Sequence variation among members of the miR-200 microRNA family is correlated with variation in the ability to induce hallmarks of mesenchymal-epithelial transition in ovarian cancer cells

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Date: Jan. 21, 2014
From: Journal of Ovarian Research(Vol. 7, Issue 1)
Publisher: BioMed Central Ltd.
Document Type: Article
Length: 4,024 words
Lexile Measure: 1490L

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Author(s): Neda Jabbari1 , Ashley N Reavis1 and John F McDonald1,2

Background

Ovarian cancer is the most lethal of all gynecologic cancers[1]. The majority of OC related deaths is attributable to the spread of cancer cells from the primary tumor to metastatic sites throughout the abdominal cavity[2]. During early stages of metastasis, a subset of primary epithelial tumor cells undergo epithelial-to-mesenchymal transition (EMT), whereby intercellular adhesion complexes are disrupted, the characteristic apico-basal polarity of the cells is lost and cells acquire elevated levels of motility, invasiveness and resistance to standard chemotherapeutic treatments[3-5]. Subsequent to attachment to secondary sites, metastasizing cells undergo a complementary mesenchymal-to-epithelial transition (MET) whereby the metastatic cells reacquire epithelial morphologies and other features characteristic of the primary tumor cells[6]. Because of the high clinical significance of metastasis in ovarian and other cancers, considerable effort is currently being directed towards the development of new classes of agents that may reduce the spread of cancer cells by inducing MET[7].

We have previously shown that mesenchymal-like OC cells undergo MET in response to ectopic over-expression of miR-429, a member of the miR-200 family of miRNAs[8]. This finding is consistent with earlier observations implicating members of the miR-200 family of miRNAs in MET[9, 10]. In this paper we report the results of a systematic examination of the effect of ectopic over expression of members of the miR-200 family of miRNAs in OC mesenchymal-like cell lines. The results indicate that although over expression of each member of the miR-200 family induces significant changes in many of the morphological and molecular hallmarks of MET, significant differences exist among family members in the expression of EMT/MET biomarkers and in induced drug sensitivity. This functional variability is associated with sequence variation mapping to both the seed and non-seed regions of individual miRNAs.

Methods

Cell culture and miRNA transfection

HEY and HEY A8 ovarian cancer cell lines were provided by Gordon B. Mills (MD Anderson Cancer Center, Houston, TX). SKOV-3 ovarian cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 (Mediatech, Manassas, VA) supplemented with 10% FBS (Fetal Bovine Serum; Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic solution (Mediatech-Cellgro, Manassas, VA). For miRNA transfections, 6 x 104 cells were seeded per well in 24-well plates. Cells at exponential phase of growth were transfected with 30 nM miRNA purchased as Pre-miR miRNA Precursors (Ambion, Austin, TX) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and according to the manufacturer's instructions. Cells were allowed to grow for 48 hours before RNA isolation. Ambion Pre-miR miRNA Precursor Negative Control was used as a negative control (nc-miR).

Image capture and morphological assessment

Morphological changes were monitored using an Olympus IX51 microscope (Olympus Optical, Melville, NY). The effect of treatment on cell morphology was objectively measured using CellProfiler cell-imaging software (2.1.0)[11].

Quantitative reverse transcription real-time PCR (qRT-PCR)

Total RNA was extracted from cells using the RNeasy mini kit (Qiagen, Valencia, CA). RNA concentrations were measured using a NanoDrop 1000 Spectrophotometer V3.2...

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Gale Document Number: GALE|A540663648