Down-regulation of DNMT3b in PC3 cells effects locus-specific DNA methylation, and represses cellular growth and migration

Abstract

Background: Aberrations in DNA methylation patterns promote changes in gene expression patterns and are invariably associated with neoplasia. DNA methylation is carried out and maintained by several DNA methyltransferases (DNMTs) among which DNMT1 functions as a maintenance methylase while DNMT3a and 3b serve as de novo enzymes. Although DNMT3b has been shown to preferentially target the methylation of DNA sequences residing in pericentric heterochromatin whether it is involved in gene specific methylation remains an open question. To address this issue, we have silenced the expression of DNMT3b in the prostate-derived PC3 cells through RNA interference and subsequently studied the accompanied cellular changes as well as the expression profiles of selected genes.

Results: Our results demonstrate that DNMT3b depletion results in increased apoptosis and reduced migration of PC3 cells compared to the untransfected control cells. Reduced DNMT3b expression resulted in hypomethylation of retinoblastoma (Rb), retinoic-acid receptor beta (RAR-beta), and adenomatous polyposis coli (APC) gene promoters, and also culminated in increased expression of CDKN3 and cytochrome b5. Although DNMT3b silenced cells were found to have reduced growth and migratory potential, there was no apparent changes in their invasive ability compared to the parental PC3 cell line.

Conclusion: Our findings reveal that DNMT3b preferentially targets certain gene promoters in PC3 cells and that its depletion significantly reduces growth and migration of PC3 cells.

Figure 1

Silencing of DNMT3b expression in PC3 cells and validation of cell lines. A. Total RNA as well as protein was extracted from mock (control), and psiDNMT3b transfected PC3 cells and subjected to semi-quantitative RT-PCR analysis and Western blotting. β-actin was used as loading control. B. Affect of DNMT3b silencing on cell proliferation. Cells were grown and their respective rate of proliferation was measured by MTT assay after 24 and 48 hours. Absorbance was measured at 595 nm. C. Apoptosis was studied quantitatively by ethidium bromide and acridine orange staining of control and DNMT3b silenced cells. D. Expression analysis of MCM2. Total protein from mock (control), and psiDNMT3b transfected PC3 cells was subjected to Western blotting to determine expression of MCM2. β-actin was used as loading control. E. Expression analysis of caspase 3. Total protein from mock (control), and psiDNMT3b transfected PC3 cells was subjected to Western blotting to determine expression of caspase 3. β-actin was used as loading control.

Figure 2

Cellular invasion and migration assays. A. Boyden chamber invasion assays were carried out on control and DNMT3b silenced cells and the number of cells invading matrigel was determined. B. Wound healing migration assays were carried out as described. Panels show wounds in control, and DNMT3b depleted cells at 0 and 48 hours.

Figure 3

Methylation specific PCR analyses of RASSF1a, APC, RAR-β, RB1, hMLH1, Survivin, DAPK, BRCA, TIMP3, VHL, p16, PTEN, CDH1, CASP8, hTERTc, and RASSF1c genes in mock and psiDNMT3b transfected cells. Presence of PCR product indicates methylated (lane M) or unmethylated (lane U) alleles.

Figure 4

Gene expression is impacted by DNMT3b silencing. A. Expression profile obtained after mRNA was isolated from control and DNMT3b silenced PC3 cells, labeled and then hybridized to prostate cancer specific GE-array. B Results of gene expression profile. Only the genes whose expression increased in DNMT3b silenced cells 2-fold, relative to control cells, are listed. C and D. Expression of CDKN3 and cytochrome b5 was analyzed in DNMT3b silenced cells and controls by semi-quantitative RT-PCR.