243. In vitro culture significantly reduces differences in gene expression profiles between myometrial and fibroid smooth muscle cells

Abstract

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMC) have been widely used as a model for the study of fibroid growth. Although it has been shown that FSMC can behave differently in culture to MSMC, it is not clear how relevant the cultured cells and their responses are to the in-vivo situation. The aim of the present study was to compare gene expression profiles of M and F tissue to cells isolated from the same tissue and cultured for up to 3 passages. M and F were collected from hysterectomy specimens (n = 6), part was snap frozen for RNA and the rest used to isolate SMC, which were cultured for 3 passages and RNA was collected at passage 0 (P0) and 3 (P3). 36 microarrays were performed on 8K human cDNA slides, 6 per each specimen (3 for M and 3 for F: tissue, cell at P0 and P3) against reference RNA. Analysis revealed significant differences between tissues and cultured cells. Independent clustering assigned tissues versus cells into two distinct groups based on their expression profiles. Parametric ANOVA with Benjamini-Hochberg correction and post-hoc testing was used to determine similarities and differences between tissues and cells. 128 genes were found to be statistically different between M and F tissue, 66 between MSMC and FSMC at P0, and only 9 at P3. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Similar numbers of genes were up regulated as were down regulated. Expression profiles of genes of interest including estrogen receptor α and progesterone receptor were also validated using real-time PCR. This is the first study to compare gene expression of in vivo and in vitro fibroid and myometrial SMC. The results demonstrate that large changes occur in SMC gene expression in culture, reducing differences between myometrial and fibroid cells. This study indicates that results of in vitro studies should be interpreted with caution as many genes have an altered gene expression profile in culture.