Yonsei Med J.  2019 Feb;60(2):163-173. 10.3349/ymj.2019.60.2.163.

MicroRNA-206 Reduces Osteosarcoma Cell Malignancy In Vitro by Targeting the PAX3-MET Axis

• Zhan FB
• Zhang XW
• Feng SL
• Cheng J
• Zhang Y
• Li B
• Xie LZ
• Deng QR

• KMID: 2431633
• DOI: http://doi.org/10.3349/ymj.2019.60.2.163

Abstract

PURPOSE
This study was undertaken to explore how miR-206 represses osteosarcoma (OS) development.
MATERIALS AND METHODS
Expression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissue specimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviral transduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor (HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verified by AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated in vitro by cell proliferation, metastasis, and apoptosis assays. PAX3 and MET gene expression in OS cells was assayed by RT-qPCR and Western blot. Activation of PI3K-AKT and MAPK-ERK in OS cells were assayed by evaluating Akt1 Ser473 phosphorylation and total threonine phosphorylation of Erk1/2, respectively.
RESULTS
Expression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts, and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNA in OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells in vitro, resulting in significant decreases in Akt1 and Erk1/2 activation, cell proliferation, and metastasis, as well as increases in cell apoptosis, while miR-206 knockdown showed the opposite effects. The effects of miR-206 overexpression on OS cells were reversed by PAX3 or MET overexpression, but only partially attenuated by HGF treatment.
CONCLUSION
miR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET gene expression.

Keyword

Osteosarcoma; miR-206; PAX3; c-Met; PI3K-AKT; MAPK-ERK

MeSH Terms

Apoptosis
Blotting, Western
Cell Line
Cell Proliferation
Gene Expression
Hepatocyte Growth Factor
Immunoprecipitation
In Vitro Techniques*
MicroRNAs
Neoplasm Metastasis
Osteosarcoma*
Phosphorylation
Plasmids
RNA, Messenger
Threonine
Transfection
Hepatocyte Growth Factor
MicroRNAs
RNA, Messenger
Threonine

Figure

• 

  Fig. 1 miRNA-206 expression is inversely correlated with PAX3 or MET mRNA expression in osteosarcoma and adjacent tissue specimens. (A–F) mRNA expression levels of miR-206, MET mRNA, and PAX3 mRNA in 25 pairs of OS and adjacent tissue specimens. (G and H) Pearson correlation analysis determining the significance of correlation between miR-206 expression and PAX3 or MET mRNA expression. (I and J) Protein levels of c-Met and Pax3 that are encoded by MET and PAX3 gene in 25 pairs of OS and adjacent tissue specimens. OS, osteosarcoma.

• 

  Fig. 2 miR-206 interacts with PAX3 or MET mRNA in OS cells. (A) AGO2-RIP assay determining the association between AGO2 protein and mRNA of indicated genes in OS cells after transfection with different miRNA mimics. (B) miRNA pulldown assay determining the association between miR-206 and mRNA of indicated genes in OS cells. miR-NC transfected group was used as control for significance test. OS, osteosarcoma.

• 

  Fig. 3 miR-206 reduces PAX3 and MET expression, as well as Akt1 and Erk1/2 activation. (A–C) RT-qPCR evaluating miR-206, PAX3 mRNA and MET mRNA in OS cells after indicated lentiviral transduction. (D–H) Western blot detecting the protein expression levels of PAX3 and MET, as well as Akt1 Ser473 and Erk1/2 total threonine phosphorylation, in OS cells after indicated transduction. NT was used as control for significance test. OS, osteosarcoma; NT, non-transduced group.

• 

  Fig. 4 miR-206 reduces OS cell proliferation while promoting apoptosis in vitro. miR-206 overexpression or knockdown in OS cells in vitro was achieved by lentiviral transduction. (A) CCK-8 cell viability assay evaluating overall cell growth in vitro. (B) Western blot evaluating the protein expression levels of the cell proliferation marker PCNA. (C) Flow cytometry detection of cell apoptosis. (D) Western blot evaluating the activation of pro-apoptotic protein Caspase-3. (E) Representative results of Western blots. NT was used as a control for significance test. *p<0.01, †p<0.001. OS, osteosarcoma; NT, non-transduced group.

• 

  Fig. 5 miR-206 reduces OS cell migration (A) and invasion (B) in vitro. miR-206 overexpression or knockdown in OS cells in vitro was achieved by lentiviral transduction. (C) Representative results of cell migration and invasion assays. NT was used as a control for significance test. OS, osteosarcoma; NT, non-transduced group.

• 

  Fig. 6 miR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET. miR-206 overexpressing OS cells were transfected with PAX3 or MET OE plasmids or treated with HGF before assay. (A) CCK-8 cell viability assay evaluating the overall cell growth in vitro. (B) Flow cytometry detection cell apoptosis. (C and D) Cell invasion and migration assay evaluating cell metastasis in vitro. (E) Representative results of cell migration and invasion assay. WT OS cells were used as a control for significance test. *p<0.01, †p<0.001. OS, osteosarcoma; OE, overexpressing; WT, wild-type, HGF, hepatocyte growth factor; NC, negative control.

• 

  Fig. 7 miR-206 reduces PAX3 and MET gene expression and IP3K-AKT, MAPK-ERK signaling in OS cells. OS cells were treated as indicated in Fig. 6 before Western blot assay (A), Western blot detecting the protein expression levels of Pax3 or c-Met in OS cells after different treatment. (B and C) Western blot detecting Akt1 Ser473 or Erk1/2 total threonine phosphorylation in OS cells after different treatments. (D) Representative Western blot results. WT OS cells were used as a control for significance test.. OS, osteosarcoma; WT, wild-type.

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