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. 2013 Aug;6(2):393-400.
doi: 10.3892/ol.2013.1380. Epub 2013 Jun 7.

A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first

Ning Liu  1 , Rui Zhang, Xiaoming Zhao, Jiaming Su, Xiaolei Bian, Jinsong Ni, Ying Yue, Yong Cai, Jingji Jin
Affiliations

A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first

Ning Liu et al. Oncol Lett. 2013 Aug.

Abstract

Human males absent on the first (hMOF), a human ortholog of the Drosophila MOF protein, is responsible for histone H4 lysine 16 (H4K16) acetylation in human cells. The depletion of hMOF leads to a global reduction in histone H4K16 acetylation in human cells, genomic instability, cell cycle defects, reduced transcription of certain genes, defective DNA damage repair and early embryonic lethality. Studies have shown that abnormal hMOF gene expression is involved in a number of primary cancers. The present study examined the involvement of hMOF expression and histone H4K16 acetylation in clinically diagnosed primary ovarian cancer tissues. Clinically diagnosed frozen primary ovarian cancer tissues were used for polymerase chain reaction (PCR), quantitative PCR (qPCR), western blotting and immunohistochemical staining approaches. A PCR analysis of mRNA expression in 47 samples revealed a downregulation of hMOF mRNA in 81% of patients, whereas only 13% of patients demonstrated upregulation. qPCR was used to validate the frequent downregulation of hMOF expression in the primary ovarian cancer tissues. As expected, the analysis of hMOF expression in 57 samples revealed that hMOF mRNA expression was significantly downregulated (>2-fold decrease) in 65% of patients, while a <2-fold reduction of hMOF was observed in 10.5% of patients. Furthermore, the expression of hMOF-regulated human leukocyte antigen (HLA) complex 5, (HCP5), was also found to be downregulated in >87% of patients with a decrease in hMOF. hMOF and its regulated gene, HCP5, are frequently downregulated in human ovarian cancer, suggesting that hMOF may be involved in the pathogenesis of the disease.

Keywords: histone acetyltransferase; human males absent on the first; ovarian cancer.

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Figures

Figure 1.
Figure 1.
A reduction in hMOF mRNA levels is observed in human ovarian cancer. (A) PCR analysis of 47 clinical ovarian cancer tissues. Total RNA was isolated from the tissues using TRIzol. The PCR assay was performed to detect the mRNA expression levels of hMOF, CA9, VEGF, HIF1α and hSTC1 in clinical ovarian cancer and normal ovarian tissues. The PCR products were then separated by electrophoresis on a 2% agarose gel. The DNA fragments were visualized and photographed under ultraviolet light with ethidium bromide. The mRNA levels from 37 ovarian cancer tissues were compared with corresponding contralateral ovarian normal tissues. However, 10 clinical ovarian cancer tissues were missing contralateral ovarian normal tissues and were compared with non-corresponding normal ovarian tissues. (B) Summarization of the PCR results. The 100% stacked column charts were used to compare the case numbers of differentially-expressed mRNAs in the ovarian cancer tissues. The total case numbers of differentially-expressed mRNAs (increased, decreased and no change) in the ovarian cancer tissues is equal to 100%. (C) Statistical analysis of quantified mRNA levels between the ovarian cancer and normal tissues. The mRNA expression signals shown in (A) were quantified by densitometry using Quantity One Basic Software. The significant difference is expressed as *P<0.05, **P<0.01. hMOF, human males absent on the first; PCR, polymerase chain reaction; CA9, carbonate anhydrate IX; VEGF, vascular endothelial growth factor; HIF1α, hypoxia-inducible factor-1α; hSTC1, human stanniocalcin 1; N, normal tissue; C, cancer tissue.
Figure 2.
Figure 2.
Downregulation of hMOF mRNA expression in ovarian cancer tissues confirmed using qPCR and immunoblotting. (A) Expression patterns of hMOF in clinical ovarian cancer tissues. Total RNA was isolated from 57 clinical ovarian cancer and 15 normal tissues (8/15 cases were the contralateral normal tissues). The relative mRNA expression levels of hMOF were analyzed using qPCR. Expression is displayed as a ratio of hMOF gene expression in the ovarian cancer versus normal tissues. Each bar is the log2 value of the ratio of hMOF expression levels between the ovarian cancer and normal tissues. Bar value >1 represents >2-fold increase, whereas bar value <1, represents >2-fold decrease. (B) Statistical analysis of the qPCR data. Each bar represents the mean of three independent experiments. The significant difference is expressed as **P<0.01. (C) Four randomly selected, pathologically diagnosed ovarian cancer and contralateral normal tissues from the same patients were used. A whole cell extract was prepared from the tissues and equivalent total protein amounts were subjected to SDS-PAGE in 12% gels. Proteins were detected by western blotting with anti-hMOF, H4K16Ac and GAPDH antibodies (lower panel). Western blotting images were quantified using Quantity One software and normalized by GAPDH levels. The significant difference is expressed as *P<0.05. (D) Immunohistochemical staining for (a and b) hMOF and (c and d) H4K16Ac in ovarian cancer tissues (×200). (a and c) High or (b and d) low hMYST1 and H4K16Ac expression levels in ovarian cancer tissues. hMOF, human males absent on the first; qPCR, quantitative polymerase chain reaction; H4K16, histone H4 lysine 16; MYST1, Moz-Ybf2/Sas3-Sas2-Tip60 1; N, normal tissue; C, cancer tissue.
Figure 3.
Figure 3.
Gene expression profiles in hMOF siRNA knockdown HeLa cells. (A) Relative mRNA levels markedly decreased following the specific knockdown of hMOF. The HeLa cells were transfected with hMOF siRNAs or a non-targeting (NT) control siRNA. At 48 h post-transfection, hMOF mRNA levels were measured by qPCR. Actin mRNA was used to assess the integrity of the RNA. (B) hMOF protein expression and its corresponding modifications were down-regulated by transfecting HeLa cells with hMOF siRNAs. Equivalent total protein amounts of whole cell extracts from the cells treated with hMOF siRNAs or NT siRNAs were subjected to SDS-PAGE in 12% gels, and the proteins were detected by western blotting using the indicated antibodies. (C) Gene expression profiles in hMOF siRNA knockdown HeLa cells. The gray bars represent the upregulated genes (log2>1), while the black bars represent the downregulated genes (log2<−1). Each bar is the log2 value of the ratio of gene expression in hMOF siRNA knockdown HeLa cells. hMOF, human males absent on the first; qPCR, quantitative polymerase chain reaction; H4K16, histone H4 lysine 16; WB, western blotting.
Figure 4.
Figure 4.
HPC5, an hMOF target gene, is frequently downregulated in ovarian cancer tissues. (A) A reduction in HCP5 mRNA expression levels in hMOF siRNA knockdown cells. HeLa cells were transfected with hMOF or NT siRNAs. Following 48 h of transfection, the mRNA levels of HCP5 and actin were measured by qPCR. Error bars represent the standard error of the mean of three independent experiments. (B and C) hMOF colocalizes with H4K16Ac at HCP5 promoter. ChIP assays using transfected hMOF or NT siRNA HeLa cells were analyzed by qPCR. Bar graphs show the ratio of ChIP signals that were normalized to the input DNA. (D) HCP5 mRNA expression patterns in ovarian cancer tissues. 28 randomly selected clinical ovarian cancer and contralateral normal tissues were used. The HCP5 and hMOF mRNA expression levels in ovarian cancer were analyzed by qPCR. The y-axis indicates the log2 value of the ratio of HCP5 and hMOF expression levels between the cancer and normal tissues from the same patients. (E) Statistical analysis of qPCR results. Each bar represents the mean of three independent experiments. The significant difference is expressed as *P<0.05 and **P<0.01. hMOF, human males absent on the first; HCP5, human leukocyte antigen (HLA) complex P5; NT, non-targeting; qPCR, quantitative polymerase chain reaction; ChIP, chromatin immunoprecipitation.
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