[Impact of stromal interaction molecule 1 silencing on cell cycle of endothelial progenitor cells]
Chun-Yan Kuang1, Lan Huang, Yang Yu
1Cardiovascular Research Institute of Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
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.
Insights
Silencing stromal interaction molecule 1 (STIM1) in endothelial progenitor cells (EPCs) halts cell cycle progression. This occurs by reducing intracellular calcium via the TRPC1-SOC pathway, impacting EPCs proliferation.
Area of Science:
- Cell Biology
- Molecular Biology
- Physiology
Background:
- Stromal interaction molecule 1 (STIM1) plays a critical role in calcium signaling.
- Endothelial progenitor cells (EPCs) are crucial for vascular repair and angiogenesis.
- The cell cycle regulation of EPCs is vital for their function.
Purpose of the Study:
- To investigate the impact of stromal interaction molecule 1 (STIM1) gene silencing on the cell cycle of endothelial progenitor cells (EPCs).
- To elucidate the underlying molecular mechanisms, including calcium signaling and protein interactions.
Main Methods:
- Isolation and culture of rat bone marrow-derived EPCs.
- Transfection with STIM1 siRNA (Ad-si/rSTIM1) or STIM1 overexpression plasmid (Ad-hSTIM1).
- Analysis of STIM1 mRNA expression (RT-PCR), cell cycle (flow cytometry), intracellular calcium (LSCM), STIM1-TRPC1 interaction (co-immunoprecipitation), and IP3 levels (ELISA).
Main Results:
- STIM1 mRNA expression and intracellular free calcium levels were significantly reduced in EPCs transfected with Ad-si/rSTIM1.
- Cell cycle arrest at the G1 phase and inhibition of store-operated calcium entry were observed following STIM1 silencing.
- Overexpression of STIM1 reversed these effects, and STIM1 was found to co-precipitate with TRPC1.
Conclusions:
- siRNA-mediated knockdown of STIM1 inhibits EPC proliferation.
- This inhibition is mediated by a reduction in intracellular free calcium through the TRPC1-SOC signaling pathway.
- STIM1 is a key regulator of EPC proliferation via calcium homeostasis.
