Impact of stromal interaction molecule 1 silencing on cell cycle of endothelial progenitor cells
10.3760/cma.j.issn.0253-3758.2011.07.011
- VernacularTitle:基质交感分子1对大鼠内皮祖细胞细胞周期的影响
- Author:
Chun-Yan KUANG
1
;
Lan HUANG
;
Yang YU
;
Meng-Yang DENG
;
Kui WANG
;
De-Hui QIAN
Author Information
1. 第三军医大学新桥医院
- Keywords:
Stem cells;
Cell cycle;
RNA interference;
Transient receptor potential channels;
Stromal interaction molecule 1
- From:
Chinese Journal of Cardiology
2011;39(7):649-653
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of stromal interaction molecule 1 (STIM1) silencing on EPCs cell cycle. Methods Rat 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. Results Forty-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). Conclusion siRNA-mediated knockdown of STIM1 inhibited EPCs proliferation by reducing intracellular free Ca2+ through TRPC1-SOC signaling pathway.
