MiR-335 Is Upregulated upon Retinoic Acid-Induced Differentiation of NTera-2 Human Embryonal Carcinoma Cell Line and Can Induce Differentiation
Sebastian Vencken, Michael Gallagher, Salah Elbaruni, Cara Martin, Orla Sheils, John O'Leary. Trinity College Dublin, Dublin, Ireland
Background: MicroRNAs (miRNAs) are endogenous, non-coding RNA molecules of 22-25 nucleotides in length and regulate various cell processes, by inhibiting mRNA translation.
NTera-2 embryonal carcinoma cells are frequently used to study both cancer stem cells (CSCs) and human embryonal stem cells (hESCs).
Like hESCs and many CSC types, NTera-2 cells are sensitive to all trans retinoic acid (ATRA) –induced differentiation. Upon subjecting these cells to ATRA, phenotypic change in cell morphology, a reduction in core stem cell markers and an increase in differentiation markers have been shown shortly after by various laboratories, including ours.
Design: Our study showed a wide range of differential miRNA expression upon incubation of NTera-2 cells in 10-5 M ATRA in growth medium for 72 hours. Hsa-miR-335 was among some of the most substantially upregulated miRNAs upon ATRA-induced differentiation of NTera-2 cells.
The highly cited miRNA target prediction software packages, TargetScan 5.1 and miRANDA, predict miR-335 binding regions within the 3' untranslated regions of various CSCMs, among which OCT3/4 and SOX2.
We proceeded to upregulate this miR-335 in NTera-2 cells by transfecting Ambion's Pre-miR miRNA precursors, stable mimics of endogenous miRNAs. As a negative control, we used the manufacturer's Pre-miR Negative Control #1, a scrambled, non-targeting Pre-miR.
Results: Results showed a clear morphological change in the NTera-2 cells after four days, with characteristics of cell differentiation and cell senescence. Cell viability studies using an MTT assay showed a significant reduction in cell proliferation.
Gene expression analysis with qRT-PCR showed a significant downregulation of OCT3/4 and NANOG CSCMs. SOX2, another CSCM frequently associated with the previous two, was significantly upregulated at the gene level, but this was not beyond the margin considered to be biologically insignificant. Protein expression analysis with Western blot showed a significant downregulation of OCT3/4 and SOX2, the latter result suggesting post-transcriptional intervention of the SOX2 mRNA, possibly by miRNA.
Conclusions: Our findings suggest miR-335 to be a miRNA involved in key regulatory mechanisms during early differentiation in NTera-2 cells. We are currently focused on validating predicted targets of miR-335 we believe are crucial to the maintenance of the pluripotent and self-renewal state.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 290, Tuesday Morning