靶向siRNA阻断Wnt/β-catenin信号通路对子宫内膜异位症患者在位子宫内膜基质细胞VEGF、MMP-9表达的影响

Gene Silencing of Wnt/β-catenin Signaling Pathway by siRNA Inhibits VEGF and MMP-9 Expression in Endometrial Stromal Cells in Patients with Endometriosis

目的通过观察人子宫内膜异位症在位子宫内膜基质细胞β-catenin的表达,及利用siRNA靶向沉默β-catenin基因以阻断Wnt/β-catenin信号通路来观察其下游靶基因血管内皮生长因子(vascular endothelial growth factor,VEGF)和基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)表达的变化,探讨Wnt/β-catenin信号通路在子宫内膜异位症发生机制中的作用。方法采用随机、对照设计,将体外分离、培养的24例子宫内膜异位症患者在位子宫内膜基质细胞分成3组(n=8):空白组、阴性组(以含阴性荧光siRNA 100pmol和转染试剂5μg的混合物转染)以及siRNA干扰组(以含β-catenin siRNA 100pmol和转染试剂5μg的混合物转染),并以10例非子宫内膜异位症患者子宫内膜基质细胞作为正常组。利用Real-time PCR和Western blot方法分别检测子宫内膜异位症在位子宫内膜空白组和非子宫内膜异位症正常组β-catenin的表达。转染24h后,用以上方法分别检测子宫内膜异位症空白组、阴性组和siRNA干扰组β-catenin、VEGF以及MMP-9的表达。结果子宫内膜异位症空白组β-catenin mRNA和蛋白的表达明显高于非子宫内膜异位症正常组(均P<0.05)。子宫内膜异位症siRNA干扰组β-catenin、VEGF以及MMP-9mRNA和蛋白的表达明显低于空白组和阴性组(均P<0.05),而空白组与阴性组之间差异无统计学意义(均P>0.05)。结论子宫内膜异位症在位子宫内膜基质细胞β-catenin的表达明显高于非子宫内膜异位症患者,阻断Wnt/β-catenin信号通路后,其下游靶基因VEGF、MMP-9的表达明显受到抑制。因此,Wnt/β-catenin信号通路可能在子宫内膜异位症的发生机制中起重要作用。

Objective To explore the role of Wnt/β-catenin signal pathway in the pathogenesis of endometriosis by detecting the expression ofβ-catenin in endometrial stromal cells of the eutopic endometrium of women with endometriosis,and the expression of its down-stream target genes vascular endothelial growth factor（VEGF）and matrix metalloproteinases-9（MMP-9）after silencingβ-catenin gene.Methods Endometrial stromal cells were isolated from the eutopic endometrium of 24 women with endometrisis and cultured in vitro.They were divided into 3groups at random：blank group（n=8）,negative group（transfection with 100 pmol negative fluorescence-labeled siRNA and 5μg transfection agent,n=8）and siRNA interference group（transfection with 100pmolβ-catenin-containing siRNA and 5μg transfection agent,n=8）.Endometrial stromal cells from 10 women without endometriosis served as controls.Real-time PCR and Western blot were used to detect the expression levels ofβ-catenin in endometrial stromal cells from women with or without endometrisis,and the expression levels of VEGF and MMP-924 hafter transfection in the three groups.Results The expression levels ofβ-catenin were significantly higher in endometrisis group than in non-endometrisis group（P〈0.05）.The expression levels ofβ-catenin,VEGF and MMP-9were significantly lower in siRNA interference group than in blank and negative groups（P〈0.05）.There were no differences between blank and negative groups（P〉0.05）.Conclusion β-catenin expression is significantly increased in endometrial stromal cells from the eutopic endometrium of women with endometriosis as compared with those without endometriosis.After blockage of Wnt/β-catenin signal pathway,the expression of VEGF and MMP-9,down-stream target genes ofβ-catenin,is suppressed dramatically.This studysuggests that Wnt/β-catenin signal pathway may play an important role in the pathogenesis of endometriosis.