PADI2 gene confers susceptibility to breast cancer and plays tumorigenic role via ACSL4, BINC3 and CA9 signaling

Abstract

Background: Peptidylarginine deiminase (PAD) post-translationally converts arginine residues to citrulline residues. Recent studies have suggested that PADI2 (PAD isoform 2), a member of the PAD family, is involved in the tumorigenic process of some tumors, especially breast cancer. However, little is known about the mechanisms of PADI2 in tumorigenesis. This study aimed to elucidate the tumorigenic role and regulatory pathway of PADI2 in breast tumors.

Methods: The Sequenom MassARRAY and TaqMan genotyping methods were used to investigate the correlation between PADI2 gene SNPs and various tumor risks. PCR array analyses, including cancer pathway finder and signal transduction PCR arrays, were performed to investigate the tumorigenic pathway of PADI2 in the MCF-7 breast cancer cell line following treatment with anti-PADI2 siRNA. Cell proliferation, apoptosis and transwell migration assays were performed to observe the effect of PADI2 in MCF-7 cells treated with anti-PADI2 siRNA.

Results: Both Sequenom MassARRAY and TaqMan genotyping assays demonstrated that SNP rs10788656 in the PADI2 gene was significantly associated with breast cancer. PCR arrays indicated that inhibiting PADI2 expression significantly increased expression of CA9 and decreased expression of ACSL4 and BIRC3 in MCF-7 cells, which was verified using real-time PCR. Inhibiting PADI2 expression also significantly decreased the migration ability of MCF-7 cells but did not affect cell proliferation or apoptosis.

Conclusions: The PADI2 gene confers susceptibility to breast cancer. PADI2 expression contributes to abnormal migration of breast tumor cells. PADI2 affects tumorigenesis in breast tumor cells by regulating the expression of ACSL4, BINC3 and CA9, which are known to promote abnormal lipid metabolism and cell invasion of tumors.

Keywords: ACSL4 (long-chain fatty acyl-CoA synthetase 4); BIRC3 (baculoviral IAP repeat containing 3); CA9 (carbonic anhydrase IX); Citrullination; PADI2 (peptidylarginine deiminase isoform 2); Peptidylarginine deiminase (PAD).

Fig. 1

Proliferation of MCF-7 cells that were treated with anti-PADI2 siRNA. a Real-time assay detected the PADI2 mRNA level in the anti-PADI2 siRNA-treated cells. b CCK-8 assay detected viable cell numbers as represented by an O.D. value at 405 nm. The cells treated with HiPerFect transfection reagent were used as normal controls, and the cells treated with Allstar siRNA were used as negative controls

Fig. 2

Apoptosis in MCF-7 cells that were treated with anti-PADI2 siRNA, as measured using an annexin V cell apoptosis assay. a The cells were treated with anti-PADI2 siRNA. b The cells treated with Allstar siRNA were used as negative controls. c The cells without siRNA treatment were used as normal controls. d The result of the apoptosis assay is shown in a graph

Fig. 3

Migration of MCF-7 cells that were treated with anti-PADI2 siRNA, as measured using a transwell migration assay. a The cells were treated with anti-PADI2 siRNA. b The cells treated with Allstar siRNA were used as negative controls. c The cells without siRNA treatment were used as normal controls. d The result of the migration measurement is shown in a graph. Original magnification: ×4.2

Fig. 4

Determination of the regulatory pathway of PADI2 using PCR arrays. The tumor cell line MCF-7 was treated with anti-PADI2 siRNA. Cells treated with Allstar siRNA were used as a negative control. a PADI2 expression level was detected using real time-PCR in MCF-7 cells following treatment with anti-PADI2 siRNA. The PADI2 transcription level in the treated cells was normalized with the mRNA level in negative control cells. b Cancer pathway finder (c) Signal Transduction PCR arrays were used to detect altered expression of tumor-related genes in the treated cells. Fold changes were calculated and expressed as log-normalized ratios of the expression level in siRNA-treated cells/the expression level in negative control. Genes with at least a fourfold change in expression were considered biologically significant

Fig. 5

Determination of the mRNA expression levels of PADI2, ACSL4, BICR3 and CA9 in MCF-7 cells using real-time PCR. MCF-7 cells were treated with anti-PADI2 siRNA. Cells treated with Allstar siRNA were used as negative controls. The transcription levels of the target genes in the treated cells were normalized with their mRNA levels in negative control cells. a PADI2 expression in MCF-7 cells, b ACSL4 expression in MCF-7 cells, c BICR3 in MCF-7 cells, d CA9 in MCF-7 cells. ***p < 0.001