Effect of overexpression of aging-association transcription factors on epithelial cells.

RNA-seq profiling of whole, intact uteri at embryonic day 3.5 (E3.5) from young and aged virgin wild-type C57BL/6 mice—each mated with vasectomized wild-type males—identified the transcription factors Foxc1 and Six1 as aging-associated markers. Immunostaining of mouse uteri across an age range of 8 to 41 weeks revealed a progressive upregulation of FOXC1, initially localized to individual stromal cells in the endometrium, followed by prominent expression in both luminal and glandular epithelial cells. To investigate the functional role of Foxc1 and Six1, their full-length open reading frames were cloned into a PiggyBac expression vector, sequence-verified, and overexpressed in Ishikawa cells. Phenotypic characterization and RNA-seq analysis of these cells provided a framework for understanding FOXC1-mediated aging-related effects. RNA-seq profiling of Ishikawa cells (human endometrial adenocarcinoma cell line) overexpressing Foxc1, Six1, alone and in combination. Ishikawa cells treated with / without progesterone-estrodiol-cyclic AMP were processed for RNA-sequencing analysis.