LncRNA FAM95B1调控胰岛素样生长因子2的表达对宫腔粘连发生的影响

The effect of lncRNA FAM95B1 on the pathogenesis of intrauterine adhesion by regulating insulin-like growth factor 2

目的探究lncRNA FAM95B1及蛋白编码基因胰岛素样生长因子2(insulin-like growth factor 2,IGF2)对宫腔粘连发生的作用,为宫腔粘连的防治提供新的分子靶点。方法在宫腔粘连组织中采用反转录实时定量聚合酶链反应(reverse transcription-quantitative polymerase chain reaction,RT-qPCR)方法检测lncRNA FAM95B1的mRNA表达水平,采用Western blotting方法检测IGF2及纤维化标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、I型胶原蛋白(collagen type I alpha 1 chain protein,COL1A1)的蛋白表达水平。在经过转化生长因子-β1(transforming growth factor-β1,TGF-β1)处理的子宫内膜基质细胞(endometrial stromal cells,ESCs)中,采用RT-qPCR方法检测lncRNA FAM95B1、IGF2、纤维化标志物(α-SMA、COL1A1、Snail)及上皮标志蛋白E-cadherin的mRNA表达水平。在ESCs中,分别下调lncRNA FAM95B1和IGF2基因,验证二者的调控关系;免疫共沉淀实验检测lncRNA FAM95B1与IGF2的结合关系。然后,si-FAM95B1转染入ESCs中并经过TGF-β1处理,进一步探究lncRNA FAM95B1及IGF2在ESCs向成纤维细胞分化的上皮-间质转化(epithelial-to-mesenchymal transition,EMT)过程中的作用。结果宫腔粘连组织中lncRNA FAM95B1、IGF2及纤维化标志物α-SMA、COL1A1的表达水平上调;经过TGF-β1处理的ESCs中lncRNA FAM95B1、IGF2、纤维化标志物(α-SMA、COL1A1、Snail)的表达水平上调,上皮标志蛋白E-cadherin的表达水平下调。IGF2随lncRNA FAM95B1的变化而变化,而下调IGF2后,lncRNA FAM95B1的表达无明显变化,lncRNA FAM95B1正向调节IGF2;lncRNA FAM95B1与IGF2在TGF-β1处理的ESCs中结合。si-FAM95B1在ESCs向成纤维细胞分化的EMT过程中抑制IGF2的表达,发挥抗纤维化作用。结论si-FAM95B1可通过下调IGF2的表达抑制宫腔粘连的发生,lncRNA FAM95B1/IGF2轴可能为宫腔粘连的靶向治疗提供新的分子靶点。

Objective To investigate the effect of long non-coding RNA(lncRNA)family with sequence similarity 95 member B1(FAM95B1)and insulin-like growth factor 2(IGF2)on the pathogenesis of intrauterine adhesion.Methods Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was used to determine the mRNA expression levels of lncRNA FAM95B1,Western blotting was used to determine the protein expression levels of IGF2,α-smooth muscle actin(α-SMA)and collagen type I alpha 1 chain protein(COL1A1)in samples of intrauterine adhesion(IUA)tissue.RT-qPCR was used to determine the mRNA expression levels of lncRNA FAM95B1,IGF2,fibrotic markers(α-SMA,COL1A1,Snail)and epithelial marker E-cadherin in endometrial stromal cells(ESCs)that had been treated with transforming growth factor-β1(TGF-β1).Then,lncRNA FAM95B1 and IGF2 genes were down-regulated respectively to verify the regulatory relationship between lncRNA FAM95B1 and IGF2;Co-immunoprecipitation assay was performed to measure the binding relationship between lncRNA FAM95B1 and IGF2.Next,ESCs were transfected with si-FAM95B1 and then were treated with TGF-β1 to investigate the effect of lncRNA FAM95B1 and IGF2 on the transdifferentiation of ESCs into myofibroblasts.Results The expression levels of lncRNA FAM95B1,IGF2,α-SMA and COL1A1 was upregulated in IUA tissues.The mRNA expression levels of lncRNA FAM95B1,IGF2,α-SMA,COL1A1 and Snail were upregulated and the mRNA level of E-cadherin was downregulated in ESCs that had been treated with TGF-β1.IGF2 was positively regulated by lncRNA FAM95B1.LncRNA FAM95B1 and IGF2 were directly bound in TGF-β1-treated ESCs.During the epithelial-to-mesenchymal transition(EMT)of ESCs transdifferentiating into myofibroblasts,si-FAM95B1 exerted antifibrotic effect by down-regulating IGF2.Conclusions si-FAM95B1 inhibits the pathogenesis of IUA by down-regulating IGF2.LncRNA FAM95B1/IGF2 axis may provide novel molecular target for the prevention and treatment of IUA.