Effects And Mechanisms Of Let-7Family On Ox-LDL-induced Human Endothelial Cell Injuries

Atherosclerosis is a chronic multifactorial disease which has become a major cause for deaths worldwide. In the pathogenesis of atherosclerosis, many cells and factors are participated, including endothelial cell, vascular smooth muscle cell, leukocyte etc. There are at least three hypothesises for the pathogenesis of atherosclerosis:lipid infiltration theory, endothelial response to injury theory, vascular smooth muscle cell proliferation and migration theory. Among them, the endothelial response to injury theory is the most accepted theory. Endothelium injury is thought to be the initial step. The risk factors for endothelium injury include ox-LDL, AngⅡ, AGEs, and sheer stress, etc, among which the ox-LDL is now considered to play a fundamental role. Ox-LDL induces endothelial cell apoptosis, changes the secretory activities of endothelium and reduces the antioxidant capability.All these effects of ox-LDL are tightly related to a novel receptor for ox-LDL, lectin-like low density lipoprotein receptor-1(LOX-1). LOX-1was first discovered as a special receptor for oxLDL in endothelial cells. However, further studies found it also expressed in smooth muscle cells, macrophage cells and cardiac cells. The ligand for LOX-1including modifiedlipoproteins (oxLDL, acetyl LDL, hypochlorite-modified HDL, etc), proteins (CRP, fibronectin, HSP), cells (activated platelets, leukocytes, apoptotic cells, etc), phospholipids and polyanions. The activation of LOX-1stimulates the inflammation, apoptosis and dysfunction of endothelial cells. However the regulation factors for LOX-1are still unclear.MicroRNAs are small, endogenous, non-coding RNAs that regulate the gene expression in post-transcriptional level. They participate in the physiological and pathological process such as biological development, cell proliferation, organ formation. It was reported that about one third of human genes were regulated by microRNA. Therefore, we hypothesised that microRNA might regulate the expression of LOX-1. On the other hand, the generation process of microRNA is similar to other genes. The encoding genes for microRNA were first transcripted and modified to mature functional microRNAs. However, there are only few studies focus on the transcriptional regulation of microRNA.Studies demonstrate a tight relationship between microRNA and transcriptional factors. About half of the microRNA achieved their function via transcriptional factors. On the other hand, in the stage of microRNA transcription from genome, transcriptional factors are working as regulators for microRNA expression.In endothelial cells, oxLDL upregulates the expression of LOX-1, and the LOX-1eventually activates NF-κB. Whether could NF-κB regulate the generation of microRNA, and exist a feedback loop is still unclear.Thus we hypothesize that in oxLDL-induced endothelial cell injuries, the microRNA participated in the upregulation of LOX-1, and influence the function of endothelial cells via LOX-1/p38MAPK/NF-κB signaling. On the other hand, NF-κB regulate the generation of microRNA, and a signal feedback loop exist among microRNA, LOX-1and NF-κB.Thus the purpose of the present study is to screening the microRNAs targeting LOX-1, to demonstrate the microRNA/LOX-1/ROS/p38MAPK/NF-KB/microRNA feedback loop, to found out the function of these microRNAs on oxLDL-induced endothelial cell injuries. The results will discover the relationship and signaling pathway of microRNA in the pathogenesis of atherosclerosis, and will provide new insights and therapeutic target for atherosclerosis.Part I Screen the microRNAs targeting LOX-1Aim:To screening the microRNAs targeting LOX-1Methods:The website of mirbase, targetscan, pictar were used to predict the microRNAs targeting LOX-13’-UTR; Real-time quantitative PCR was used to determine the changes of predicted microRNAs in oxLDL (150μg/ml) treated endothelial cells; Construct the LOX-13’-UTR reporter plasmids, and the effects of selected microRNAs on these reporter plasmids were measured by dual luciferase reporter assays; The expression of let-7members in HUVEC were measured by real-time qPCR; The effects of let-7a、let-7b、let-7c mimics on endogenous LOX-1mRNA and protein expression in HUVEC were detected by real-time qPCR and Western-blot methods.Results:Bioinformatic analysis predicted several microRNAs targeting LOX-13’-UTR, and among them, miR-590、miR-18b、let-7family were predicted by more than two website. When treated with different concentration of oxLDL (50,100,150, and200μg/ml) for24hours, the cell viability of HUVEC were declined to98.3%,73.1%,55.1%and44.3%of control. Moreover, the expression of miR-590, miR-18b, let-7family in HUVEC were inhibited after treatment with150μg/ml of oxLDL for24hours. And the expression of miR-590and let-7b were down-regulated to the24.4%and12.1%of control. Dual luciferase assays found let-7a, let-7b, let-7c, let-7g and miR-98inhibited the LOX-13’-UTR reporter gene expression, while miR-590didn’t. In normal HUVEC cell line, the expression of let-7a, let-7b and let-7c are more abundant than other let-7members. When infected with75nM let-7a, let-7b and let-7c mimics, the expression of LOX-1mRNA and protein were inhibited in HUVEC.Conclusion:1. Let-7a, let-7b, let-7c, let-7g and miR-99a are miRNAs targeting LOX-1;2. In HUVECs, the let-7a, let-7b and let-7c expression are higher than other members of let-7family;3. Let-7a, let-7b and let-7c downregulate the expression of LOX-1in HUVEC. Part II The feedback loop between let-7, LOX-1and NF-κBAim:To investigate the regulatory mechanisms between let-7and NF-kB, and to verify the let-7c/LOX-1/NF-κB negative feedback loop.Methods:Transient transfect let-7a, let-7b and let-7c to HUVECs, investigate the p38MAPK, p-p38MAPK, NF-κB p65protein expression by Western-blot analysis. After treatment of HUVECs by siLOX-1, PDTC (NF-κB inhibitor), SB203580(p38MAPK inhibitor), the alteration of let-7a, let-7b and let-7c expression were measured by real-time PCR. Different length (-1.5kb,-2.0kb,-2.0kb-mut)of miR-99a/let-7c promoter plasmids, and co-transfected these promoter plasmids with NF-kB (p65and/or p50) subunits separately, dural luciferase reporter analysis were used to explore the directly regulatory effects of NF-kB on miR-99a/let-7c promoter. Electrophoretic-mobility-shift assays (EMSA) were used to confirm the specific binding of NF-κB on-1643to-1652sites of miR-99a/let-7c promoter. Further studies transfected NF-κB (p65and/or p50) into HUVECs, and measured the precursor and mature miR-99a and let-7c alteration by real-time qPCR to validate the effects of NF-κB on miR-99a/let-7c promoter.Results:let-7a, let-7b, let-7c overexpression inhibited the phosphorylation of p38MAPK, the p-p38MAPK/p38MAPK were down-regulated to the0.65,0.58, and0.62folds of NC group respectively; The nuclear translocation of NF-κB (p65) were inhibited, it is48.5%for NC group, but declined to34.8%,32.5%, and20.9%after let-7a, let-7b and let-7c overexpression. siLOX-1inhibited the LOX-1mRNA and protein expression dramatically. After treatment with siLOX-1, PDTC and SB203580for24hours in HUVEC, the let-7a, let-7b and let-7c expression decreased significantly. Dual luciferase reporter analysis indicated that NF-kB p65and/or p50promote the-2.0kb reporter gene expression. However, the promote effects were much less for-1643～-1652mutant-2.0kb reporter gene and-1.5kb reporter gene. The binding of NF-κB on-1643to-1652sites of miR-99a/let-7c promoter were further confirmed by EMSA. NF-kB p65and/or p50over-expression also promote the endogenous pre-miR-99a, pre-let-7c, miR-99a, let-7c expression.Conclusion:NF-κB promotes the expression of let-7c directly by binding to-1702bp of let-7c promoter; A negative feedback loop exists between LOX-1, NF-kB and let-7c.Part Ⅲ⑧The effects and mechanisms of let-7a/b/c on oxLDL-induced human endothelial cell injuriesAim:To investigate the effects and mechanisms of let-7a, let-7b and let-7c on oxLD-induced endothelial cell injuries.Methods:The real-time quantatitive PCR was used to detect the expression of let-7a, let-7b and let-7c in HUVECs after treatment with oxLDL for different concentration and different time. Transfect the HUVEC with let-7a, let-7b and let-7c mimics, and measured the cell viability by MTS method, determined the LDH and NO secretion by assay kits. The morphological changes of apoptosis cells were observed by Hoechst33342staining, the apoptosis rate was detected by flow cytometry. The reactive oxygen species were measured by DCFH-DA. We also tested the LOX-1and eNOS mRNA levels by real-time quantatitive PCR, and tested the LOX-1, eNOS, Bcl-2, caspase-3, p38MAPK, NF-κB protein expression as well as NF-κB nuclear translocation by Western-blot.Results:Treatment of HUVEC with different concentration of oxLDL for24hours, or treatment with150μg/ml of oxLDL for different time decreased the expression of let-7a, let-7b and let-7c in a concentration-or time-dependent manner. Ox-LDL(150μg/ml) significantly decreased cell viability, NO secreation and increased the LDH release, as well as the apoptotic rate (P<0.01). Over-expression of let-7a, let-7b and let-7c resulted in remarkable inhibition of such effects induced by oxLDL Besides, let-7a, let-7b and let-7c over-expression alone promote the production of NO. Moreover, ox-LDL caused the up-regulation of LOX-1, the overproduction of ROS, the phosphorylation of p38MAPK, the dephosphorylation of PKB, the translocation of NF-κB. let-7a, let-7b, let-7c over-expression reduced these effects introduced by ox-LDL.Conclusion:1) let-7a, let-7b, let-7c expression were inhibited by oxLDL in a concentration-and time-dependent manner;2) let-7a, let-7b, let-7c over-expression inhibited the oxLDL-induced endothelial cell apoptosis and secreation dysfunction;3) The protective effects of let-7a, let-7b, and let-7c were associated with LOX-1/ROS/p38MAPK/NF-κB pathway and LOX-1/ROS/PKB/eNOS pathway.