LINC00662 Long Non-Coding RNA Knockdown Attenuates the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Regulating the microRNA-15a-5p/Notch2 Axis

Abstract

Purpose: Osteosarcoma (OS) is a frequently occurring malignancy in children and adolescents. In this study, we aimed to investigate the effects of the long non-coding RNA (lncRNA) LINC00662 (LINC00662) in OS and the underlying molecular mechanism.

Methods: The expression of LINC00662, microRNA-15a-5p (miR-15a-5p), and Notch2 in OS was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration, and invasion of OS cells were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound-healing, and transwell assay. The interactions among LINC00662, miR-15a-5p, and Notch2 were determined by dual-luciferase reporter assays. A tumor xenograft model was established in mice for evaluating tumor growth in vivo.

Results: The expression of LINC00662 and Notch2 was found to be upregulated in OS, but the expression of miR-15a-5p was downregulated. The results demonstrated that LINC00662 knockdown attenuated the proliferation, migration, and invasion of OS cells and suppressed tumor growth in mice. The study further demonstrated that LINC00662 directly interacted with miR-15a-5p, and that Notch2 was a target of miR-15a-5p. The inhibition of miR-15a-5p or Notch2 overexpression markedly reversed the suppressive effect of sh-LINC00662 on the proliferation, migration, and invasion of OS cells.

Conclusion: The study demonstrated that LINC00662 could be a potential biomarker for OS therapy, and LINC00662 knockdown suppressed the proliferation, migration, and invasion of OS cells by regulating the miR-15a-5p/Notch2 axis.

Keywords: Notch2; long non-coding RNA LINC00662; microRNA-15a-5p; osteosarcoma; proliferation.

Figure 1

LINC00662 expression increased in osteosarcoma (OS). (A) The expression of LINC00662 in adjacent tissues and tumor tissues of OS patients was detected by qRT-PCR. P < 0.001 vs Adjacent tissues; (B) The expression of LINC00662 in OS patients at the tumor-node-metastasis (TNM) I/II and TNM III/IV was detected by qRT-PCR. P < 0.001 vs I/II; (C) The expression of LINC00662 in tumors with and without metastasis was detected by qRT-PCR. P < 0.001 vs NO; (D) The relationship between LINC00662 and overall survival of OS patients was measured by Gene Expression Profiling Interactive Analysis (GEPIA) with The Cancer Genome Atlas (TCGA) samples.

Figure 2

LINC00662 knockdown inhibited tumorigenesis of osteosarcoma (OS) cells. (A) The expression of LINC00662 in HOS, MG63, SJSA-1, U2OS and hFOB cells was detected by qRT-PCR. **P < 0.01 vs hFOB; (B) The expression of LINC00662 in MG63 and U2OS cells was detected by qRT-PCR. **P < 0.01 vs sh-NC; (C) The proliferation of MG63 and U2OS cells was detected by MTT assay. *P < 0.05, **P < 0.01 vs sh-NC; (D) The wound healing rate of MG63 and U2OS cells was detected by wound healing assay. **P < 0.01 vs sh-NC; (E) The invasion rate of MG63 and U2OS cells was detected by transwell assay. **P < 0.01 vs sh-NC; (F) Tumor in nude mice. (G) Tumor volume and tumor weight. *P < 0.05, **P < 0.01 vs sh-NC.

Figure 3

LINC00662 was negatively correlated with miR-15a-5p expression. (A) The putative binding site of miR-15a-5p in LINC00662 was predicted by starBase v3.0; (B) The expression of miR-15a-5p in MG63 and U2OS cells was detected by qRT-PCR. **P < 0.01 vs sh-NC; (C) RIP assay was performed to determine the enrichment degrees of LINC00662 and miR-15a-5p in IgG or AGO2 immunoprecipitate. **P < 0.01 vs Anti-IgG; (D) Relative luciferase activity in MG63 and U2OS cells was measured by dual-luciferase reporter assay. **P < 0.01 vs miR-NC; (E) The expression of miR-15a-5p in adjacent tissues and tumor tissues in OS patients was detected by qRT-PCR; (F) The expression of LINC00662 was negatively correlated with miR-15a-5p.

Figure 4

MiR-15a-5p inhibited proliferation, migration and invasion of osteosarcoma (OS) cells. (A) The expression of miR-15a-5p in MG63 and U2OS cells was detected by qRT-PCR. **P < 0.01 vs miR-NC; (B) The proliferation of MG63 and U2OS cells was detected by MTT assay. **P < 0.01 vs miR-NC; (C) The wound healing rate of MG63 and U2OS cells was detected by wound healing assay. **P < 0.01 vs miR-NC; (D) The invasion rate of MG63 and U2OS cells was detected by transwell assay. **P < 0.01 vs miR-NC.

Figure 5

Notch2 was directly targeted by miR-15a-5p. (A) The binding site for miR-15a-5p on the 3ʹ UTR of Notch2 was predicted by targetScan; (B) Relative luciferase activity in MG63 and U2OS cells was measured by dual-luciferase reporter assay. **P < 0.01 vs miR-NC; (C) The expression of Notch2 in adjacent tissues and tumor tissues in OS patients was detected by qRT-PCR. P < 0.001 vs Adjacent tissues; (D) The expression of Notch2 was negatively correlated with miR-15a-5p; (E) The protein expression of Notch2 in MG63 and U2OS cells was measured by Western blot. **P < 0.01 vs miR-NC.

Figure 6

LINC00662 knockdown attenuated the proliferation, migration and invasion of osteosarcoma (OS) cells by targeting miR-15a-5p/Notch2 axis. (A) The expression of Notch2 in hFOB, MG63, U2OS, HOS and SJSA-1 cells was detected by qRT-PCR. **P < 0.01 vs hFOB; (B) Notch2 expression in U2OS cells was measured by qRT-PCR. **P < 0.01 vs pcDNA-NC; (C) The proliferation of U2OS cells was detected by MTT assay. *P < 0.05, **P < 0.01 vs sh-NC; ^#P < 0.05 vs sh-LINC00662; (D) The wound healing rate of U2OS cells was detected by wound healing assay. *P < 0.05, **P < 0.01 vs sh-NC; ^#P < 0.05 vs sh-LINC00662; (E) The invasion rate of U2OS cells was detected by transwell assay. *P < 0.05, **P < 0.01 vs sh-NC; ^#P < 0.05 vs sh-LINC00662; (F) The protein expression of Notch2 in U2OS cells was detected by Western blot. *P < 0.05, **P < 0.01 vs sh-NC; ^#P < 0.05 vs sh-LINC00662.