RING finger gene 180 inhibits osteosarcoma progression through regulating chromobox homolog 4 ubiquitination

Abstract

Osteosarcoma (OS) is still the most common malignant bone tumor whose etiology remains largely unclear. Here, we aimed to investigate the role of a novel E3 ubiquitin ligase RING finger gene 180 (RNF180) in OS progression. RNF180 was significantly down-regulated in both OS tissues and cell lines. We up-regulated RNF180 using over-expression vector and knocked down RNF180 using specific short hairpin RNAs in OS cell lines. RNF180 over-expression inhibited the viability and proliferation yet promoted apoptosis in OS cells, while RNF180 knockdown showed the opposite effects. RNF180 also suppressed tumor growth and lung metastasis in mouse model, accompanied with elevated E-cadherin level and decreased ki-67 level. Besides, chromobox homolog 4 (CBX4) was predicted as a substrate of RNF180. RNF180 and CBX4 were both localized mainly in nucleus and their interaction was validated. RNF180 aggravated the decline of CBX4 level after cycloheximide treatment. RNF180 also promoted the ubiquitination of CBX4 in OS cells. Furthermore, CBX4 was significantly up-regulated in OS tissues. RNF180 also up-regulated Kruppel like factor 6 (KLF6) yet down-regulated RUNX family transcription factor 2 (Runx2) in OS, which served as downstream targets of CBX4. In addition, RNF180 inhibited migration, invasion and epithelial-mesenchymal transition (EMT) in OS cells, which were partially abolished by CBX4 over-expression. In conclusion, our findings demonstrated that RNF180 inhibits OS development via regulating CBX4 ubiquitination, and RNF180-CBX4 axis is a potential therapeutic target for OS treatment.

Keywords: CBX4; Osteosarcoma; RNF180; migration and invasion; ubiquitination.

Figure 1.

rnf180 displays lower expression and promoter hypermethylation in OS. (a) The expression and promoter methylation level of RNF180 in sarcoma tissues based on race, gender and TP53 mutation status were available on UALCAN database. (b) the RNF180 mRNA level in OS and adjacent normal tissues was measured using quantitative real-time PCR (n = 72). (c, d) the protein level of RNF180 in OS and adjacent normal tissues was detected by western blotting and immunohistochemistry (bar = 200 μm, 100 μm) assays. (e, f) Methylation-specific PCR was used to evaluate methylation level in OS tissues (n = 5) and cell lines. (g) Methylation-specific PCR was used to evaluate methylation level in human osteoblast cells (hFOB1.19) and OS cells (MnnG/HOS, Saos-2, MG-63, and U2OS). OS: osteosarcoma; RNF180: Ring Finger Protein 180; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

RNF180 inhibits OS cell proliferation in vitro. U2OS and Saos-2 cells were transfected with vector, pcDNA3.1-RNF180, scramble shRNA or RNF180 shRnas. (a) the RNF180 mRNA level in human osteoblast cell line hFOB 1.19 and four OS cell lines were measured using quantitative real-time PCR. (b) RNF180 protein level was detected using western blotting. (c) Cell viability was evaluated using a commercial cell counting kit-8. (d, e) Cell proliferation was assessed using clone forming assay and Edu staining (bar = 100 μm). (f) Cell apoptosis was detected using Tunel staining (bar = 100 μm). RNF180: Ring Finger Protein 180; Edu: Ethynyl-2’-deoxyuridine; Tunel: TdT-mediated dUTP Nick-End Labeling; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

RNF180 inhibits migration and invasion of OS cells. U2OS and Saos-2 cells were transfected with vector, pcDNA3.1-RNF180, scramble shRNA or RNF180 shRnas. (a) Cell migration was evaluated using wound healing assay (bar = 100 μm). (b) Cell invasive ability was assessed by Transwell assay (bar = 100 μm). (c) the expression of E-cadherin and N-cadherin was detected using immunofluorescence assay (bar = 20 μm). RNF180: Ring Finger Protein 180; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

RNF180 is a putative E3 ubiquitin enzyme of CBX4. (a) the predicted targets of RNF180 on Ubibrowser database were shown. (b) the localization of RNF180 and CBX4 in HEK293T and U2OS cells was observed using immunofluorescence assay (bar = 50 μm). (c) U2OS and Saos-2 cells were transfected with Flag-RNF180, Myc-CBX4 or Flag-RNF180+Myc-CBX4. The interaction between RNF180 and CBX4 was detected using co-immunoprecipitation. (d) Cells were transfected with vector or pcDNA3.1-RNF180. The protein levels of CBX4 after CHX treatment was detected using western blotting. (e) the ubiquitylation of CBX4 was evaluated by immunoprecipitation using HA tag, followed by western blotting detection. RNF180: Ring Finger Protein 180; CBX4: chromobox homolog 4; CHX: cycloheximide. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

RNF180 regulates KLF6 and Runx2 expression through targeting CBX4. (a) the mRNA level of CBX4 in OS and adjacent normal tissues were measured using quantitative real-time PCR (n = 72). (b) the correlation between RNF180 level and CBX4 level in OS tissues were analyzed. (b) the protein level of CBX4 in OS and adjacent normal tissues was evaluated using immunohistochemistry (bar = 200 μm, 100 μm). (c) Relative mRNA level of CBX4 in both U2OS and Saoa-2 cells transfected with vector, pcDNA3.1-RNF180, scramble shRNA, or RNF180 shRNA. (d, e) U2OS and Saos-2 cells were transfected with vector, pcDNA3.1-RNF180, scramble shRNA, RNF180 shRnas, vector + control, pcDNA3.1-RNF180 + control, vector + pcDNA3.1-CBX4, pcDNA3.1-RNF180 + pcDNA3.1-CBX4. Protein levels of CBX4, KLF6 and Runx2 were detected by western blotting. OS: osteosarcoma; RNF180: Ring Finger Protein 180; CBX4: chromobox homolog 4; KLF6: Kruppel like factor 6; Runx2: RUNX family transcription factor 2; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

RNF180 inhibits proliferation, migration and invasion in OS cells by targeting CBX4. U2OS and Saos-2 cells were transfected with vector + control, pcDNA3.1-RNF180 + control, vector + pcDNA3.1-CBX4, pcDNA3.1-RNF180 + pcDNA3.1-CBX4. (a) Cell viability was measured using cell counting kit-8 assay. (b, c) Cell proliferation was evaluated using clone forming assay. (d, e) Tunel assay was carried out to assess cell apoptosis (bar = 100 μm). (f, g) Wound healing was performed to evaluate cell migration (bar = 100 μm). (h, i) Transwell was performed to assess cell invasion (bar = 100 μm). (j) the protein levels of E-cadherin and N-cadherin were detected using immunofluorescence assay (bar = 20 μm). OS: osteosarcoma; RNF180: Ring Finger Protein 180; CBX4: chromobox homolog 4; Tunel: TdT-mediated dUTP Nick-End Labeling; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

RNF180 inhibits OS tumor growth in mouse model. Balb/c node mice were injected with U2OS or Saos-2 cells via tail vein. Cells were transfected with vector or EGFP tagged pcDNA3.1-RNF180. (a) Xenograft tumors were shown (n = 5). (b, c) Tumor volume and tumor weight were measured and analyzed 22 days after implantation. (d) Immunohistochemistry was performed to evaluate protein levels of RNF180, E-cadherin, Ki67 and CBX4 (bar = 100 μm). (e) Tumor metastasis was shown by in vivo imaging (bar = 100 μm). (f) Lung metastasis nodes was observed after hematoxylin-eosin staining. OS: osteosarcoma; RNF180: Ring Finger Protein 180; *p < 0.05, **p < 0.01, ***p < 0.001.