Effects And Mechanisms Of MicroRNA On Rapamycin-induced Suppression Of Endothelial Proliferation And Migration

Rapamycin (sirolimus) is a macrocyclic lactone antibiotic. Because it can suppress the proliferation and migration of vascular smooth muscle cells (VSMCs), rapamycin is widely used in drug eluting stents (DES) to prevent in-stent restenosis (ISR) after percutaneous coronary intervention. However, recent clinical trials revealed that patients who received rapamycin-eluting stents implantation presented with higher rates of late stent thrombosis (LST), compared to patients who received bare-metal stents. This is due to the ability of rapamycin to impair the proliferation and migration of vascular endothelial cells, which leads to a delayed re-endothelialization of stent struts. This disadvantage has limited the use of rapamycin in DES.The mechanism of the inhibitory effects of rapamycin on endothelial proliferation and migration has been previously discussed. The mammalian target of rapamycin complex1(mTORC1) is comprised of GβL, mammalian target of rapamycin (mTOR) and Raptor. mTORC2is comprised similarly of G β L and mTOR, though Rictor is substituted for Raptor. Once bound to FKBP12, rapamycin can directly inhibit mTORC1signalling, which leads to a total arrest of transcription, ribosome biogenesis, translation initiation, and stop cell cycle at the G0/G1phase in endothelial cells. mTORC2was once thought to be insensitive to rapamycin and to control the actin cytoskeleton; however, later studies revealed that rapamycin could also inhibit mTORC2signalling and that long-term rapamycin treatment (>24h) could impair the mobility of endothelial cells through the mTORC2-p27(kip) pathway. Although all these studies seemed to provide answers to the question of how rapamycin mediated the prevention of endothelial cell proliferation or migration, more recently, new and deeper insights into these mechanisms have been indicated.MicroRNAs (miRNAs, miRs) are a class of endogenous small non-coding RNAs that are approximately19-22bases in length. MiRs can regulate gene expression widely at the post-transcriptional level, mostly by targeting the3’-UTR of mRNAs. miRs have been reported to play large roles in several functions of endothelial cells, including proliferation, migration, angiogenesis, senescence and survival. Recent studies have revealed direct connections between miRs and mTORC. Uesugi et al. reported that miR-218targets the mTORC component Rictor in oral cancer cells. Chen et al. found that miR-101mediates the suppressive effects of laminar shear stress on mTOR expression in vascular endothelial cells. These reports of feedback between miRs and the mTOR complex indicate that miRs may be involved in rapamycin-induced regulation in endothelial cells.Based on these considerations, we hypothesized that miRs may mediate the suppression effect of rapamycin on endothelial proliferation and migration. The research on this topic will not only give new explanations of rapamycin-induced inhibitory effect on angiogenesis, but also provide new insight of methods to solve LST problem. In the present study, we first observed the effect of rapamycin on endothelial proliferation and migration, found out the miR whose expression was deeply affected by rapamycin treatment. Then, we tested the effect of this miR on rapamycin induced suppression of endothelial proliferation and migration, and investigated the mechanism as well. Now we will explain our study by two parts.Part1:Effects of rapamycin on human umbilical vein endothelial proliferation and migration, and the expressive difference of microRNAs under rapamycin treatmentObjective:To investigate the effect of rapamycin on human umbilical vein endothelial proliferation and migration, find out the most effective concentration and treated time of rapamycin on endothelial growth and mobility, and investigate the expressive difference of microRNA under this rapamycin treatment.Methods:HUVEC cell line was purchased and cultured. Various concentration (1-1000ng/ml) of rapamycin were added and treated for different durations (12-72h). The proliferation ability of HUVECs was then detected by using CCK-8test and EDU proliferation assay. Wound healing test as well as Boyden chamber assay were used for detecting the mobility of HUVECs. By searching Pubmed with Mesh words "microRNA" and "endothelial cells", we chose several miRs, evaluating the expression levels of these miRs by qRT-PCR array. miR whose level was changed greatestly was further analyzed and checked by qRT-PCR.Result:Rapamycin treatment could significantly inhibit the proliferation of HUVECs in a time-dose dependent manner, and had a peak at100ng/ml and24h, as revealed by CCK-8test and EDU proliferation assay. Wound healing test as well as Boyden chamber assay revealed that24h rapamycin treatment could suppress the migration of HUVECs in a dose dependent manner. qRT-PCR array showed that among all the tested miRs, only the level of miR-21was significantly upregulated by rapamycin treatment (100ng/ml,24h). Further test confirmed that the expression of miR-21was augmentated by rapamycin treatment in a time-dose depedent manner with a peak at100ng/ml and24h.Conclusion:Rapamycin treatment can inhibit the proliferation and migration of HUVEC, and can upregulate the level of miR-21in a time-dose dependent manner. Part2:Effects and mechanisms of miR-21on rapamycin-induced suppression of endothelial proliferation and migrationObjective:To investigate the Effects and mechanisms of miR-21on rapamycin-induced suppression of endothelial proliferation and migration.Methods:By using miR-21mimic and inhibitor, we upregulation/downregulation the level of miR-21in HUVECs. CCK-8test, EDU proliferation assay, wound healing test and Boyden chamber assay were used for detecting the proliferation and migration of HUVECs respectively. Using bioinformatic method, we found out the putative target gene of miR-21, and confirm this target gene by using qRT-PCR, western blot and luciferase reporter assay. Finally, we knock-down the expression of Raptor or Rictor with siRNA technology, and investigated the level of miR-21after that.Result:Downregulation of miR-21by its inhibitor totally abolished the negative effects of rapamycin on HUVEC proliferation and migration. RhoB was confirmed as a direct target gene of miR-21. Knockdown of Raptor by siRNA mimicked the effects of rapamycin on miR-21expression.Conclusion:Downregulation of miR-21can abolished the negative effects of rapamycin on HUVEC proliferation and migration. RhoB is a direct target gene of miR-21. The upregulation of miR-21induced by rapamycin may be a result of Raptor inhibition.