Loss of miR-200c: A Marker of Aggressiveness and Chemoresistance in Female Reproductive Cancers

Abstract

We focus on unique roles of miR-200c in breast, ovarian, and endometrial cancers. Members of the miR-200 family target ZEB1, a transcription factor which represses E-cadherin and other genes involved in polarity. We demonstrate that the double negative feedback loop between miR-200c and ZEB1 is functional in some, but not all cell lines. Restoration of miR-200c to aggressive cancer cells causes a decrease in migration and invasion. These effects are independent of E-cadherin status. Additionally, we observe that restoration of miR-200c to ovarian cancer cells causes a decrease in adhesion to laminin. We have previously reported that reintroduction of miR-200c to aggressive cells that lack miR-200c expression restores sensitivity to paclitaxel. We now prove that this ability is a result of direct targeting of class III beta-tubulin (TUBB3). Introduction of a TUBB3 expression construct lacking the miR-200c target site into cells transfected with miR-200c mimic results in no change in sensitivity to paclitaxel. Lastly, we observe a decrease in proliferation in cells transfected with miR-200c mimic, and cells where ZEB1 is knocked down stably, demonstrating that the ability of miR-200c to enhance sensitivity to paclitaxel is not due to an increased proliferation rate.

Figure 1

Reciprocal repression between ZEB1 and miR-200c occurs in some, but not all cell types. (a) Schematic of the mutual repression of ZEB1 and miR-200c. (b) Western blotting for ZEB1 and α-tubulin loading control, and real time RT-PCR for miR-200c in Hey ovarian cells transiently transfected with a miR-200c mimic. Real time RT-PCR for miR-200c and western blotting for ZEB1, E-cadherin, and α-tubulin in MDA-MB-231 breast cancer cells (c), and Hec50 endometrial cancer cells (d) stably transfected with shRNA lentiviral vector targeting ZEB1 (shZEB), luciferase (shLuciferase), or the empty vector (shRNA EV).

Figure 2

Increased miR-200c decreases migration and invasion, not necessarily dependent on restoration of E-cadherin. Migration (a) and invasion (b) assays for MDA-MB-231 cells stably expressing an shRNA targeting ZEB1 with representative images below. Migration (c) and invasion (d) assays in Hey cells transiently transfected with a miR-200c mimic. Asterisks indicate a statistically significant difference (P < .05, Student's t-test) versus negative controls.

Figure 3

Increased miR-200c levels decrease adhesion to substrates. Fluorescent adhesion assays of Hey cells to (a) basement membrane complex, (b) laminin, (c) collagen type IV, and (d) fibronectin. Adhesion of MDA-MB-231 cells to (e) basement membrane complex and (f) laminin. Asterisks indicate a statistically significant difference (P < .05, Student's t-test) versus the negative controls.

Figure 4

Chemosensitivity to paclitaxel induced by miR-200c expression. (a) Clonogenic assay in Hey cells either mock transfected, transiently transfected with a negative control, or miR-200c mimic and treated with 0–5 nM paclitaxel. The total area (b) and number of colonies (c) at 5 nM paclitaxel are quantified. Asterisks indicate statistically significant difference (P < .05, Student's t-test) versus negative controls.

Figure 5

Restoration of TUBB3 reverses miR-200c-mediated enhanced chemosensitivity to paclitaxel. (a) Western blots for ZEB1, E-cadherin, TUBB3, and α-tubulin in Hec50 cells transiently transfected with a miR-200c mimic or negative controls and an expression vector for TUBB3 or empty vector. (b) Real time RT-PCR for miR-200c. Cell death ELISA for cells transfected with a miR-200c mimic and an empty vector (c) or TUBB3 expression vector (d) treated with various concentrations of paclitaxel. Asterisks indicate statistically significant difference (P < .05, Student's t-test) versus negative controls.

Figure 6

Proliferation assay in Hey cells transiently transfected with a miR-200c mimic (a), or Hec50 cells (b), and MDA-MB-231 cells (c) stably expressing an shRNA against ZEB1. Asterisks indicate statistically significant difference (P < .05, Student's t-test) versus negative controls.