Aberrant expression of the PHF14 gene in biliary tract cancer cells

Abstract

DNA copy number aberrations in human biliary tract cancer (BTC) cell lines were investigated using a high-density oligonucleotide microarray. A novel homozygous deletion was detected at chromosomal region 7p21.3 in the OZ cell line. Further validation experiments using genomic PCR revealed a homozygous deletion of a single gene, plant homeodomain (PHD) finger protein 14 (PHF14). No PHF14 mRNA or protein expression was detected, thus demonstrating the absence of PHF14 expression in the OZ cell line. Although the PHD finger protein is considered to be involved in chromatin-mediated transcriptional regulation, little is known about the function of PHF14 in cancer. The present study observed that the knock down of PHF14 using small interfering RNA (siRNA) enhanced the growth of the BTC cells. These observations suggest that aberrant PHF14 expression may have a role in the tumorigenesis of BTC.

Keywords: PHF14; biliary tract cancer; deletion.

Figure 1

Summary of the genetic imbalances detected in eight BTC cell lines using SNP array analyses. The 22 autosomes and X chromosome are represented by ideograms showing G-banding patterns. Copy number gains are indicated by red horizontal lines above the chromosome ideogram; high-level gains (amplifications) are shown as bright red lines, whereas simple gains are shown as dark red lines. Copy number losses are indicated by green lines under the chromosome ideogram. Each horizontal line represents an aberration detected in a single BTC cell line. BTC, biliary tract cancer; SNP, single nucleotide polymorphism.

Figure 2

Homozygous deletion of the PHF14 gene in the OZ cell line. (A) Chromosome 7 cytoband map and copy numbers were determined via SNP arrays of OZ cells. The arrow indicates the locus of the homozygous deletion at position 7p21.3. (B) PCR analysis of the exons in the PHF14 gene from genomic DNA templates derived from two BTC cell lines and normal lymphocytes. (C) Copy numbers of the PHF14 gene in the BTC cell lines, as measured by real-time quantitative PCR with reference to the LINE-1 control. Values are normalized such that the copy number in the genomic DNA derived from the normal lymphocytes was assigned a value of 2. (D) Relative expression levels of PHF14 mRNA as evaluated by real-time quantitative RT-PCR. Results are presented as the expression level of the PHF14 gene relative to a reference gene (GAPDH) in order to correct for variations in RNA amounts. Values are normalized such that the mRNA derived from normal liver samples had a value of 1. (E) Immunoblot analyses of PHF14 protein levels in the indicated cell lines, with β-actin as an internal control. PHF14, plant homeodomain finger protein 14; SNP, single nucleotide polymorphism.

Figure 3

Enhanced growth of BTC cells by PHF14-knockdown. The BTC cell line OCUG1 was treated with two independent siRNAs targeting PHF14 (siRNAb and sRNAc), control siRNA or left untreated. (A) Expression levels of PHF14 mRNA were determined by real-time quantitative RT-PCR. The cells were harvested at 48 h post-transfection. (B) The effect of the siRNA targeting of PHF14 on cell proliferation was measured with the MTT assay at the indicated times following transfection. Each assay was performed in triplicate. Values are represented as the mean ± SD. Differences were evaluated with the Student’s t-test (^*P<0.05, ^**P<0.01). BTC, billiary tract cancer; PHF14, plant homeodomain finger protein 14; siRNA, small interfering RNA.