犬UC-MSC-Exo来源miRNA表达分析及其cfa-miR-34a/-143对血管内皮细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2020.03.004

Abstract

Abstract: This study was aimed to isolate and identify exosomes secreted by canine umbilical cord mesenchymal stem cells(UC-MSC-Exos) and study the expression of miRNAs derived from UC-MSC-Exo and its effect on proliferation of vascular endothelial cells (VECs).Exosomes were isolated from the culture supernatant of canine umbilical cord mesenchymal stem cells by ultra-high speed centrifugation and identified by Western blotting,transmission electron microscopy and nanoparticle tracking analysis (NTA).Two miRNAs (cfa-miR-34a/-143) were screened by sequencing miRNAs in canine UC-MSC-Exo,and the corresponding mimic and inhibitor were synthesized and then transfected into canine VECs.The mimic and inhibitor transfections and their transfection efficiency were detected by fluorescence microscopy and Real-time PCR,and its effect on the proliferation of canine VECs was detected by CCK-8 method.Finally,the target genes were predicted.Canine UC-MSC-Exo expressed specific exosomal surface proteins CD9,CD63 and CD81 with a particle size of 100-200 nm and a typical cup shape.The results of immunofluorescence assay showed that both miRNA mimic and inhibitor were transfected into cells and accumulated in the cytoplasm;Real-time PCR results showed that the expression of cfa-miR-34a in cells increased about 400-fold after transfection of cfa-miR-34a mimic,while cfa-miR-143 increased about 78-fold;Whereas transfection of cfa-miR-34a inhibitor,the expression of cfa-miR-34a in cells decreased by 77%,while that of cfa-miR-143 decreased by 83%,and the cfa-miR-34a and cfa-miR-143 could extremely significantly promote the proliferation of VECs in vitro by CCK-8.The target genes of cfa-miR-34a predicted that there were 195 conserved target sites,and 69 cross-target genes were shared with miRDB prediction results,while cfa-miR-143 had 447 conserved target genes,128 cross-target genes were shared with miRDB prediction results.Canine UC-MSC-Exo was successfully isolated,and it was confirmed that the target miRNAs cfa-miR-34a and cfa-miR-143 could extremely significantly promote VECs proliferation in vitro.

Key words: umbilical cord mesenchymal stem cells (UC-MSCs); exosomes; miRNA; vascular endothelial cells; proliferation

CLC Number:

S829.2

LUO Huina, LUO Dongzhang, FAN Quanbao, WANG Bingyun, ZHAN Xiaoshu, CHEN Shengfeng, CHEN Zhisheng, BAI Yinshan, LIU Canying, JI Huiqin. Expression of miRNA from Canine UC-MSC-Exo and the Effect of cfa-miR-34a/-143 on Proliferation of Vascular Endothelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 676-685.

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Expression of miRNA from Canine UC-MSC-Exo and the Effect of cfa-miR-34a/-143 on Proliferation of Vascular Endothelial Cells

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