CircORC2 promoted proliferation and inhibited the sensitivity of osteosarcoma cell lines to cisplatin by regulating the miR-485-3p/TRIM2 axis

Abstract

Resistance to chemotherapy leads to poor prognosis for osteosarcoma (OS) patients. However, due to the high metastasis of tumor and the decrease in sensitivity of tumor cells to cisplatin (DDP), the 5-year survival rate of OS patients is still unsatisfactory. This study explored a mechanism for improving the sensitivity of OS cells to DDP. A DDP-resistant OS cell model was established, and we have found that circORC2 and TRIM2 were upregulated in DDP-resistant OS cells, but miR-485-3p was downregulated. The cell viability and proliferation of the OS cells decreased gradually with the increase of DDP dose, but a gradual increase in apoptosis was noted. CircORC2 promoted OS cell proliferation and DDP resistance and upregulated TRIM2 expression by targeting miR-485-3p. Functionally, circORC2 downregulated miR-485-3p to promote OS cell proliferation and inhibit DDP sensitivity. Additionally, it promoted cell proliferation and inhibited the sensitivity of DDP by regulating the miR-485-3p/TRIM2 axis. In conclusion, circORC2 promoted cell proliferation and inhibited the DDP sensitivity in OS cells via the miR-485-3p/TRIM2 axis. These findings indicated the role of circORC2 in regulating the sensitivity of OS cells to DDP.

Keywords: MiR‐485‐3p; TRIM2; circORC2; cisplatin resistance; osteosarcoma.

FIGURE 1

CircORC2 and TRIM2 were upregulated, but miR‐485‐3p was downregulated in OS cells. (A). Quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) was used to detect the circORC2 level in OS (Saos‐2, SW1353, U‐2Os, SJSA‐1, and HOS) and HFOB1.19 cells. (B). miR‐485‐3p level in OS cells assessed by qRT‐PCR. (C). QRT‐PCR was used to detect the TRIM2 mRNA levels in OS cells. (D). Western blot was employed to measure the TRIM2 protein level in OS cells. U‐2Os and SJSA‐1 cells were exposed to DDP at various concentrations (0, 1, 2, 4, and 8 μM). (E). MTS assay was used to detect the cell viability. (F). Cell proliferation was assessed using the colony formation assay. (G). Flow cytometry was used to detect cell apoptosis. Data are presented as mean ± SD of three replicate experiments (n = 3), *p < 0.05, **p < 0.01 and ***p < 0. 001.

FIGURE 2

CircORC2 promoted OS cells proliferation and DDP resistance. (A). Quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) detected circORC2, miR‐485‐3p, and TRIM2 levels in the U‐2Os, U‐2Os‐DDP, SJSA‐1, and SJSA‐1‐DDP cells. (B). U‐2Os, U‐2Os‐DDP, SJSA‐1, and SJSA‐1‐DDP cells were transfected with oe‐circORC2 or sh‐circORC2, and qRT‐PCR was used to estimate the circORC2 expression levels in these cells. Subsequently, the cells were exposed to DDP (concentration, 4 μM). (C). The MTS assay was performed to assess the cell viability. (D). Cell proliferation was evaluated using the colony formation assay. (E). Flow cytometry was employed to assess cell apoptosis. Data are presented as mean ± SD of three replicate experiments (n = 3), *p < 0.05, **p < 0.01 and ***p < 0. 001.

FIGURE 3

CircORC2 upregulated TRIM2 expression by targeting miR‐485‐3p. (A). The Circinteractome database was used to predict the binding relationship between circORC2 and miR‐485‐3p. (B). The luciferase activity was evaluated via the dual‐luciferase assay. (C). Quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) was employed to detect the miR‐485‐3p level after transfecting the U‐2Os and SJSA‐1 cells with sh‐circORC2 and oe‐circORC2. (D). The starBase database was used to predict the binding sites between miR‐485‐3p and TRIM2. (E). The luciferase activity was evaluated using the dual‐luciferase assay. (F). TRIM2 levels detected by qRT‐PCR after transfecting the U‐2Os and SJSA‐1 cells with miR‐485‐3p mimics and miR‐485‐3p inhibitor. Data are presented as mean ± SD of three replicate experiments (n = 3), **p < 0.01 and ***p < 0. 001.

FIGURE 4

CircORC2 downregulated miR‐485‐3p to promote OS cell proliferation and DPP resistance. U‐2Os and SJSA‐1 cells were transfected with miR‐485‐3p mimics and oe‐circORC2. (A). The miR‐485‐3p level was assessed via Quantitative reverse transcriptase‐polymerase chain reaction. U‐2Os and SJSA‐1 cells were transfected with miR‐485‐3p mimics and oe‐circORC2 and then exposed to DDP. (B). The MTS assay was used to assess the cell viability. (C). Colony formation assay was performed to evaluate cell proliferation. (D). Cell apoptosis was detected by flow cytometry. Data are presented as mean ± SD of three replicate experiments (n = 3), *p < 0.05, **p < 0.01 and ***p < 0. 001.

FIGURE 5

CircORC2 promoted OS cell proliferation and DPP resistance by regulating the miR‐485‐3p/TRIM2 axis. OS cells were transfected with oe‐TRIM2, oe‐TRIM2 with miR‐485‐3p mimics, and/or oe‐circORC2 and then administrated with DDP. (A). Quantitative reverse transcriptase‐polymerase chain reaction was used to detect the TRIM2 level. (B). MTS assay was performed to assess cell viability. (C). Colony formation assay was used to evaluate cell proliferation. (D). Cell apoptosis was detected by flow cytometry. Data are presented as mean ± SD of three replicate experiments (n = 3), *p < 0.05, **p < 0.01, and ***p < 0. 001.