Impact of stromal interaction molecule 1 silencing on cell cycle of endothelial progenitor cells.
- Author:
Chun-Yan KUANG
1
;
Lan HUANG
;
Yang YU
;
Meng-Yang DENG
;
Kui WANG
;
De-Hui QIAN
Author Information
- Publication Type:Journal Article
- MeSH: Adenoviridae; genetics; Animals; Cell Cycle; Cell Proliferation; Cells, Cultured; Endothelial Cells; cytology; Gene Silencing; Genetic Vectors; Membrane Proteins; genetics; Neoplasm Proteins; genetics; RNA, Small Interfering; Rats; Stem Cells; cytology; Stromal Interaction Molecule 1; Transfection; Transient Receptor Potential Channels; metabolism
- From: Chinese Journal of Cardiology 2011;39(7):649-653
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the effect of stromal interaction molecule 1 (STIM1) silencing on EPCs cell cycle.
METHODSRat bone marrow derived endothelial progenitor cells (EPCs) were isolated and cultured in L-DMEM with 20% FBS. Ad-si/rSTIM1 and Ad-hSTIM1 were then transfected into EPCs and the expression of STIM1 mRNA was detected by RT-PCR. The cell cycle was determined using flow cytometry analysis and intracellular free Ca2+ was measured using LSCM. Co-immunoprecipitation was performed to examine the interaction between STIM1 and TRPC1. Protein levels of inositol 1, 4, 5-trisphosphate were analyzed with ELISA assay.
RESULTSForty-eight hours after transfection, the expression of STIM1 mRNA was significantly downregulated (0.37 +/- 0.02 vs. 1.00 +/- 0.02, P < 0.05) and intracellular free Ca2+ level was significantly reduced (34.07 +/- 4.10 vs. 86.51 +/- 14.12, P < 0.05) in Ad-si/rSTIM1 group compared with control group. The cell cycle was arrested at G1 phase [(90.91 +/- 1.10)% vs. (77.10 +/- 0.56)%, P < 0.05] and the store-operated channel entry was strikingly inhibited in EPCs after treatment with Ad-si/rSTIM1. However, cotransfection of Ad-hSTIM1 with Ad-si/rSTIM1 significantly reversed these responses. Interestingly, co-immunoprecipitation study showed that STIM1 co-precipitated with TRPC1, and IP3 levels measured by ELISA were similar among three groups (P > 0.05).
CONCLUSIONsiRNA-mediated knockdown of STIM1 inhibited EPCs proliferation by reducing intracellular free Ca2+ through TRPC1-SOC signaling pathway.
