J Gastric Cancer.  2017 Dec;17(4):295-305. 10.5230/jgc.2017.17.e33.

ATM Signaling Pathway Is Implicated in the SMYD3-mediated Proliferation and Migration of Gastric Cancer Cells

• Wang L
• Wang QT
• Liu YP
• Dong QQ
• Hu HJ
• Miao Z
• Li S
• Liu Y
• Zhou H
• Zhang TC
• Ma WJ
• Luo XG

• KMID: 2398796
• DOI: http://doi.org/10.5230/jgc.2017.17.e33

Abstract

PURPOSE
We previously found that the histone methyltransferase suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3 (SMYD3) is a potential independent predictive factor or prognostic factor for overall survival in gastric cancer patients, but its roles seem to differ from those in other cancers. Therefore, in this study, the detailed functions of SMYD3 in cell proliferation and migration in gastric cancer were examined.
MATERIALS AND METHODS
SMYD3 was overexpressed or suppressed by transfection with an expression plasmid or siRNA, and a wound healing migration assay and Transwell assay were performed to detect the migration and invasion ability of gastric cancer cells. Additionally, an MTT assay and clonogenic assay were performed to evaluate cell proliferation, and a cell cycle analysis was performed by propidium iodide staining. Furthermore, the expression of genes implicated in the ataxia telangiectasia mutated (ATM) pathway and proteins involved in cell cycle regulation were detected by polymerase chain reaction and western blot analyses.
RESULTS
Compared with control cells, gastric cancer cells transfected with si-SMYD3 showed lower migration and invasion abilities (P<0.05), and the absence of SMYD3 halted cells in G2/M phase and activated the ATM pathway. Furthermore, the opposite patterns were observed when SMYD3 was elevated in normal gastric cells.
CONCLUSIONS
To the best of our knowledge, this study provides the first evidence that the absence of SMYD3 could inhibit the migration, invasion, and proliferation of gastric cancer cells and halt cells in G2/M phase via the ATM-CHK2/p53-Cdc25C pathway. These findings indicated that SMYD3 plays crucial roles in the proliferation, migration, and invasion of gastric cancer cells and may be a useful therapeutic target in human gastric carcinomas.

Keyword

SMYD3 protein, human; Stomach neoplasms; G2 phase cell cycle checkpoints; ATM pathway

MeSH Terms

Ataxia Telangiectasia
Blotting, Western
Cell Cycle
Cell Proliferation
G2 Phase Cell Cycle Checkpoints
Histones
Humans
Plasmids
Polymerase Chain Reaction
Propidium
RNA, Small Interfering
Stomach Neoplasms*
Transfection
Wound Healing
Histones
Propidium
RNA, Small Interfering

Figure

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  Fig. 1 Effect of SMYD3 on the migration and invasion of gastric cells. After transfection with SMYD3-specific siRNAs or an overexpression plasmid, the migration ability of cells was detected by a wound healing assay (A) and a Transwell assay (B), and the invasion ability was analyzed by a Matrigel-coated Transwell assay (C). The interference efficiency of SMYD3 at various time points was detected by RT-qPCR (D). SMYD3 = suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3; RT-qPCR = reverse transcription quantitative polymerase chain reaction; si-SMYD3 = siRNA targeting SMYD3. *P<0.05; †P<0.01; ‡P<0.001 vs. control cells.

• 

  Fig. 2 Knockdown of SMYD3 could inhibit the proliferation of gastric cancer cells. MGC-803 and AGS gastric cancer cells were transfected with si-SMYD3 and cell viability was analyzed by MTT assays at various time points (A). A clonogenic assay was performed after transfection for 10 days (B). The same experiments were performed using GES-1 normal gastric cells transfected with a control vector or SMYD3 overexpression vector. SMYD3 = suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3; MTT = 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; si-SMYD3 = siRNA targeting SMYD3. *P<0.05; †P<0.01; ‡P<0.001 vs. control cells.

• 

  Fig. 3 The anti-proliferation effect of SMYD3-targeted siRNA might be due to G2/M cell cycle arrest. MGC-803 and AGS gastric cancer cells were transfected with si-SMYD3, and cell cycle progression was evaluated by flow cytometry after 24 hours (A); the cell cycle distribution was further analyzed (B). SMYD3 = suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3; si-SMYD3 = siRNA targeting SMYD3. *P<0.05; †P<0.01; ‡P<0.001 vs. control cells.

• 

  Fig. 4 The absence of SMYD3 could activate the ATM- CHK2/p53 pathway and stimulate the phosphorylation of Cdc25C. The mRNA levels of ATM, CHK2, Cdc25C, Cyclin B, CDK1, and p53 were determined by real-time qPCR in MGC-803 cells and AGS cells after 24 hours (A). The expression level of ATM and proteins associated with G2/M phase were further evaluated by western blotting after 48 hours (B). GAPDH was used as an internal control. SMYD3 = suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3; ATM = ataxia telangiectasia mutated; CHK2 = checkpoint kinase 2; CDK1 = cyclin-dependent kinase 1; qPCR = quantitative polymerase chain reaction; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; si-SMYD3 = siRNA targeting SMYD3. *P<0.05.

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