Retinal pigment mesenchyme transition

Retinal pigment mesenchyme transition

Time course ID: human_ARPE-19
Sample provider: Soichi Ogishima, Hiroshi Tanaka, Ken Miyaguchi, Afsaneh Eslami

Introduction

Epithelial–mesenchymal transition (EMT) is a process of transition from epithelial cells to mesenchymal cells characterized by loss of cell adhesion and polarity, repression of E-cadherin expression, gain of cell motility and nvasive properties. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT is also essential for wound healing, organ fibrosis and initiation of metastasis for cancer progression. During the process of epithelial to mesenchymal transition, epithelial cells transforms to mesenchymal cells, and change their shapes. Mesenchymal cells get to gather and form focus. To clarify the transcriptional networks regulating EMT, we obtained time-course sample of human ARPE-19 epithelial to mesenchymal transition. After preculture for 7 days on 10cm dishes and cell seeding and cell culture for 5 days on 6-well glass bottom plate, we induced epithelial to mesenchymal transition on human ARPE-19 cells by TT-mixture of TNFα and TGFβ2. Triplication for RNA extraction and 1 for imaging. We sampled total RNA of human ARPE-19 cells along with time-course of epithelial to mesenchymal transition.

Samples

ARPE-19 cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 medium (Sigma-Aldrich) with 10% fetal bovine serum in a CO2 incubator at 37 °C. After 5 days of preculture, cells were treated with human recombinant TGF-β2 and human recombinant TNF-α and continuously incubated in serum free medium at 37 °C to induce EMT. At 21 different time points (0 min, 15 min, 30 min, 45 min, 60 min, 80 min, 100 min, 2 hr, 2.5 hr, 3 hr, 3.5 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 12 hr, 16 hr, 24 hr, 42 hr and 60 hr), cells were dissolved in lysis reagent and collected. For the samples of 0 min, cells were treated with PBS without TGF-β2 and TNF-α. Using miRNeasy Mini Kit (QIAGEN), total RNA was extracted from each cell lysate according to a manufacturer’s protocol.

Figure 1: Sample protocol

Figure 2: Cell morphology

Quality control

Figure 3: CAGE expression of marker genes in TPM.

References

[1] Tumor necrosis factor-alpha regulates transforming growth factor-beta-dependent epithelial-mesenchymal transition by promoting hyaluronan-CD44-moesin interaction. Takahachi E et al, J Biol Chem, 2010, 285(6):4060-73. PMID:19965872

[2] Epithelial-mesenchymal transitions in development and disease, Thiery JP et al, Cell, 2009, 139(5):871–890. PMID:19945376

Related samples

We have RNA samples of all time points for validation.

Data

Expression profiles

• Gene and CAGE cluster expression for the retinal epithelium series
• https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/human_ARPE-19/expression_tables/

Zenbu configuration and status

• • ARPE-19
  • ARPE-19 timecourse - update Oct 26th

MARA based network results

MARA

• https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/human_ARPE-19/mara/

ISMARA analysis results

All samples: http://ismara.unibas.ch/timecourses/retinal_pigment_EMT/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/ARPE19-avgd/averaged_report/index.html

For more information, see ISMARA.