tatistical difference in DNA methylation levels between the uterine leiomyoma and matched myometrial tissues. The Illumina platform covers 50 bp regions, whereas bisulfite sequencing evaluates a 250 300 bp region, which overlaps with the 50 bp sequence of interest. ” These longer fragments enhance fidelity. Impact of DNA methylation on gene expression in human uterine leiomyoma and matched adjacent myometrial tissues To validate that DNA methylation leads to gene silencing of the tumor suppressor genes KLF11, DLEC1, and KRT19, we assessed mRNA levels in vivo using real-time RT-PCR in uterine leiomyoma and matched myometrial tissues. We performed realtime RT-PCR on all 18 samples originally used in the microarray analysis plus we incorporated 710 new samples from Caucasian subjects. KLF11 mRNA levels in uterine leiomyoma were considerably lower than those in matched adjacent normal myometrial tissues. DLEC1 mRNA levels in uterine leiomyoma tissues were also significantly lower than those in matched myometrial tissues. KRT19 mRNA levels in uterine leiomyoma were considerably lower than those in matched adjacent normal myometrial tissues. We have not observed any differences in mRNA levels between samples from African- and CaucasianAmerican subjects. Genome-Wide DNA Methylation in Uterine Leiomyoma The effects of chemical demethylation of CpG dinucleotides on mRNA levels To determine whether the decrease in mRNA expression of these three tumor suppressor genes is regulated by DNA methylation, primary cultured leiomyoma smooth muscle cells isolated from 7 new subjects not previously used in microarrays were treated with the DNMT inhibitor, 5-aza-dC at different concentrations and time points. Realtime RT-PCR was performed to measure KLF11, DLEC1, and KRT19 mRNA levels. We observed that 5-aza-dC treatments at various doses had a similar effect on “
23370967“restoring mRNA levels. We chose the 3 mM dose to perform the subsequent experiments because it was potentially less toxic to the cells while being maximally effective. After analyzing mRNA expression after 1, 3 and 5 days, we determined that the effect is most effective at restoring mRNA expression levels after 5 days of treatment. As shown in Protein expression in human uterine leiomyoma and matched adjacent myometrial tissues To understand the in vivo relevance of how DNA methylation affects gene function, we analyzed protein levels of KLF11, DLEC1 and KRT19 in human leiomyoma and matched normal myometrial tissues using western blot. KLF11 protein levels in all 6 subjects were significantly lower in leiomyoma compared with myometrial tissues. MedChemExpress TL32711 Overall, DLEC1 protein levels were also significantly lower in leiomyoma than in myometrial tissues, and only 2 out of 9 subjects had no difference in DLEC1 expression in leiomyoma compared 
with myometrial tissues. KRT19 protein levels in 8 subjects were lower in leiomyoma than myometrial tissues, and only 1 subject had higher KRT19 protein levels in leiomyoma ” compared with myometrial tissues. All protein studies were performed with 69 new pairs of matched samples not previously used in the microarray experiments; 5 subjects were African American and 4 subjects were Caucasian. Overall, western blots showed that KLF11, DLEC1 and KRT19 . Protein levels in leiomyoma tissues were significantly lower compared with matched normal myometrial tissues. 4 Genome-Wide DNA Methylation in Uterine Leiomyoma DNA Methylation1 P-value HTATIP2 SOX18 CREG1 PECAM1 CRIM1 1 2 Gene n