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Cited by 115Extracellular vesicles in diseaseCancer-related molecular mechanisms researchCircular RNAs in diseases

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Long non-coding RNA PVT1 encapsulated in bone marrow mesenchymal stem cell-derived exosomes promotes osteosarcoma growth and metastasis by stabilizing ERG and sponging miR-183-5p

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Zhao, Wei, Qin, Pan, Zhang, Da, Cui, Xichun, Gao, Jing, Yu, Zhenzhu, Chai, Yuting, Wang, Jiaxiang, Li, Juan·2019·Aging
DOI 10.18632/aging.102406PMC PMC6874467
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Abstract

Exosomes secreted by bone marrow mesenchymal stem cells (BMSCs) promote osteosarcoma cell proliferation and migration, while the underlying mechanism remains unknown. Since the long non-coding RNA PVT1 has been reported to be upregulated in osteosarcoma cells and contributes to its growth and metastasis, we aim to investigate whether BMSC-derived exosomes promote osteosarcoma growth and metastasis via transporting PVT1 into osteosarcoma cells. The PVT1 expression in BMSC-derived exosomes was markedly higher than that in osteosarcoma cell-derived exosomes. The co-culturing of BMSC-derived exosomes and osteosarcoma cells (Saos-2, MG-63, and MNNG/HOS cell lines) significantly raised PVT1 expression of osteosarcoma cells. The direct binding between PVT1 and the oncogenic protein ERG was confirmed using RNA immunoprecipitation and RNA pull-down assays, and the transported PVT1 promotes osteosarcoma cell proliferation and migration via inhibiting degradation and ubiquitination of ERG. PVT1 also increased ERG expression through sponging miR-183-5p. In summary, our findings indicated that BMSC-derived exosomes encapsulate PVTl and transport it into osteosarcoma cells, and the transported PVT1 promotes tumor growth and metastasis by inhibiting ubiquitination and promoting expression of ERG in osteosarcoma cells. These data provide a novel insight into the mechanism of BMSC-derived exosomes in affecting osteosarcoma progression.

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Figure 1 BMSC-derived exosomes transport lncRNA PVT1 into osteosarcoma cells. The BMSC-derived exosomes (BMSC-EXO) and the osteosarcoma cell MNNG/HOS-derived exosomes (MNNG-EXO) in the culture medium were respectively isolated. (A) They were observed under the transmission electron microscopy. (B) The expression of CD81 and CD63 proteins, both of which were exosome markers, was detected using western blot analysis. (C) The expression of PVT1 was detected using qRT-PCR. The osteosarcoma cell lines, including Saos-2, MG-63, and MNNG/HOS, were co-cultured with 40 μg/mL BMSC-EXO or MNNG-EXO for 48 h. (D) The expression of PVT1 in osteosarcoma cells. (E) BMSCs were transfected with si-PVT1 for 48 h. The expression of PVT1 in exosomes which were isolated from PVT1-interfering BMSCs was detected. (F) The expression of PVT1 in osteosarcoma cells, which were co-cultured with BMSC-EXOsi-PVT1 or BMSC-EXOsi-control for 48 h. Three independent experiments. **p<0.01 vs MNNG-EXO or si-control. PBS, phosphate buffer saline was used as the control of both kinds of exosomes. si-PVT1, small interfering RNA of PVT1.
Figure 1 BMSC-derived exosomes transport lncRNA PVT1 into osteosarcoma cells. The BMSC-derived exosomes (BMSC-EXO) and the osteosarcoma cell MNNG/HOS-derived exosomes (MNNG-EXO) in the culture medium were respectively isolated. (A) They were observed under the transmission electron microscopy. (B) The expression of CD81 and CD63 proteins, both of which were exosome markers, was detected using western blot analysis. (C) The expression of PVT1 was detected using qRT-PCR. The osteosarcoma cell lines, including Saos-2, MG-63, and MNNG/HOS, were co-cultured with 40 μg/mL BMSC-EXO or MNNG-EXO for 48 h. (D) The expression of PVT1 in osteosarcoma cells. (E) BMSCs were transfected with si-PVT1 for 48 h. The expression of PVT1 in exosomes which were isolated from PVT1-interfering BMSCs was detected. (F) The expression of PVT1 in osteosarcoma cells, which were co-cultured with BMSC-EXOsi-PVT1 or BMSC-EXOsi-control for 48 h. Three independent experiments. **p<0.01 vs MNNG-EXO or si-control. PBS, phosphate buffer saline was used as the control of both kinds of exosomes. si-PVT1, small interfering RNA of PVT1.CC BY · PubMed Central
Figure 2 PVT1 in exosomes inhibits degradation and ubiquitination of ERG in osteosarcoma cells. Ssos-2 and MNNG/HOS cells were transfected with siRNA of PVT1 (si-PVT1) for 48 h. (A) The expression of ERG protein. (B) The degradation of ERG protein at 3, 6, and 9 hours after the treatment of the protein synthesis inhibitor, CHX (125 μg/mL). (C) The SPOP and ERG proteins were detected in PVT1-protein complex using RNA pull-down assay. Input was used as the positive control; antisense RNA was used as the negative control. (D) PVT1 was detected in ERG-RNA binding complex using RIP assay. Input was used as the positive control; IgG was used as the negative control. (E) Ubiquitination assay: Ssos-2, MG-63, and MNNG/HOS cells were transfected with pcDNA-PVT1, HA-Ub and His-SPOP for 24 h followed by the immunoprecipitation with HA antibody and immunoblotting with ERG antibody. (F) The ubiquitination assay was also performed in PVT1-interfering osteosarcoma cells after being co-cultured with BMSC-EXO. Three independent experiments. *p<0.05, **p<0.01 vs si-control. CHX, cycloheximide. pcDNA-PVT1, the PVT1 overexpressing vector. HA, hemagglutinin. Ub, ubiquitin.
Figure 2 PVT1 in exosomes inhibits degradation and ubiquitination of ERG in osteosarcoma cells. Ssos-2 and MNNG/HOS cells were transfected with siRNA of PVT1 (si-PVT1) for 48 h. (A) The expression of ERG protein. (B) The degradation of ERG protein at 3, 6, and 9 hours after the treatment of the protein synthesis inhibitor, CHX (125 μg/mL). (C) The SPOP and ERG proteins were detected in PVT1-protein complex using RNA pull-down assay. Input was used as the positive control; antisense RNA was used as the negative control. (D) PVT1 was detected in ERG-RNA binding complex using RIP assay. Input was used as the positive control; IgG was used as the negative control. (E) Ubiquitination assay: Ssos-2, MG-63, and MNNG/HOS cells were transfected with pcDNA-PVT1, HA-Ub and His-SPOP for 24 h followed by the immunoprecipitation with HA antibody and immunoblotting with ERG antibody. (F) The ubiquitination assay was also performed in PVT1-interfering osteosarcoma cells after being co-cultured with BMSC-EXO. Three independent experiments. *p<0.05, **p<0.01 vs si-control. CHX, cycloheximide. pcDNA-PVT1, the PVT1 overexpressing vector. HA, hemagglutinin. Ub, ubiquitin.CC BY · PubMed Central
Figure 3 PVT1 promotes ERG expression via sponging miR-183-5p. (A) The online database predicted the potential binding between PVT1 and miR-183-5p. (B) The luciferase activity after the co-transfection of miR-183-5p mimic and pGL3-PVT1-wt or miR-183-5p mimic and pGL3-PVT1-mut. (C) The expression of miR-183-5p in PVT1-interfering osteosarcoma cells. (D) The online database predicted the interaction between miR-183-5p and ERG 3’-UTR. The luciferase activity of ERG 3’-UTR and the protein level of ERG were detected after overexpressing miR-183-5p. (E) The ERG protein expression after the transfection of si-PVT1 or the co-transfection with miR-183-5p inhibitor for 48 h in three osteosarcoma cell lines. Three independent experiments. **p<0.01 vs negative control (pre-NC) or si-control. wt, wild type. mut, mutant. si-PVT1, the siRNA of PVT1. Pre-NC, the control of miR-183-5p mimic. NC, the control of imR-183-5p inhibitor.
Figure 3 PVT1 promotes ERG expression via sponging miR-183-5p. (A) The online database predicted the potential binding between PVT1 and miR-183-5p. (B) The luciferase activity after the co-transfection of miR-183-5p mimic and pGL3-PVT1-wt or miR-183-5p mimic and pGL3-PVT1-mut. (C) The expression of miR-183-5p in PVT1-interfering osteosarcoma cells. (D) The online database predicted the interaction between miR-183-5p and ERG 3’-UTR. The luciferase activity of ERG 3’-UTR and the protein level of ERG were detected after overexpressing miR-183-5p. (E) The ERG protein expression after the transfection of si-PVT1 or the co-transfection with miR-183-5p inhibitor for 48 h in three osteosarcoma cell lines. Three independent experiments. **p<0.01 vs negative control (pre-NC) or si-control. wt, wild type. mut, mutant. si-PVT1, the siRNA of PVT1. Pre-NC, the control of miR-183-5p mimic. NC, the control of imR-183-5p inhibitor.CC BY · PubMed Central
Figure 4 PVT1 in BMSC-EXO promotes osteosarcoma cell proliferation and migration via increasing ERG. (A) The BMSC-EXOsi-PVT1 was co-cultured with MNNG/HOS cells for 48 h. After the co-culturing, cell proliferation and migration were detected using CCK-8, colony formation, scratch and Transwell migration assays. (B) The MNNG/HOSsi-ERG cells and MNNG/HOSsi-Ets-1 cells were co-cultured with or without BMSC-EXO for 48 h. The cell proliferation and migration were detected. (C) The BMSC-EXOsi-PVT1 was co-cultured with MNNG/HOS cells or MNNG/HOSAd-ERG cells for 72 h. The cell proliferation, migration, and invasion were detected. Three independent experiments. *p<0.05, **p<0.01 vs negative control or exosomes+si-control or si-control. #p<0.05, ##p<0.01 vs exosomes+si-control or si-PVT1+Ad-GFP. BMSC-EXOsi-PVT1, exosomes isolated from PVT1-interfering BMSC. MNNG/HOSsi-ERG cells, the MNNG/HOS cells which were transfected with siRNA of ERG. MNNG/HOSsi-Ets-1 cells, the MNNG/HOS cells which were transfected with siRNA of Ets-1. MNNG/HOSAd-ERG, the MNNG/HOS cells which were transfected with ERG-overexpressing vector. CCK-8, cell counting kit-8 assay.
Figure 4 PVT1 in BMSC-EXO promotes osteosarcoma cell proliferation and migration via increasing ERG. (A) The BMSC-EXOsi-PVT1 was co-cultured with MNNG/HOS cells for 48 h. After the co-culturing, cell proliferation and migration were detected using CCK-8, colony formation, scratch and Transwell migration assays. (B) The MNNG/HOSsi-ERG cells and MNNG/HOSsi-Ets-1 cells were co-cultured with or without BMSC-EXO for 48 h. The cell proliferation and migration were detected. (C) The BMSC-EXOsi-PVT1 was co-cultured with MNNG/HOS cells or MNNG/HOSAd-ERG cells for 72 h. The cell proliferation, migration, and invasion were detected. Three independent experiments. *p<0.05, **p<0.01 vs negative control or exosomes+si-control or si-control. #p<0.05, ##p<0.01 vs exosomes+si-control or si-PVT1+Ad-GFP. BMSC-EXOsi-PVT1, exosomes isolated from PVT1-interfering BMSC. MNNG/HOSsi-ERG cells, the MNNG/HOS cells which were transfected with siRNA of ERG. MNNG/HOSsi-Ets-1 cells, the MNNG/HOS cells which were transfected with siRNA of Ets-1. MNNG/HOSAd-ERG, the MNNG/HOS cells which were transfected with ERG-overexpressing vector. CCK-8, cell counting kit-8 assay.CC BY · PubMed Central
Figure 5 PVT1 in BMSC-EXO promotes osteosarcoma growth and metastasis via increasing ERG in vivo. The mouse xenograft (n=18) was established by subcutaneous inoculation of MNNG/HOS cells, and the pulmonary metastatic model (n=18) was established by tail vein injection of MNNG/HOS cells. Eight days after the establishment of xenograft and 3 weeks after the establishment of metastatic model, mice were divided into 3 groups, the control group (with PBS injection in tail vein, n=6), the exosome group (with 10 μg of BMSC-EXO injection in tail vein, n=6), and the exosomes+si-PVT1 group (with PVT1-interfering BMSC derived exosome injection in tail vein, n=6). (A) The tumor volume was detected every 4 days. (B) The expression of PVT1 and ERG in tumor tissues after 3 weeks. (C) The number and the H&E staining of lung metastatic nodules (red arrows). *p<0.05, **p<0.01 vs control. #p<0.05, ##p<0.01 vs exosomes.
Figure 5 PVT1 in BMSC-EXO promotes osteosarcoma growth and metastasis via increasing ERG in vivo. The mouse xenograft (n=18) was established by subcutaneous inoculation of MNNG/HOS cells, and the pulmonary metastatic model (n=18) was established by tail vein injection of MNNG/HOS cells. Eight days after the establishment of xenograft and 3 weeks after the establishment of metastatic model, mice were divided into 3 groups, the control group (with PBS injection in tail vein, n=6), the exosome group (with 10 μg of BMSC-EXO injection in tail vein, n=6), and the exosomes+si-PVT1 group (with PVT1-interfering BMSC derived exosome injection in tail vein, n=6). (A) The tumor volume was detected every 4 days. (B) The expression of PVT1 and ERG in tumor tissues after 3 weeks. (C) The number and the H&E staining of lung metastatic nodules (red arrows). *p<0.05, **p<0.01 vs control. #p<0.05, ##p<0.01 vs exosomes.CC BY · PubMed Central

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Metadata

Authors
Zhao, W., Qin, P., Zhang, D., Cui, X., Gao, J., Yu, Z., Chai, Y., Wang, J., & Li, J.
Affiliations
First Affiliated Hospital of Zhengzhou University (CN)
Corresponding author
Li, Juan
ORCID
Venue
Aging
Publisher
Impact Journals, LLC
Published
2019-11-07
Volume
11
Issue
21
Pages
9581-9596
ISSN
1945-4589 (ISSN-L)
DOI
10.18632/aging.102406
PMC
PMC6874467
OpenAlex
W2984884798
Cited by
115
References
42
Citations by year
2026: 5 · 2025: 14 · 2024: 18 · 2023: 16 · 2022: 26 · 2021: 19 · 2020: 17
FWCI
5.13
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Aging (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Circular RNAs in diseases
Keywords
bone marrow mesenchymal stem cells, exosomes, osteosarcoma, ERG, miR-183-5p, tumor growth, metastasis, protein stabilization, ubiquitination, tumor microenvironment, cell proliferation, cell migration
MeSH terms
Transcriptional Regulator ERG, Animals, Bone Neoplasms, Humans, Mice, Inbred BALB C, Mice, Nude, Neoplasm Metastasis, Neoplasm Transplantation, Osteosarcoma, MicroRNAs, Cell Line, Tumor, Exosomes, Primary Cell Culture, RNA, Long Noncoding

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