Figure 7.

Effect of 27nt RNA duplex on eNOS mRNA expression and stability. (A) Human umbilical venous endothelial cells (HUVECs) at fourth passage homozygous for 5x27nt repeats or homozygous for 4x27nt repeats in the eNOS intron 4 were cultured to 100% confluence before harvested for RNA extraction. The RNA samples were electrophoresized on 1% acrylamid/8 M urea gel and analyzed by Northern blot for eNOS mRNA using the exon4 specific mRNA probe (top lane). The cyclophilin RNA was used as the loading control. The bottom of bar graph represents average eNOS RNA levels from HUVECs of three individuals homozygous for 4x27nt three individuals homozygous for 5x27nt. *p < 0.05 by the Student test. (B) Effects of 27nt small RNA on eNOS mRNA stability in HAECs. HAECs were plated onto six-well plates and cultured up to 90% confluence. We then transfected 27nt RNA duplexes to these cells using liposome. The transcription was blocked by adding actinomycin D (2 μg/ml). Cells were then harvested for RNS extraction at 0, 6, 12, 24, 48 and 72 h after treatment. The relative levels of eNOS mRNA were determined by quantitative realtime RT-PCR adjusted by housekeeping gene β-actin. ***p < 0.001 by the Student's t test comparing to the levels at 0 and 6 h.