Effects of RNAi-mediated Gene Silencing of LRIG3 Expression on Cell Cycle and Survival of Glioma Cells
JOURNAL OF HUAZHONG UNIVERSITY OF SCIENCE AND TECHNOLOGY-MEDICAL SCIENCES
Authors: Cai, Mingjun; Xie, Ruifan; Han, Lin; Chen, Rudong; Wang, Baofeng; Ye, Fei; Guo, Dongsheng; Lei, Ting
Abstract
The effects of RNAi-mediated gene silencing of LRIG3 expression on cell cycle and survival of human glioma cell line GL15 and the possible mechanisms were explored. The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 were transfected into GL15 glioma cells respectively by using Metafectine, and the transfected cells that stably suppressed LRIG3 expression were selected by G418. The control cells were transfected with negative control shRNA. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blot. The apoptosis rate and cell cycle were analyzed by flow cytometry. As compared with the negative shRNA-transfected GL15 cells, LRIG3 mRNA expression in GL15 cells transfected with pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 was silenced by 52.4%, 63.8%, and LRIG3 protein expression was reduced by 50.9% and 67.4% respectively. The LRIG3-specific siRNA transfected cells had higher proliferation rate than control cells. Cell cycle analysis showed that silencing LRIG3 increased the percentage of G(2)/M phase cells and the proliferation index significantly (P<0.01). Silencing LRIG3 could inhibit the apoptosis of GL15 cells (P<0.05). These findings suggest that the siRNA targeting LRIG3 gene shows a dramatic inhibitory effect on RNA transcription and protein expression, then promoting the proliferation of GL15 cells, arresting GL15 cells in G2/M phase, and suppressing apoptosis of GL15 cells.
Activin and BMP4 Synergistically Promote Formation of Definitive Endoderm in Human Embryonic Stem Cells
STEM CELLS
Authors: Teo, Adrian K. K.; Ali, Yusuf; Wong, Kee Yew; Chipperfield, Hiram; Sadasivam, Akila; Poobalan, Yogavalli; Tan, Ee Kim; Wang, Siew Tein; Abraham, Suman; Tsuneyoshi, Norihiro; Stanton, Lawrence W.; Dunn, N. Ray
Abstract
Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic beta cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage. STEM CELLS 2012; 30:631642