[Impact of stromal interaction molecule 1 silencing on cell cycle of endothelial progenitor cells]

Zhonghua Xin Xue Guan Bing Za Zhi. 2011 Jul;39(7):649-53.
[Article in 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Membrane Proteins
  • Neoplasm Proteins
  • RNA, Small Interfering
  • STIM1 protein, human
  • Stromal Interaction Molecule 1
  • Transient Receptor Potential Channels