RhoGDI2调控TGF-β1对气道上皮-间质转化的研究

Role of Rho guanosine diphosphate dissociation inhibitor 2 on the airway epithelial mesenchymal transformation via TGF-β1 pathway

目的:探讨鸟嘌呤核苷酸解离抑制因子2(Rho guanosine diphosphate dissociation inhibitor 2,RhoGDI2)在气道上皮-间质转化(epithelial mesenchymal transformation,EMT)中的作用,以及可能的作用机制,有助于揭示气道重塑的分子生物学机制并提供新的治疗靶点。方法:构建RhoGDI2过表达气道上皮细胞(16HBE)模型。Western Blot检测RhoGDI2蛋白的表达。构建成功后,Western Blot检测细胞中E-cadherin、N-cadherin、Snail及转化生长因子β1(transforming growth factor-β1,TGF-β1)的表达;同时收集细胞培养上清液,双抗体夹心酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测上清液中TGF-β1的表达水平。在RhoGDI2过表达16HBE细胞模型中加入TGF-β1抑制剂,进一步明确TGF-β1在RhoGDI2调控EMT过程中的作用。结果:(1)Western Blot检测16HBE过表达组RhoGDI2表达水平较对照组明显升高(P<0.01)。(2)过表达组16HBE中N-cadherin、Snail表达较对照组明显升高(P<0.05、P<0.01),而E-cadherin表达明显下降(P<0.01);RhoGDI2过表达组细胞中、上清液中TGF-β1的表达水平亦较对照组明显升高(P<0.05、P<0.01)。(3)过表达组中加入TGF-β1抑制剂后,TGF-β1、N-cadherin、Snail表示水平下降,E-cadherin表达水平升高(P<0.05)。结论:RhoGDI2可通过调控TGF-β1的表达促进人气道EMT过程。

Objective: To explore the role of Rho guanosine diphosphate dissociation inhibitor 2(RhoGDI2) on the airway epithelial mesenchymal transformation(EMT) and the potential mechanism of this process, in order to reveal the molecular biological mechanism of airway remodeling and provide new therapeutic target. Methods: The RhoGDI2 overexpression human airway epithelial cells(16 HBE) were first constructed with plasmid. The expression levels of RhoGDI2 protein in each group were detected by Western Blot to determine whether the RhoGDI2-overexpressed 16 HBE cell model was successfully constructed.After successful transfection, the expression levels of key EMT genes(E-cadherin, N-cadherin, Snail) and transforming growth factor-β(TGF-β1) were analyzed by using Western Blot and enzyme-linked immunosorbent assay(ELISA). And then, the TGF-β1 inhibitor was added to RhoGDI2-overexpressed 16 HBE cells to further clarify the relationship between RhoGDI2 and TGF-β1 involved in the airway epithelial mesenchymal transformation. Results:(1)Western Blot analysis indicated the RhoGDI2 expression level in overexpression group was statistically higher than that in the control group(P<0.01).(2)RhoGDI2 promoted the ex-pression of EMT promoting genes N-cadherin, Snail and repressed EMT marker gene E-cadherin in 16 HBE cells(P<0.05, P<0.01, P<0.01). The same RhoGDI2 also promoted the expression of TGF-β1.(3)Inhibition of TGF-β1 signalling prevented the RhoGDI2 driven EMT-like changes above. The differences were statistically significant(P<0.05). Conclusion: RhoGDI2 can promote the airway EMT by regulating the TGF-β1 pathway.