Different DNA methylome, transcriptome and histological features in uterine fibroids with and without MED12 mutations

Abstract

Somatic mutations in Mediator complex subunit 12 (MED12m) have been reported as a biomarker of uterine fibroids (UFs). However, the role of MED12m is still unclear in the pathogenesis of UFs. Therefore, we investigated the differences in DNA methylome, transcriptome, and histological features between MED12m-positive and -negative UFs. DNA methylomes and transcriptomes were obtained from MED12m-positive and -negative UFs and myometrium, and hierarchically clustered. Differentially expressed genes in comparison with the myometrium and co-expressed genes detected by weighted gene co-expression network analysis were subjected to gene ontology enrichment analyses. The amounts of collagen fibers and the number of blood vessels and smooth muscle cells were histologically evaluated. Hierarchical clustering based on DNA methylation clearly separated the myometrium, MED12m-positive, and MED12m-negative UFs. MED12m-positive UFs had the increased activities of extracellular matrix formation, whereas MED12m-negative UFs had the increased angiogenic activities and smooth muscle cell proliferation. The MED12m-positive and -negative UFs had different DNA methylation, gene expression, and histological features. The MED12m-positive UFs form the tumor with a rich extracellular matrix and poor blood vessels and smooth muscle cells compared to the MED12m-negative UFs, suggesting MED12 mutations affect the tissue composition of UFs.

Figure 1

DNA methylation profiling of the MED12m-positive and -negative uterine fibroids, and myometrium. (a) DNA methylation profiles of the MED12m-positive and -negative uterine fibroids, and myometrium were compared using hierarchical clustering analyses. Distances of DNA methylation pattern are indicated as height. Each color indicates the myometrium (light blue), the MED12m-positive uterine fibroids (red), and the MED12m-negative uterine fibroids (green). The MED12m-negative uterine fibroids were further classified into three different clusters, Subtype-1, Subtye-2, and Subtype-3. (b) Chromosomal distribution of hyper- or hypomethylated CpGs in the MED12m-positive and -negative uterine fibroids (Subtype-1, -2, and -3) compared to the myometrium are shown. The locations of CpG sites, which have p < 0.05 and beta-value difference > 0.2 compared to the myometrium, are indicated with red (hypermethylated CpGs) or blue (hypomethylated CpGs). Autosomal and sex chromosome numbers are shown on the top.

Figure 2

The scatterplot of GO terms in DEGs. The plots and tables show the GO terms after the redundancy reduction in the MED12m-positive-increased (a), -decreased (b), the MED12m-negative-increased (c), and -decreased (d) DEGs. The colors indicate the log10(p-value) of the summarized GO terms. The size of the circle indicates the frequency of the GO term in the underlying GO database. The circles of more general terms are plotted larger. The color and the size of circles are plotted according to the default setting of REVIGO. (e) Summary of GO analysis in the MED12m-positive and -negative uterine fibroids (drawn with Microsoft PowerPoint). The specific terms to the MED12m-positive uterine fibroids, the MED12m-negative uterine fibroids, and commonly detected terms are shown. Red and blue mean higher and lower expression compared to the myometrium.

Figure 2

The scatterplot of GO terms in DEGs. The plots and tables show the GO terms after the redundancy reduction in the MED12m-positive-increased (a), -decreased (b), the MED12m-negative-increased (c), and -decreased (d) DEGs. The colors indicate the log10(p-value) of the summarized GO terms. The size of the circle indicates the frequency of the GO term in the underlying GO database. The circles of more general terms are plotted larger. The color and the size of circles are plotted according to the default setting of REVIGO. (e) Summary of GO analysis in the MED12m-positive and -negative uterine fibroids (drawn with Microsoft PowerPoint). The specific terms to the MED12m-positive uterine fibroids, the MED12m-negative uterine fibroids, and commonly detected terms are shown. Red and blue mean higher and lower expression compared to the myometrium.

Figure 2

The scatterplot of GO terms in DEGs. The plots and tables show the GO terms after the redundancy reduction in the MED12m-positive-increased (a), -decreased (b), the MED12m-negative-increased (c), and -decreased (d) DEGs. The colors indicate the log10(p-value) of the summarized GO terms. The size of the circle indicates the frequency of the GO term in the underlying GO database. The circles of more general terms are plotted larger. The color and the size of circles are plotted according to the default setting of REVIGO. (e) Summary of GO analysis in the MED12m-positive and -negative uterine fibroids (drawn with Microsoft PowerPoint). The specific terms to the MED12m-positive uterine fibroids, the MED12m-negative uterine fibroids, and commonly detected terms are shown. Red and blue mean higher and lower expression compared to the myometrium.

Figure 3

Expression levels of representative genes in the detected biophysical processes. According to Figs. 2e, Fig. 3 shows the expression statuses of the representative genes not only from the commonly detected terms between the MED12m-positive and MED12m-negative uterine fibroids (a), but also from the terms specific to the MED12m-positive or the MED12m-negative uterine fibroids (b). We selected the genes in which the difference in expression levels was clearly significant among the MED12m-positive uterine fibroids, the MED12m-negative uterine fibroids, and the normal myometrium. ^ap < 0.01 (myometrium (n = 6) vs. MED12m-positive uterine fibroids (n = 6), student t-test). ^bp < 0.01 (myometrium (n = 6) vs. MED12m-negative uterine fibroids (n = 9), student t-test).

Figure 4

Enriched GO terms identified by weighted gene co-expression network analysis (WGCNA) in the MED12m-positive and -negative uterine fibroids. Twenty-six and 14 COGs groups in the MED12m-positive and -negative uterine fibroids, respectively, were introduced into the pathway and GO enrichment analyses. Three and five COGs groups in the MED12m-positive and -negative uterine fibroids were significantly enriched with GO terms. The other 23 and 9 COGs groups in the MED12m-positive and -negative uterine fibroids, respectively, were not significantly enriched with GO terms. The ratio of the number of identified genes to all genes in each term is shown as "geneRatio". P-values were adjusted with the BH method by clusterProfiler and indicated with colors.

Figure 5

Histological examination in the uterine fibroids and myometrium. (a) Immunofluorescent staining for collagen fibers in the MED12m-positive, -negative uterine fibroids, and myometrium. Collagen fibers are detected as blue by a trichrome staining kit (TRM-1, ScyTec Laboratories inc). (b) Boxplots show the occupation rate of collagen fiber. The collagen fiber area was quantified by Image J. The percentage per view field was calculated on 15 randomly chosen areas at × 200 magnification, and average percentages were indicated in each tissue section. *p < 0.05. (c). Immunofluorescent staining for smooth muscle cells (αSMA, green) and vascular endothelial cells (CD31, red) in the MED12m-positive, -negative uterine fibroids, and myometrium. (d). Boxplots show the number of blood vessels, which was counted by Image J. The number per view field was calculated on 5 randomly chosen areas at × 100 magnification, and the average numbers were indicated in each tissue section. *p < 0.05. (e) Immunofluorescent staining for smooth muscle cells (αSMA, red) and nucleus (DAPI, blue) in the MED12m-positive, -negative uterine fibroids, and myometrium. The cells stained with αSMA were considered as smooth muscle cells, whereas the cells that were not stained with αSMA was considered as non-smooth muscle cells. (f) Boxplots show the percentage of the smooth muscle cells in the smooth and non-smooth muscle cells. The number per view field was calculated on 5 randomly chosen areas at × 200 magnification, and the average numbers were indicated in each tissue section. *p < 0.05.

Figure 5

Histological examination in the uterine fibroids and myometrium. (a) Immunofluorescent staining for collagen fibers in the MED12m-positive, -negative uterine fibroids, and myometrium. Collagen fibers are detected as blue by a trichrome staining kit (TRM-1, ScyTec Laboratories inc). (b) Boxplots show the occupation rate of collagen fiber. The collagen fiber area was quantified by Image J. The percentage per view field was calculated on 15 randomly chosen areas at × 200 magnification, and average percentages were indicated in each tissue section. *p < 0.05. (c). Immunofluorescent staining for smooth muscle cells (αSMA, green) and vascular endothelial cells (CD31, red) in the MED12m-positive, -negative uterine fibroids, and myometrium. (d). Boxplots show the number of blood vessels, which was counted by Image J. The number per view field was calculated on 5 randomly chosen areas at × 100 magnification, and the average numbers were indicated in each tissue section. *p < 0.05. (e) Immunofluorescent staining for smooth muscle cells (αSMA, red) and nucleus (DAPI, blue) in the MED12m-positive, -negative uterine fibroids, and myometrium. The cells stained with αSMA were considered as smooth muscle cells, whereas the cells that were not stained with αSMA was considered as non-smooth muscle cells. (f) Boxplots show the percentage of the smooth muscle cells in the smooth and non-smooth muscle cells. The number per view field was calculated on 5 randomly chosen areas at × 200 magnification, and the average numbers were indicated in each tissue section. *p < 0.05.

Figure 6

DNA methylation and mRNA expression statuses of SATB2 and NRG1 genes. (a,b) The DNA methylation levels of SATB2 (a) and NRG1 (b) genes are shown in dot plots. The vertical axis indicates the DNA methylation levels in the MED12m-positive uterine fibroids (n = 9), -negative uterine fibroids (n = 12), and the corresponding myometrium. The DNA methylation levels were examined by COBRA and range from 0 to 100%. (c,d) The expression levels of SATB2 (c) and NRG1 (d) genes in the MED12m-positive uterine fibroids, -negative uterine fibroids, and the myometrium analyzed by qRT-PCR are shown in dot plots. The expression levels are corrected for myometrium expression as 1. ^ap < 0.01 (higher expression in uterine fibroids compared to myometrium, student t-test). ^bp < 0.01 (lower expression in uterine fibroids compared to myometrium, student t-test).