and overall survival of OS patients was evaluated using the Kaplan-Meier method. The effects of LINC01128 knockdown and overexpression were evaluated through in vitro and in vivo assays. The LINC01128/miR-299-3p/ MMP2 axis was verified using dual-luciferase reporter assay and qRT-PCR assays. GEO data sets analysis revealed that the expression of LINC01128 was increased in OS. Elevated LINC01128 expression was accompanied by shorter overall survival in OS patients. Functional studies revealed that LINC01128 knockdown reduced the proliferation, migration and invasion of OS cells both in vitro and in vivo. Mechanistically, LINC01128 sponged miR-299-3p to increase MMP2 expression. Rescue assays determined the role of the LINC01128/miR-299-3p/MMP2 axis in the proliferation, migration and invasion of OS cells. Additionally, the Wnt/β-catenin signalling pathway was activated by LINC01128 and MMP2 in OS cell lines. In summary, this study demonstrates that LINC01128 facilitates OS by functioning as a sponge of miR-299-3p, thus promoting MMP2 expression and activating the Wnt/β-catenin signalling pathway.

Keywords: bone tumour; ceRNA; invasion; lncRNA; proliferation.

FIGURE 1

LINC01128 expression is associated with poor prognosis in OS patients. (A) Three osteosarcoma GEO data sets (GSE21257, GSE36001, GSE42352) were analysed and ten highly expressed genes were identified from the intersection. (B) QRT‐PCR analysis of LINC01128 expression in 50 pairs of OS and normal tissues obtained from OS patients. (C) LINC01128 expression in OS cell lines and in hFOB1.19. (D) Kaplan‐Meier curve showing the overall survival of OS patients stratified based on LINC01128 expression. ^* P < .05, ^** P < .01 and ^*** P < .001

FIGURE 2

Effect of LINC01128 on the proliferation of OS cells. (A) Transfection efficiency of sh‐LINC01128 and LINC01128 overexpression vector. (B) CCK‐8, (C) EdU (bar = 100 μm) and (D) colony formation assays confirmed the effect of LINC01128 knockdown and overexpression on the proliferation of OS cells. ^* P < .05, ^** P < .01 and ^*** P < .001

FIGURE 3

Effect of LINC01128 on migration and invasion of OS cells. (A) Migration of Saos2 and U2OS cells were analysed using wound healing assay (bar = 100 μm). (B) Invasion of Saos2 and U2OS cells were analysed using Transwell assay (bar = 100 μm). ^** P < .01 and ^*** P < .001

FIGURE 4

Sh‐LINC01128 inhibits the OS tumour growth and metastasis. (A) Xenograft OS tumours. (B) Growth of xenograft tumours formed by sh‐LINC01128 cells is slower than that of xenograft tumours formed by sh‐NC cells. (C) Mean weight of xenograft tumours. (D) Sh‐LINC01128 markedly reduced MMP2 and Ki‐67 in tumours compared with negative control (bar = 50 μm). (E) Representative macroscopic and microscopic images (H&E staining) of the lung tissue sections. **P < .01 and ***P < .001

FIGURE 5

LINC01128 serves as a molecular sponge of miR‐299‐3p. (A) The localization of LINC01128 in OS cells was assessed using subcellular fractionation assay. (B) The putative binding sites between LINC01128 and miR‐299‐3p. (C) Dual‐luciferase reporter assay in 293T cells. (D) MiR‐299‐3p expression in OS tissues. (E) LINC01128 expression negatively correlates with miR‐299‐3p expression in OS tissues (r = −0.6737). (F) MiR‐299‐3p expression in OS cell lines and in hFOB1.19. (G) LINC01128 negatively regulates the expression of miR‐299‐3p in OS cells. ^** P < .01 and ^*** P < .001

FIGURE 6

MMP2 is a target of miR‐299‐3p. (A) The binding sites of miR‐299‐3p in the 3'UTR of MMP2 were predicted. (B) Dual‐luciferase reporter assay in 293T cells. (C) MMP2 expression level in OS tissues. (D) Pearson correlation analysis showed that the expression of MMP2 is inversely correlated with miR‐299‐3p expression in OS tissues (r = −0.5430). (E) MMP2 expression is positively correlated with LINC01128 expression in OS tissues (r = 0.6380). (F) MMP2 expression in OS cell lines and in hFOB1.19. (G) Transfection efficiency of miR‐299‐3p mimics and miR‐299‐3p inhibitor. (H, I) RNA and protein level of MMP2 in OS cells after transfection with miR‐299‐3p mimics or inhibitors. ^** P < .01 and ^*** P < .001

FIGURE 7

LINC01128 affects proliferation and invasion of OS cells through collaborating with miR‐299‐3p and MMP2. (A) Transfection efficiency of sh‐MMP2 and MMP2 overexpression vector. (B, C) CCK‐8 and EdU assays showing proliferation of Saos2(sh‐LINC01128) and U2OS(LINC01128) cells after transfection (bar = 100 μm). (D) Transwell assay showing invasion of Saos2(sh‐LINC01128) and U2OS(LINC01128) cells after transfection (bar = 100 μm). ^* P < .05, ^** P < .01 and ^*** P < .001

FIGURE 8

LINC01128 and MMP2 jointly activate the Wnt/β‐catenin signalling pathway. (A) Levels of Wnt/β‐catenin signalling pathway‐associated proteins in OS cells transfected with sh‐MMP2 or MMP2 overexpression vector. (B) Protein levels of Wnt/β‐catenin signalling pathway‐associated proteins in OS cells. (C) Proposed mechanism of LINC01128 in OS LINC01128 functions as a sponge of miR‐299‐3p, thus promoting MMP2 expression and activating the Wnt/β‐catenin signalling pathway, and finally facilitates OS. ^* P < .05, ^** P < .01 and ^*** P < .001