miR-200c调控RhoA基因表达介导RMP7增加血肿瘤屏障通透性机制的研究

摘要

Objective To study the mechanism of miR⁃200c in regulating RMP7⁃induced increases of blood⁃tumor barrier(BTB)permeability by targeting Ras homolog gene family member A(RhoA). Methods Endogenous expression of miR⁃200c was detected by real⁃time PCR in hu⁃man cerebral microvascular endothelial cell line hCMEC/D3(ECs)after RMP7 treatment. miR⁃200c mimic and miR⁃200c inhibitor were transfect⁃ed into GECs(ECs with U87 glioma cells co⁃culturing),respectively. Transfection efficiency of miR⁃200c mimic and miR⁃200c inhibitor were de⁃termined by real⁃time PCR. HRP flux and TEER assays revealed BTB permeability. The protein expression level of RhoA was assessed by West⁃ern blotting. The distribution of RhoA was assessed by immunofluorescence microscopy. RhoA luciferase assays were performed using the Dual⁃Lucif⁃erase reporter assay system. Results RMP7 significantly induced a decrease in miR⁃200c expression in GECs of BTB. miR⁃200c mimic and miR⁃200c inhibitor were successfully transfected into GECs. Overexpression of miR⁃200c inhibited endothelial leakage and restored normal transendo⁃thelial electric resistance values. Simultaneously ,overexpression of miR⁃200c significantly reduced the protein expression level of RhoA. In addi⁃tion,immunofluorescence analysis revealed that the distribution of RhoA in the cytoplasm and nuclei of GECs were decreased in miR⁃200c mimic group. RhoA was one of the direct targets of miR⁃200c with the specific binding site being located at the seed sequence. The results of miR⁃200c si⁃lencing were opposite to that of the miR⁃200c overexpression group. Conclusion miRNA⁃200c regulated RMP7⁃induced increases in BTB perme⁃ability by targeting RhoA.%目的：研究miR⁃200c调控Ras基因家族成员A（RhoA）表达介导RMP7增加血肿瘤屏障（BTB）通透性的机制。方法应用real⁃time PCR检测RMP7作用BTB后人脑微血管内皮细（ECs）miR⁃200c的表达；应用miR⁃200c模拟物和miR⁃200c抑制物分别转染GECs（ECs和U87脑胶质瘤细胞共培养的细胞），测量跨内皮阻抗值（TEER）、辣根过氧化物酶（HRP）渗漏量，分析BTB通透性；应用Western blotting检测RhoA的表达；应用免疫荧光方法观察GECs中RhoA表达和分布；应用双荧光素酶报告基因检测miR⁃200c转录后水平调控RhoA机制。结果 RMP7作用GECs后，使GECs内源性miR⁃200c表达显著降低；miR⁃200c模拟物和miR⁃200c抑制物成功转染到GECs中；miR⁃200c模拟物显著抑制RMP7诱导TEER值的降低、HRP的升高；miR⁃200c模拟物显著减少RhoA的表达，促使RhoA在GECs细胞质和细胞核分布减少；miR⁃200c模拟物转录后水平负性调控RhoA基因表达。miR⁃200c抑制物与miR⁃200c模拟物实验结果相反。结论 miR⁃200c转录后水平负性调控RhoA表达，介导RMP7增加血肿瘤屏障通透性机制。