Culture on PAO is relatively simple, with the facility to gain quantitative data on microcolony growth with changes in the environment

onductor GeneAnswers package, and found that based on their p-values level, the top two most significantly enriched gene functions are cancer processes or reproductive system diseases . The genes involved in cancer were DLEC1, KRT19, KLF11, SERPINF1, TEK, APOLD1, LYVE1, CCL2, IL17B, and TNFS10, and genes 11885967” involved in reproductive system diseases were CRIM1, PCP4, CHRDL2, HOXA5, PLP1, COL9A2, 21278085” SOX18, BMP, CALCRL, SFRP1. Results Analysis of DNA PTK/ZK methylation and mRNA expression in uterine leiomyoma and matched adjacent myometrial tissue Validation of differential DNA methylation using bisulfite genomic sequencing We hypothesized that the 55 overlapping genes with differential DNA methylation and mRNA expression in uterine leiomyoma compared with normal myometrium were likely to be true targets of epigenetic regulation in uterine leiomyoma. Initially, we examined the regulatory CpG islands in the promoter regions of selected genes from the 55 candidates, and characterized the positions of 59 CpG islands and transcriptional start sites using available genome databases. From this set, we then selected three of the hypermethylated genes, Kruppel-like transcription factor 11, deleted in lung and esophageal cancer 1, keratin 19 for further analysis based on their known tumor suppressor functions. First, we studied the KLF11 promoter via sequencing of bisulfite-treated genomic DNA from uterine leiomyoma and myometrial tissues from 8 subjects. Four of these were African American that were included in our original genome-wide DNA methylation study, and we incorporated four new matched samples from Caucasian subjects. We analyzed the DNA methylation status of a cluster of 16 CpG dinucleotides across a 249-bp region of a CpG island, located approximately 2900 bp to 2500 bp upstream of the KLF11 promoter region. Four to six clones were sequenced from each subject. The detailed CpG methylation level of primary leiomyoma and matched myometrial tissues verified the hypermethylated state of the KLF11 promoter in uterine leiomyoma compared with adjacent normal myometrium. Six of the eight uterine leiomyoma samples showed increased DNA methylation of the KLF11 promoter. There was a significant statistical difference in DNA methylation levels between the uterine leiomyoma and matched myometrial tissues. Then, we analyzed the promoter region of another tumor suppressor gene, DLEC1, in uterine leiomyoma and myometrial samples from 7 subjects. Three subjects were African Americans included in our original genome-wide DNA methylation study, and we incorporated four new matched samples from Caucasian subjects. We sequenced a cluster of 18 CpG dinucleotides across a 252-bp region of a CpG island located within a 2100 to 150 bp region of the DLEC1 promoter. Uterine leiomyoma tissues demonstrated a dense methylation pattern at the DLEC1 promoter region in 5 of the 7 subjects. The majority of the 18 CpG dinucleotides in the DLEC1 promoter were consistently methylated in uterine leiomyoma, but not in normal myometrial tissues. Overall, there was a significant statistical difference in methylation levels between uterine leiomyoma and matched normal myometrial tissues. Finally, we studied the KRT19 promoter via sequencing of bisulfite-treated genomic DNA from uterine leiomyoma and myometrial tissues from 7 subjects. Four subjects were African Americans included in our original genome-wide DNA methylation study, and we incorporated three new matched samples from Caucasian s