miR-200c Regulates RMP7-mediated Increases of Blood-tumor Barrier Permeability by Targeting RhoA
10.12007/j.issn.0258-4646.2016.12.001
- VernacularTitle: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
- Author:
Teng MA
;
Libo LIU
;
Yang LIN
;
Jun MA
;
Yixue XUE
- Keywords:
miR-200c;
RhoA;
RMP7;
blood tumor-barrier;
gene expression regulation
- From:
Journal of China Medical University
2016;45(12):1057-1062
- CountryChina
- Language:Chinese
-
Abstract:
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.
