Identification of Polycomb Group Protein EZH2-Mediated DNA Mismatch Repair Gene MSH2 in Human Uterine Fibroids

Abstract

Uterine fibroids (UFs) are benign smooth muscle neoplasms affecting up to 70% of reproductive age women. Treatment of symptomatic UFs places a significant economic burden on the US health-care system. Several specific genetic abnormalities have been described as etiologic factors of UFs, suggesting that a low DNA damage repair capacity may be involved in the formation of UF. In this study, we used human fibroid and adjacent myometrial tissues, as well as an in vitro cell culture model, to evaluate the expression of MutS homolog 2 (MSH2), which encodes a protein belongs to the mismatch repair system. In addition, we deciphered the mechanism by which polycomb repressive complex 2 protein, EZH2, deregulates MSH2 in UFs. The RNA expression analysis demonstrated the deregulation of MSH2 expression in UF tissues in comparison to its adjacent myometrium. Notably, protein levels of MSH2 were upregulated in 90% of fibroid tissues (9 of 10) as compared to matched adjacent myometrial tissues. Human fibroid primary cells treated with 3-deazaneplanocin A (DZNep), chemical inhibitor of EZH2, exhibited a significant increase in MSH2 expression (P < .05). Overexpression of EZH2 using an adenoviral vector approach significantly downregulated the expression of MSH2 (P < .05). Chromatin immunoprecipitation assay demonstrated that enrichment of H3K27me3 in promoter regions of MSH2 was significantly decreased in DZNep-treated fibroid cells as compared to vehicle control. These data suggest that EZH2-H3K27me3 regulatory mechanism dynamically changes the expression levels of DNA mismatch repair gene MSH2, through epigenetic mark H3K27me3. MSH2 may be considered as a marker for early detection of UFs.

Keywords: DNA mismatch repair; EZH2; H3K27me3; MSH2; fibroid; uterine fibroid.

Figure 1.

The expression levels of DNA mismatch repair gene MSH2 in human fibroid and adjacent myometrial tissues. A, The RNA expression of MSH2 was determined in fibroid tumors as compared to matched adjacent myometrial samples by real-time polymerase chain reaction (n = 8). 18S was used as an endogenous control. B, The protein lysates were prepared from fibroids (F; n = 10) and matched myometrium tissues (M). The protein expression of MSH2 was determined by Western blot analysis. MSH2 protein bands were quantified and normalized to β-actin, and relative values were used to generate data graphs (bottom panel). Each short horizontal line indicates a pair of M and F from same patient.

Figure 2.

Inhibition of EZH2 increases the expression of MSH2 in a dose-dependent manner in human fibroid cells. A, Inhibition of EZH2 by 3-deazaneplanocin A (DZNep) decreased levels of H3K27me3 by Western blot analysis. Primary fibroid cells (PFCs) were plated in 60-mm dish. Cells were treated with varying concentrations of EZH2 inhibitor DZNep for 3 days. Dimethyl sulfoxide (DMSO) was used as a vehicle control. B, The protein lysates were prepared from PFCs treated with vehicle or 1 μmol/L DZNep for 3 days. The protein levels of MSH2 were determined by Western blot analysis. In addition, cells were treated with varying concentrations of EZH2 inhibitor DZNep for 3 days. Cells were collected and subjected to RNA extraction and complementary DNA (cDNA) synthesis. Quantitative polymerase chain reaction was performed to measure the expression levels of MSH2 (C), SUZ12 (D), EED (E), and P27 (F). 18S was used as an endogenous control.

Figure 3.

Overexpression of EZH2 increases HuLM cell proliferation. HuLM cells were plated in 96-well dish. Cellular confluence was approximately 30%. Equal amounts of virus (20 plaque-forming unit [pfu]/cell) containing GFP or EZH2 gene were added to the medium, respectively. Eight hours later, the viral supernatant was removed and fresh medium was added. After 3 days and 6 days of infection, MTT assay was performed to determine absorbance at 570 nm. *P < .05 compared with the control.

Figure 4.

Overexpression of EZH2 using adenoviral vector decreases MSH2 expression in HuLM cells. A, EZH2 was overexpressed in HuLM cells infected with adenovirus containing EZH2 as compared to cells infected with adenovirus containing GFP. Levels of epigenetic mark H3K27me3 were also increased in HuLM cells infected with virus containing EZH2. B, EZH2 was found in both cytoplasm and nucleus; however, higher expression of EZH2 was observed in the nucleus as compared to the cytoplasm. C, MSH2 RNA expression was downregulated in HuLM cells overexpressed with EZH2 as compared to GFP control. D, Cell cycle regulatory gene p27 is downregulated in EZH2-overexpressed cells as compared to GFP control cells. *P < .05 compared with the control. Total cell lysates (E), nucleus (F), and cytoplasm (G) from HuLM cells infected with Ad-EZH2 or Ad-GFP were analyzed by Western blot using Anti-MSH2 antibody. Western blot with anti–β-actin antibody was used as the loading control. MSH2 protein bands were quantified and normalized to β-actin, and relative values were used to generate data graphs (right panels).

Figure 5.

DZNep treatment restores expression levels of MSH2 through epigenetic mark H3K27me3. A, Location of regions analyzed by chromatin immunoprecipitation (ChIP)/polymerase chain reaction (PCR) along human MSH2 promoter. The position of the transcriptional start site is designated as +1. Short horizontal lines indicate regions analyzed by ChIP/PCR. B, ChIP/quantitative PCR (q-PCR) was performed with anti-H3K27me3 antibody in the distal promoter region of MSH2 in human fibroid primary cells in the presence or absence of DZNep. C, ChIP/q-PCR was performed with anti-H3K27me3 antibody in the proximal promoter region of MSH2 in human fibroid primary cells in the presence or absence of DZNep. *P < .05 compared with the control.