Yonsei Med J.  2019 Sep;60(9):832-841. 10.3349/ymj.2019.60.9.832.

miR-1301/TRIAP1 Axis Participates in Epirubicin-Mediated Anti-Proliferation and Pro-Apoptosis in Osteosarcoma

Affiliations
  • 1Department of Pharmacy, Gansu Provincial Hospital, Lanzhou, Gansu, China.
  • 2Department III of Orthopedic, Gansu Provincial Hospital, Lanzhou, Gansu, China. budalagong305@sina.com

Abstract

PURPOSE
Epirubicin is one of the most effective drugs against osteosarcoma. miR-1301 is involved in the occurrence and development of osteosarcoma. Whether miR-1301 is responsible for the chemosensitivity of osteosarcoma cells to epirubicin remains largely unknown.
MATERIALS AND METHODS
U2OS and SAOS-2 cells were treated with various concentrations of epirubicin. Flow cytometry was employed to evaluate cell apoptotic rate. Cell proliferation was measured by Cell Counting Kit-8 assay. Western blot and quantitative real-time polymerase chain reaction were utilized to detect the expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 assaciated X protein (Bax), cleaved-caspase-3, cleaved-poly (ADP-ribose) polymerases (PARP1), TP53-regulated inhibitor of apoptosis 1 (TRIAP1), and microRNA-1301 (miR-1301). The relationship between miR-1301 and TRIAP1 was determined by luciferase reporter assay.
RESULTS
Epirubicin inhibited proliferation in a dose-dependent manner, induced apoptosis, decreased the expression of Bcl-2, and increased the expressions of Bax, cleaved-caspase-3, and cleaved-PARP1 in osteosarcoma cells. miR-1301 was downregulated in U2OS and SAOS-2 cells. Importantly, epirubicin significantly increased the levels of miR-1301. Overexpression of miR-1301 suppressed proliferation and promoted apoptosis. Interestingly, those effects were enhanced by epirubicin. In contrast, miR-1301 depletion attenuated the epirubicin-mediated anti-osteosarcoma effect. miR-1301 negatively regulated the expression of TRIAP1 in U2OS and SAOS-2 cells. Furthermore, epirubicin inhibited the mRNA and protein levels of TRIAP1 by upregulating miR-1301 levels. Epirubicin suppressed cell proliferation by downregulating TRIAP1.
CONCLUSION
miR-1301 was implicated in the chemosensitivity of osteosarcoma to epirubicin by modulating TRIAP1.

Keyword

miR-1301; TRIAP1; epirubicin; osteosarcoma

MeSH Terms

Apoptosis
B-Lymphocytes
Blotting, Western
Cell Count
Cell Proliferation
Epirubicin
Flow Cytometry
Luciferases
Osteosarcoma*
Real-Time Polymerase Chain Reaction
RNA, Messenger
Epirubicin
Luciferases
RNA, Messenger

Figure

  • Fig. 1 Effects of epirubicin on proliferation and apoptosis in osteosarcoma cells. (A) Osteosarcoma cell lines U2OS and SAOS-2 were treated with different concentrations of epirubicin for 48 h, and Cell Counting Kit-8 assay was performed to evaluate cell proliferation. (B) U2OS and SAOS-2 cells were exposed to 0 µg/mL or 1 µg/mL of epirubicin for 48 h, respectively. Flow cytometry was introduced to determine cell apoptotic rate. (C) Western blot was conducted to examine the expressions of Bcl-2, Bax, cleaved-caspase-3, and cleaved-poly (ADP-ribose) polymerase-1 (cleaved-PARP1) in U2OS and SAOS-2 cells with 48 h of 0 µg/mL or 1 µg/mL epirubicin exposure. *p<0.05. Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 assaciated X protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 2 Epirubicin stimulates the expression of microRNA-1301 (miR-1301) in U2OS and SAOS-2 cells. (A) qRT-PCR was employed to measure the expression of miR-1301 in human osteoblast cells hFOB1.19, U2OS cells, and SAOS-2 cells. (B) The expression of miR-1301 in U2OS or SAOS-2 cells treated with 1 µg/mL of epirubicin for 24 h or 48 h. *p<0.05. qRT-PCR, quantitative real-time polymerase chain reaction.

  • Fig. 3 Upregulation of microRNA-1301 (miR-1301) contributes to the anti-cancer effect of epirubicin in osteosarcoma. U2OS and SAOS-2 cells were transfected with miR-NC mimics or miR-1301 mimics, and after 24-h transfection, cells were treated with epirubicin (1 µg/mL) for 48 h. Cells were divided into five groups: Ctrl, miR-NC mimics, miR-1301 mimics, Epirubicin, or Epirubicin+miR-1301 mimics. (A) qRT-PCR was introduced to detect the levels of miR-1301. (B) Cell Counting Kit-8 was applied to analyze cell proliferation in U2OS and SAOS-2 cells. (C) Flow cytometry was utilized to evaluate cell apoptotic rate. (D) The expressions of Bcl-2, Bax, cleaved-caspase-3, and cleaved-poly (ADP-ribose) polymerase-1 (cleaved-PARP1) in U2OS and SAOS-2 cells were measured using Western blot. The + respensents Epirubicin or mimic was used to treat cells, while the meaning of - was opposite with +. *p<0.05. Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 assaciated X protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 4 MicroRNA-1301 (MiR-1301) depletion weakens the anti-cancer effect of epirubicin in osteosarcoma. U2OS and SAOS-2 cells were transfected with miR-NC inhibitors or miR-1301 inhibitors, and after 24-h transfection, cells were treated with epirubicin (1 µg/mL) for 48 h. Cells were divided into four groups: Ctrl, Epirubicin, Epirubicin+miR-NC inhibitors, or Epirubicin+miR-1301 inhibitors. (A) The expression levels of miR-1301 in cells were determined using qRT-PCR. (B) Cell proliferation was measured in U2OS and SAOS-2 cells. (C) Flow cytometry was utilized to analyze the cell apoptotic rate. (D) Western blot was employed to examine the expressions of Bcl-2, Bax, cleaved-caspase-3, and polymerases (PARP1) in U2OS and SAOS-2 cells. The + respensents Epirubicin or mimic was used to treat cells, while the meaning of - was opposite with +. *p<0.05. Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 assaciated X protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 5 TP53-regulated inhibitor of apoptosis 1 (TRIAP1) is a target of microRNA-1301 (miR-1301). (A) The binding sites between miR-1301 and TRIAP1 were predicted by the TargetScan online database, and luciferase reporter plasmids containing the wild-type (WT) or mutated (MUT) TRIAPT1 binding sites of miR-1301 were established. (B) The luciferase activity was measured in U2OS cells co-transfected with TRIAP1-WT or TRIAP1-MUT luciferase reporter plasmids and miR-1301 mimics or miR-NC mimics. (C) The luciferase activity was examined in SAOS-2 cells co-transfected with TRIAP1-WT or TRIAP1-MUT luciferase reporter plasmids and miR-1301 mimics or miR-NC mimics. (D) The expression of TRIAPT in U2OS and SAOS-2 cells transfected with miR-1301 mimics or miR-NC mimics. (E) The expression of TRIAP1 in U2OS and SAOS-2 cells transfected with miR-1301 inhibitors or miR-NC inhibitors. (D and E) The + respensents Epirubicin or mimic was used to treat cells, while the meaning of − was opposite with +. *p<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 6 Epirubicin regulates TP53-regulated inhibitor of apoptosis 1 (TRIAP1) expression by modulating microRNA-1301 (miR-1301). (A-C) U2OS and SAOS-2 cells were transfected with miR-NC inhibitors or miR-1301 inhibitors, and after 24-h transfection, cells were treated with epirubicin (1 µg/mL) for 48 h. Cells were divided into four groups: Ctrl, Epirubicin, Epirubicin+miR-NC inhibitors, or Epirubicin+miR-1301 inhibitors. (D-G) U2OS and SAOS-2 cells were transfected with vector or TRIAP1 overexpression plasmid (TRIAP1), and after 24-h transfection, cells were treated with epirubicin (1 µg/mL) for 48 h. Cells were divided into four groups: Ctrl, Epirubicin, Epirubicin+vector, or Epirubicin+TRIAP1. The mRNA levels of TRIAP1 in U2OS and SAOS-2 cells were evaluated (A and D). Western blot was applied to examine the levels of TRIAP1 in U2OS (B and E) and SAOS-2 cells (C and F). (G) Cell viability was determined by Cell Counting Kit-8 assay. (B, C, E, and F) The + respensents Epirubicin or mimic was used to treat cells, while the meaning of - was opposite with +. *p<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


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