5-Hydroxymethylcytosine promotes proliferation of human uterine leiomyoma: a biological link to a new epigenetic modification in benign tumors

Abstract

Context: Uterine leiomyoma, or fibroids, represent the most common benign tumors of the female reproductive tract. A newly discovered epigenetic modification, 5-hydroxymethylation (5-hmC), and its regulators, the TET (Ten Eleven Translocation) enzymes, were implicated in the pathology of malignant tumors; however, their roles in benign tumors, including uterine fibroids, remain unknown.

Objective: To determine the role of 5-hmC and TET proteins in the pathogenesis of leiomyoma using human uterine leiomyoma and normal matched myometrial tissues and primary cells.

Design: 5-hmC levels were determined by ELISA and immunofluorescent staining in matched myometrial and leiomyoma tissues. TET expression was analyzed by quantitative RT-PCR and immunoblotting. TET1 or TET3 were silenced or inhibited by small interfering RNA or 2-hydroxyglutarate to study their effects on 5-hmC content and cell proliferation.

Results: We demonstrated significantly higher 5-hmC levels in the genomic DNA of leiomyoma tissue compared to normal myometrial tissue. The increase in 5-hmC levels was associated with the up-regulation of TET1 or TET3 mRNA and protein expression in leiomyoma tissue. TET1 or TET3 knockdown significantly reduced 5-hmC levels in leiomyoma cells and decreased cell proliferation. Treatment with 2-hydroxyglutarate, a competitive TET enzyme inhibitor, significantly decreased both 5-hmC content and cell proliferation of leiomyoma cells.

Conclusion: An epigenetic imbalance in the 5-hmC content of leiomyoma tissue, caused by up-regulation of the TET1 and TET3 enzymes, might lead to discovery of new therapeutic targets in leiomyoma.

Figure 1.. Higher 5-hmC levels in uterine leiomyoma compared to normal myometrium.

A, Leiomyoma samples contained a higher percentage of global 5-hmC DNA compared to normal myometrial tissue, as determined by ELISA (n = 12). Data are reported as mean ± SD. **, P < .01, by Student's t test. B and C, Representative images of 5-hmC immunofluorescence (red, Cy3 labeling) in normal myometrial cells and leiomyoma cells confirmed the increased levels of 5-hmC in leiomyoma (n = 9). Nuclei are stained with DAPI (blue). Scale bar = 50 μm.

Figure 2.. TET1 and TET3 mRNA and protein are overexpressed in leiomyoma compared to normal myometrium.

A, mRNA levels by qRT-PCR demonstrate that TET1 and TET3 are up-regulated in leiomyoma compared to normal myometrial tissue; there was no difference in TET2 mRNA level (n = 12). **, P < .01. B and C, Representative immunoblots and densitometry analysis show higher expression of TET1 and TET3 proteins in leiomyoma (L) relative to myometrial tissue (M; n = 6). TET2 was slightly overexpressed, but it did not reach statistical significance. Values are reported as the mean ± SD. **, P < .01, by Student's t test. D, Representative images of TET1 and TET3 immunofluorescence confirmed the higher levels of TET1 and TET3 (red) in leiomyoma (L) compared with normal myometrial cells (M; n = 5). Nuclei are stained with DAPI (blue). Scale bar = 50 μm. TBP, TATA-binding protein.

Figure 3.. Silencing of TET1 and TET3 decreases 5-hmC levels.

A, mRNA levels of TET1 and TET3 confirm silencing by siRNAs. TET1 and TET3 were significantly silenced compared to control transfected leiomyoma cells (n = 4). B, Representative images of TET staining confirm the reduction of TET1 and TET3 protein levels after siRNA targeting of TET1 and TET3 (n = 4). C, Representative image of IF staining shows the decrease in 5-hmC in leiomyoma cells after siRNA-mediated decrease of TET1 and TET3 (red) relative to cells transfected with control siRNA. Nuclei are stained with DAPI (blue). Scale bar = 50 μm (n = 5). D, ELISA showing the percentage of global 5-hmC in leiomyoma cells after silencing of TET1 and TET3 relative to control siRNA (n = 4). Values are reported as mean ± SD. **, P < .01 by Student's t test. siCTL, control siRNA.

Figure 4.. Silencing of TET1 and TET3 decreases cell proliferation.

A, Representative immunoblot confirming siRNA knockdowns of TET1 and TET3 in leiomyoma cells compared to cells transfected with control siRNA (siCTL). B, Densitometry analysis of the immunoblots indicate that TET1 and TET3 protein expression is reduced (n = 4). C, Densitometric analysis of PCNA immunoblots indicate that it is significantly reduced in leiomyoma transfected with TET1 or TET3 siRNA (n = 4). D, qRT-PCR confirming down-regulation of TET1 and TET3 mRNA in leiomyoma cells (n = 4). E, BrdU-incorporation cell proliferation assay demonstrates a significant diminution in BrdU incorporation after siRNA knockdown of TET1 and TET3 in leiomyoma cells (n = 8). Values are reported as the mean ± SD. *, P < .05; **, P < .01; ***, P < .001, by Student's t test or two-way ANOVA analysis with Tukey comparison post-test.

Figure 5.. (S)-2HG reduces 5-hmC levels and proliferation in leiomyoma cells.

A, Representative IF images from two subjects show a notable reduction in 5-hmC in (S)-2HG-treated cells with no reduction in cells treated with vehicle. The images show a strong presence of 5-hmC (red) in cells treated with vehicle, which becomes faint or absent in (S)-2HG-treated cells (n = 5). Leiomyoma cells were treated with 700 μm (S)-2HG for 48 hours. Nuclei are stained with DAPI (blue). Scale bar = 50 μm. B, ELISA showing the percentage of global 5-hmC in leiomyoma cells after treatment with (S)-2HG relative to vehicle-treated cells (n = 5). C, Cell proliferation assay showing a significant decrease in BrdU incorporation (n = 4) after (S)-2HG treatment compared to cells treated with vehicle. D, Cell viability was assessed in terms of LDH activity; treatment with (S)-2HG does not promote leiomyoma cell death, with no difference in LDH activity compared with cells treated with vehicle (n = 4). Values are reported as the mean ± SD. **, P < .01, Student's t test or two-way ANOVA analysis with Tukey comparison post-test.