Characterization of MicroRNA-200 pathway in ovarian cancer and serous intraepithelial carcinoma of fallopian tube

Abstract

Background: Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors.

Methods: RNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors.

Results: Ovarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFβ1 and TGFβ2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection.

Conclusions: The activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

Keywords: Expression analysis; Fallopian tube tumors; MicroRNA; Ovarian tumors.

Fig. 1

Heat maps showing the expression of miR-200 family and miR-205 and target genes in a ovarian and b fallopian tube cells

Fig. 2

Heat maps showing the expression of effector genes in a ovarian and b fallopian tube cells. A box was drawn in the MUC16 lane to show the expression levels of mucinous ovarian tumors within the Non-serous malignant tumor samples

Fig. 3

Heat maps showing the expression of target and effector genes after transfection of control and different combinations of miR-200 and miR-205 precursors into a normal OSE and b normal FTE cells

Fig. 4

Induction of effector gene expression in FTE cells after miR-200 transfection. a Box plot to show the differential expression of Muc16 mRNA in FTE cells and OSE cells after the transfection. b Immunostaining of CA125 and EpCAM proteins after the transfection of either nontarget control RNA or miR-200 precursor RNA into FTE cells. Positive protein targets were pseudo-colored in red, and nuclear staining was in blue