LncRNA RUSC1-AS1 promotes osteosarcoma progression through regulating the miR-340-5p and PI3K/AKT pathway

Abstract

Dysregulation of long noncoding RNA (lncRNA) is frequently involved in the progression and development of osteosarcoma. LncRNA RUSC1-AS1 is reported to be upregulated and acts as an oncogene in hepatocellular carcinoma, cervical cancer and breast cancer. However, its role in osteosarcoma has not been studied yet. In the present study, we investigated the role of RUSC1-AS1 in osteosarcoma both in vitro and in vivo. The results showed that the expression of RUSC1-AS1 was significantly upregulated in osteosarcoma cell line U2OS and HOS compared to that in human osteoblast cell line hFOB1.19. Similar results were found in human samples. Silencing RUSC1-AS1 by siRNA significantly inhibited U2OS and HOS cell proliferation and invasion, measured by CCK-8 and transwell assay. Besides, knockdown of RUSC1-AS1 increased cell apoptosis in osteosarcoma cell lines. In addition, RUSC1-AS1 promoted the epithelial-mesenchymal transition (EMT) process of osteosarcoma cells. In vivo experiments confirmed that RUSC1-AS1 knockdown had an inhibitory effect on osteosarcoma tumor growth. Mechanically, we showed that RUSC1-AS1 directly binds to and inhibits miR-340-5p and activates the PI3K/AKT signaling pathway. In conclusion, our study demonstrated that RUSC1-AS1 promoted osteosarcoma development both in vitro and in vivo through sponging to miR-340-5p and activating the PI3K/AKT signaling pathway. Therefore, RUSC1-AS1 becomes a potential therapeutic target for osteosarcoma.

Keywords: PI3K/AKT; RUSC1-AS1; miR-340-5p; osteosarcoma.

Figure 1

RUSC1-AS1 was upregulated in osteosarcoma samples and cell lines. (A) The expression of RUSC1-AS1 in osteosarcoma tissues and adjacent normal tissues. (B) The expression of RUSC1-AS1 in osteosarcoma cell lines MG63, U2OS, HOS and Saos-2 and osteoblast cell line hFOB1.19. (C) Kaplan-Meier curve of osteosarcoma patients with high/low RUSC1-AS1 expression. (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Figure 2

RUSC1-AS1 promoted cell viability, invasion and inhibits cell apoptosis in osteosarcoma cell lines. (A) RUSC1-AS1 expression after si-RUSC1-AS1 or si-NC transfection. (B) CCK-8 assay, (C, E) transwell invasion assay, (D, F) flow cytometry apoptosis assay, and (G, H) wound healing assay in U2OS and HOS cells after si-RUSC1-AS1 or si-NC transfection.

Figure 3

miR-340-5p was a direct target of RUSC1-AS1. (A) ENCORI, Starbase and miRDB predicted miRNAs that could bind to RUSC1-AS1. (B) Predicted binding sites of miR-340-5p and RUSC1-AS1. (C) The miR-340-5p expression after mimics-NC and miR-mimics transfection. (D–E) Luciferase reporter assay in U2OS and HOS cells showed direct binding of RUSC1-AS1 and miR-340-5p. (F–G) RIP assay in U2OS and HOS cells showed direct binding of RUSC1-AS1 and miR-340-5p. (H) The miR-340-5p expression after si-RUSC1-AS1 or si-NC transfection. (I) miR-340-5p expression in osteosarcoma tissues and adjacent normal tissues. (J) Linear correlation between miR-340-5p and RUSC1-AS1.

Figure 4

RUSC1-AS1 exerted its role through miR-340-5p. (A) CCK-8 proliferation assay, (B, D) flow cytometry apoptosis assay, (C, E) wound healing assay in U2OS/HOS cells or U2OS/HOS cells transfected with RUSC1-AS1 plasmid, miR-mimics or RUSC1-AS1+miR-mimics.

Figure 5

RUSC1-AS1/miR-340-5p activated PI3K/AKT signaling pathway. (A) KEGG analysis of the target genes of miR-340-5p predicted by miRDB website tool. (B–C) Western blot analysis of proteins in PI3K/AKT pathway in U2OS/HOS cells or U2OS/HOS cells transfected with RUSC1-AS1 plasmid, miR-mimics or RUSC1-AS1+miR-mimics. (D–E) CCK-8 assay in U2OS/HOS cells transfected with RUSC1-AS1 plasmid with/without wortmannin treatment.

Figure 6

RUSC1-AS1/miR-340-5p promoted EMT in osteosarcoma cells. The expression of (A) N-Cadherin, (B) E-Cadherin, (C) Vimentin, (D) Snail and (E) ZEB1 in U2OS/HOS cells or U2OS/HOS cells transfected with RUSC1-AS1 plasmid, miR-mimics or RUSC1-AS1+miR-mimics.

Figure 7

RUSC1-AS1 promoted osteosarcoma cell growth in vivo. (A) Tumor growth, (B) tumor weight and (C) tumor volume in U2OS injected nude mice treated with si-NC and si-RUSC1-AS1. (D) The protein expression of p-AKT, AKT, p-PI3K and PI3K in tumor cells from si-NC or si-RUSC1-AS1 treated mice. (E) The expression of EMT-related genes in mice treated with si-NC and si-RUSC1-AS1.