Silencing SMYD3 in hepatoma demethylates RIZI promoter induces apoptosis and inhibits cell proliferation and migration

Abstract

Aim: To investigate the role of SMYD3 in hepatocellular carcinoma (HCC) development and progression and to verify whether its regulation activity was through RIZ1 inactivation.

Methods: Expression of SMYD3 in HCC cell lines and tissues were measured; silencing of SMYD3 by RNA interference (RNAi) was effectuated, hepatoma cell proliferation, migration and apoptosis were tested, with RIZ1 CpG promoter methylation, and corresponding mRNA expression were investigated.

Results: SMYD3 over-expression in HCC was associated with RIZ1 hypermethylation and mRNA down-expression. Suppression of SMYD3 expression de-methylated RIZ1 CpG promoter (P < 0.01) and increased RIZ1 mRNA expression (P < 0.01). Consequently, SMYD3 down-expression with RIZ1 de-methylation strongly inhibited hepatoma cell growth (MTT inhibitory rates: Pgenesil-1-s1 60.95% +/- 7.97%, Pgenesil-1-s2 72.14% +/- 9.68% vs Pgenesil-1-hk 6.89% +/- 4.12%, P < 0.01) and migration (Pgenesil-1-s1 4.24% +/- 1.58%, Pgenesil-1-s1 4.87% +/- 0.73% vs Pgenesil-1 19.03% +/- 4.63%, Pgenesil-1-hk 19.95% +/- 5.21%, P < 0.01) and induced apoptosis (FCM subG1 phase Pgenesil-1-s1 19.07% +/- 1.78%, Pgenesil-1-s2 17.68% +/- 2.36% vs Pgenesil-1 0.47% +/- 0.12%, Pgenesil-1-hk 1.46% +/- 0.28%, P < 0.01. TUNEL-positive cells: Pgenesil-1-s1 40.24% +/- 5.18%, Pgenesil-1-s2 38.48% +/- 4.65% vs Pgenesil-1 2.18% +/- 1.34%, Pgenesil-1-hk 2.84% +/- 1.22%, P < 0.01) in HepG2 cells.

Conclusion: These results demonstrate that SMYD3 plays a critical role in the carcinogenesis and progression of HCC. The proliferation, migration induction and apoptosis inhibition activities of SMYD3 may be mediated through RIZ1 CpG promoter hypermethylation.

Figure 1

Enhanced expression of SMYD3 in HCC. A: The expression of SMYD3 genes was determined by RT-PCR and GAPDH served as an internal control; B: Representative images of immunohistochemical staining of accumulated SMYD3 protein in HCC tissue, but not in peri-cancerous tissue.

Figure 2

RNAi specifically inhibits SMYD3 expression in HCC cell lines. A: Schematic drawing of the Pgenesil-1 vector; B: The predicted secondary structures of the Pgenesil-1-s1 and Pgenesil-1-s2 transcripts target SMYD3 are shown; C: 48h after transfection, Western blot analysis showed the inhibitory effect of plasmids expressing SMYD3 shRNAs in HepG2 and SMMC-7721 cells.

Figure 3

Inhibition of SMYD3 reduces hepatoma cell proliferation (MTT assays).

Figure 4

Inhibition of SMYD3 reduces hepatoma cell migration. A: Representative cells (blue) invade through Matrigel and membrane; B: The percentage of cells successfully passing through the matrigel and membrane (P < 0.01).

Figure 5

Inhibition of SMYD3 promotes apoptosis in hepatoma cells upon serum deprivation. A: FCM analysis of HepG2 cells 48 h after transfection (P < 0.01). B: TUNEL assay of cell apoptosis with siRNA suppression of SMYD3 expression in HepG2 (brown staining cells) (P < 0.01).

Figure 6

Inhibition of SMYD3 de-methylates RIZ1 promoter CpG islands and reexpresses RIZ1 in hepatoma Cells. A: Methylation specific PCR (MSP) assay of RIZ1 promoter. M: Methylated; U: Unmethylated; B: RIZ1 mRNA expression, GAPDH served as internal control (RT-PCR).