小分子RNA与心血管疾病演示文稿.pptVIP

The next question is What are the cellular mechanisms involved in miR-21 mediated effect on neointimal formation? As we know, VSMC proliferation is the key cellular event responsible for neointimal lesion formation. To identify the cellular mechanism involved in miR-21-mediated effect on neointimal lesion formation, we therefore determined the effect of miR-21 inhibitor 2OMe-miR-21 on cell proliferation in cultured VSMCs in the following experiments. It is well known that freshly isolated VSMCs mimic differentiated VSMCs in normal uninjured vascular wall, whereas serum cultured VSMCs mimic dedifferentiated VSMCs in vascular neointimal lesions. We thus determined the miR-21 levels in freshly isolated, differentiated VSMCs and dedifferentiated VSMCs cultured in DMEM containing 10% FBS. We found that the expression of miR-21 in dedifferentiated VSMCs was significantly higher than that in freshly isolated, differentiated VSMCs.The in vitro result is consistent with the in vivo results in normal and balloon injured arteries. 24. To further determine the potential roles of miRNAs in VSMC proliferation, we applied antisense oligonucleotide-mediated miRNA depletion to knock down the miR-21 overexpression by using its inhibitor, 2OMe-miR-21. Oligonucleotide transfection was performed according to an established protocol. As shown in the left Fig, miR-21 inhibitor was successfully transfected into the cultured VSMCs. Consistent with the transfection, the 2OMe-miR-21 decreased the miR-21 expression levelin a dose-dependent manner, with a significant decrease observed at a concentration of 3 nM and the maximum effect at 100 nM. In contrast, the control oligos, 2OMe-EGFP, had no effect on miR-21 level, even at the highest concentration (100 nM). ? 25. In subsequent experiments, we determined the effect of miR-21 inhibitor on VSMC proliferation by using two different methods: cell counting and BrdU incorporation assay. Consistent with the decrease of miR-21, miR-21 inhibitor