Down-regulation of circular RNA hsa_circ_0007534 suppresses cell growth by regulating miR-219a-5p/SOX5 axis in osteosarcoma

Abstract

Introduction: Circular RNA circ_0007534 and microRNA-219a (miR-219a-5p) were reported to be involved in osteosarcoma (OS) development. Osteosarcoma (OS) is one of the most common malignant bone tumors, which was more prone to occur in the metaphysis of long bones, including distal femur and proximal tibia. However, the detailed mechanisms were not fully clear. The purpose of this research was to reveal the functional mechanisms of circ_0007534 and miR-219a-5p in OS.

Methods: The levels of genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assay. Cell proliferation ability was detected by cell counting kit-8 (CCK-8) and colony formation assay. Cell migration and invasion abilities were measured using the transwell assay. Furthermore, the interaction between miR-219a-5p and circ_0007534 or SRY (sex-determining region Y)-box 5 (SOX5) was predicted by starbaseV3.0, and confirmed by the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Besides, tumor xenograft experiment was performed to analyze the effect of circ_0007534 depletion on tumor growth in vivo.

Results: The levels of circ_0007534 and SOX5 were increased, while the miR-219a-5p level was decreased in OS tissues and cells. Circ_0007534 knockdown repressed the proliferation, colony formation, migration, and invasion in OS cells. Circ_0007534 targeted miR-219a-5p, and miR-219a-5p interacted with SOX5. Furthermore, circ_0007534 regulated the growth of OS cells through modulating the levels of miR-219a-5p and SOX5.

Conclusion: Our finding demonstrated that circ_0007534 knockdown suppressed the growth of OS cells via regulating miR-219a-5p/SOX5 axis, providing a potential target for OS treatment and diagnosis.

Keywords: ATCC, American Type Culture Collection; CCK-8, cell counting kit-8; Circ_0007534; EMT, epithelial mesenchymal transformation; EZH2, zeste homolog 2; OS, osteosarcoma; Osteosarcoma; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; RIP, RNA immunoprecipitation; SD, standard deviation; SOX5; UTR, untranslated region; hFOB1.19, human osteoblast cell line; mRNA, message RNA; miR-219a-5p; qRT-PCR, quantitative real-time polymerase chain reaction.

Fig. 1

The level of circ_0007534 in OS tissues. (A–C) The expression level of circ_0007534 was detected by qRT-PCR assay in OS tissues and paratumor tissues (A), OS tissues from patients at different clinical stage (B), and OS tissues from lymph node metastasis patients (C). ***P < 0.001, n = 3.

Fig. 2

The effect of circ_0007534 on OS cell progression. (A) Circ_0007534 expression was determined in normal cells (hFOB1.19) and OS cells (143B, MG63, HOS, and U2OS). (B) Circ_0007534 expression was examined in U2OS and MG63 cells transfected with sh-NC or sh-circ_0007534. (C and D) CCK-8 was used to assess cell proliferation ability in U2OS (48 h, P = 0.004; 72 h, P < 0.001) and MG63 cells (48 h, P = 0.004; 72 h, P < 0.001). (E) Colony formation assay was performed to measure cell clone formation ability. (F and G) Cell migration and invasive abilities were determined using transwell assay. (H and I) Western blot assay was employed to investigate the levels of three EMT markers. *P < 0.05, n = 3.

Fig. 3

The relationship between circ_0007534 and miR-219a-5p. (A) The interaction between circ_0007534 and miR-219a-5p was predicted by bioinformatics tool starbaseV3.0. (B and C) The luciferase activity of U2OS and MG63 cells transfected with WT-circ_0007534 or MUT-circ_0007534 and miR-219a-5p or miR-NC was determined. (D and E) RIP assay was employed to confirm the interaction between circ_0007534 and miR-219a-5p. (F) The level of circ_0007534 was analyzed in U2OS and MG63 cells transfected with Vector or circ_0007534. (G) MiR-219a-5p level was measured in U2OS and MG63 cells transfected with sh-NC, sh-circ_0007534, Vector, or circ_0007534, respectively. (H and I) MiR-219a-5p level was examined in OS tissues and paratumor tissues (H) as well as OS cells and normal cells (I). (J) QRT-PCR assay was carried out to explore the association between circ_0007534 and miR-219a-5p. *P < 0.05, n = 3.

Fig. 4

The function of miR-219a-5p in circ_0007534-regulated OS cell progression. (A) MiR-219a-5p expression was detected in U2OS and MG63 cells transfected with sh-NC, sh-circ_0007534, sh-circ_0007534 + anti-NC, or sh-circ_0007534 + anti-miR-219a-5p, respectively. (B and C) Cell proliferation ability was measured using CKK-8. (D) Clone formation ability was examined by colony formation assay. (E and F) Transwell assay was employed to determine cell migration and invasive abilities. (G and H) The levels of EMT markers were investigated by western blot assay. *P < 0.05, n = 3.

Fig. 5

The relationship between miR-219a-5p and SOX5. (A) The interaction between miR-219a-5p and SOX5 was predicted by bioinformatics tool starbaseV3.0. (B and C) The luciferase activity was determined in U2OS and MG63 cells transfected with WT-SOX5 or MUT-SOX5 and miR-219a-5p or miR-NC. (D–F) The levels of miR-219a-5p (D) and SOX5 (E and F) were detected in U2OS and MG63 cells transfected with miR-NC, miR-219a-5p, miR-219a-5p + Vector, or miR-219a-5p + circ_0007534, respectively. (G–J) The mRNA level and protein level of SOX5 were examined in OS tissues and paratumor tissues (G and H) as well as OS cells and normal cells (I and J). (K) The relationship between SOX5 and miR-219a-5p was analyzed. (L) The relationship between SOX5 and circ_0007534 was investigated. *P < 0.05, n = 3.

Fig. 6

The function of SOX5 in circ_0007534-regulated OS cell progression. (A and B) The mRNA level and protein level of SOX5 were detected in U2OS and MG63 cells transfected with sh-NC, sh-circ_0007534, sh-circ_0007534 + Vector, or sh-circ_0007534 + SOX5, respectively. (C and D) CCK-8 was employed to determine cell proliferation ability (P < 0.001). (E) Colony formation assay was used to assess the clone formation ability. (F and G) Transwell assay was performed to measure cell migratory and invasive abilities. (H and I) The levels of EMT markers were examined by western blot assay. *P < 0.05, n = 3.

Fig. 7

The effect of circ_0007534 depletion on tumor growth in vivo. (A) Tumor volume was calculated in circ_0007534-depleted mice every 5 d. (B) 32 days upon injection, the mice were euthanized, and tumor tissues were photographed and weighted. (C) The proliferation ability of transplanted tumor tissues in nude mice was determined by immunohistochemical analysis of Ki-67. (D–F) The mRNA levels of circ_0007534, miR-219a-5p, and SOX5 were detected by qRT-PCR assay. (G) The protein levels of SOX5 and EMT markers were determined using western blot assay. *P < 0.05, n = 3.

Fig. 8

Circ_0007534 promoted proliferation, migration, invasion by regulating the miR-219a-5p/ SOX5 in OS cells.