SET and MYND domain-containing protein 3 inhibits tumor cell sensitivity to cisplatin

Abstract

Cisplatin resistance has been a major factor limiting its clinical use as a chemotherapy drug. The present study aimed to investigate whether SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase closely associated with tumors can affect the sensitivity of tumors to cisplatin chemotherapy. Real time-qPCR, western blotting, the luciferase reporter, MTT and clonogenic assays were performed to detect the effects of SMYD3 on the chemotherapy capacity of cisplatin. In the present study, SMYD3 exhibited different expression patterns in MCF-7 and T47D breast cancer cells. In addition, this differential expression was associated with tumor cell resistance to cisplatin. Furthermore, SMYD3 knockdown following small interfering RNA transfection increased cisplatin sensitivity, whereas SMYD3 overexpression decreased cisplatin sensitivity. In addition, SMYD3 knockdown synergistically enhanced cisplatin-induced cell apoptosis. SMYD3 expression was downregulated during cisplatin treatment. In addition, transcriptional regulatory activities of SMYD3 3'-untranslated region were also downregulated. These results suggested that SMYD3 may affect cell sensitivity to cisplatin and participate in the development of cisplatin resistance, which is a process that may involve microRNA-124-mediated regulation.

Keywords: SET and MYND domain containing 3; cisplatin sensitivity; tumor cells.

Figure 1.

Cisplatin sensitivity may be inversely related to endogenous SMYD3 expression. (A) Effect of cisplatin treatment on MCF-7 and T47D cell viability after 24 or 48 h was determined. (B) Cisplatin IC₅₀ in MCF-7 and T47D cell lines. (C) SMYD3 expression in MCF-7 and T47D cells assessed by reverse transcription quantitative PCR and western blotting. *P<0.05 vs. MCF7 cells. SMYD3, SET and MYND domain-containing protein 3.

Figure 2.

SMYD3 knockdown increases cisplatin sensitivity in tumor cells. (A) MCF-7 and T47D cells were transfected with si-SMYD3 or si-control before exposure to different concentrations of cisplatin. Changes in SMYD3 mRNA level were detected by reverse transcription quantitative PCR. Effect of SMYD3 knockdown on (B) MCF-7 and (C) T47D cell sensitivity to cisplatin sensitivity measured with the MTT assay and (D) colony-formation assay. (E) The membrane potential of mitochondria was assessed by Rhodamine 123 efflux assays with MCF-7 cells. Mean ± SD. n=3. **P<0.01, ***P<0.001 vs. si-control. si, small interfering; SMYD3, SET and MYND domain-containing protein 3; nc, negative control.

Figure 3.

SMYD3 overexpression decreases cell cisplatin sensitivity. MCF-7 cells were transfected with SMYD3 overexpression plasmids and control vector. (A) Semi-qPCR was performed to detect SMYD3 mRNA level, and (B) the effect of SMYD3 overexpression on cell sensitivity to cisplatin was measured with the MTT assay and (C) colony-formation assay. Mean ± SD. n=4. ***P<0.001 vs. pcDNA3.1. SMYD3, SET and MYND domain-containing protein 3.

Figure 4.

Cisplatin inhibits SMYD3 expression. SMYD3 transcription in cisplatin-treated MCF-7 cells was analyzed by (A) semi-qPCR and (B) reverse transcription-qPCR. (C) SMYD3 expression in cisplatin-treated MCF-7 and T47D cells was analyzed by western blotting. Mean ± SD, n=3, **P<0.01, ***P<0.001 vs. 0 µM cisplatin. qPCR, quantitative PCR; SMYD3, SET and MYND domain-containing protein 3.

Figure 5.

miR-124 may be involved in the cisplatin-mediated SMYD3 expression inhibition. (A) Effect of cisplatin on SMYD3 3′UTR activity assessed by luciferase assays. MCF-7 cells (left panel) and T47D cells (right panel). (B) Effect of cisplatin on miR-124 level. Mean ± SD. n=3. *P<0.05, **P<0.01, ***P<0.001 vs. 0 µM cisplatin. SMYD3, SET and MYND domain-containing protein 3; 3′UTR, 3′-untranslated region.