Altered Microrna Expression Profile In Human Umbilical Vein Endothelial Cells Inhibited By Curcumin

Objective:(1) To explore how to get and identify human umbilical vein endothelial cell (HUVECs) in vitro. To investigate the effect of curcumin on proliferation of human umbilical vein endothelial cell (HUVECs).(2) To investigate the effect of curcumin on the differential expression of microRNAs of HUVECs and to establish the relationship between HUVEC proliferation and the expression of microRNAs;Methods:(1) 0.25% Trypsin and 0.02%EDTA perfusion method was used to isolate HUVEC from fresh umbilical cord, and the cells were cultured and amplified.The composition of culture medium was simplified by not adding vascular endothelial cell growth factor and heparin.HUVECs were identified by morphological observation and immunofluorescencal method. Then HUVECs proliferation were examined at different curcumin concentrations(0,20,40,80,160 umol/l)by different time course (0,24,48,72,96h) by method of MTT;(2)RNA was extracted from 1 control samples of HUVECs and 1 curcumin (40umol/l) treated sample,and small RNA was isolated.Then we detected microRNA profiles in these samples by microRNA microarray.Meanwhile, examined the expression of microRNAs were examined in these samples by fluorescence based real-time quantitative PCR.Those microRNAs that were identified as differentially expressed by both microRNA microarray and RT-PCR were considered as significant microRNA; Possible target genes of these microRNAs were Predieted through bioinformaties analysis.Results:(1) The endothelial cells spread on the bottom of the dishes in 2h,then coalesced and grew to from confluent monolayers of polygonal cells within 24h.The cultured cells were identified as HUVECs. the effect of curcumin on proliferation of human umbilical vein endothelial cell (HUVECs) is in a time-dependent manner and a dose-dependent manner; (2) After treated with curcumin at a concentration of 40μmol/l for 24h,there are 30 significant differential expressions of microRNAs,11 microRNAs were up-regulated,19 microRNAs were down-regulated;Among them mi-1275,mi-3127 and mi-1246 were significantly up-regulated,Bioinformatie analysis showed that thesemieroRNAs with significantly different expression maybe closely related to many genes regulating conceal neovascularition,sueh as miR-1275 targeting to PEDFb.Conclusion:(1) Trypsin perfusion is a simple and effective method for collection of human umbilical vein endothelial cells. Cells harvest with protocol can be used as models on research of vascular endothelial in vitro.(2)Our results find that the expression of mi-1275,mi-3127,mi-1246 is up-regulated in HUVECs after treated with curcumin at a concentration of 40μmol/l for 24h for the first time,and VEGF may be the potential target genes that are involved in mi-1275-mediated protective effects.This findings will provide further qualification of microRNAs'in neovascularition.