CDH17调节TGF-β自分泌影响胃癌细胞侵袭作用的研究

Study on role of CDH17 regulating TGF-β autocrine for affecting invasion of gastric cancer cells

目的探讨转化生长因子(TGF)-β自分泌在钙黏蛋白17(CDH17)调节胃癌细胞侵袭性中的作用及可能机制。方法构建siRNA-CDH17并转染入胃癌细胞系MKN-45,沉默其表达。通过免疫荧光染色、Western blot、ELISA检测CDH17沉默前后细胞TGF-β表达及培养上清液中TGF-β水平变化,观察TGF-β的自分泌情况。同时采用Western blot检测TGF-β/Smad3信号通路活化情况,并通过Transwell侵袭实验观察在给予信号通路抑制剂后,胃癌细胞侵袭力的变化,评价TGF-β自分泌及相关信号通路活化在CDH17调节胃癌细胞侵袭性中的作用。结果转染siRNA-CDH17沉默MKN-45细胞CDH17表达后,细胞TGF-β表达较未转染组明显下降,其培养上清液中TGF-β水平也显著减少[(510±55.0)pg/mL vs.(115±20)pg/mL,P<0.05]。Western blot显示CDH17沉默后Smad3磷酸化水平也明显降低,而给予TGF-β/Smad3信号通路抑制剂SIS3(10μmol/L)也可抑制CDH17高表达时的Smad3磷酸化水平。同时,沉默CDH17及抑制Smad3磷酸化均可明显降低MKN-45细胞侵袭性(P<0.05)。结论 CDH17可能通过促进自分泌TGF-β活化TGF-β/Smad3信号通路参与调节胃癌细胞侵袭性。

Objective To explore the role and possible mechanism of transforming growth factor（TGF）-βautocrine in CDH17 regulating invasion of gastric cancer cells.Methods Construction and transfection of siRNA-CDH17 into MKN-45 gastric cancer cell line to silence the expression of CDH17.Expression of TGF-βand concentrations of TGF-βin supernatants were detected before and after CDH17 silence by immunofluorescence,immunoblotting and ELISA.The autocrine situation of TGF-βwas observed.Meanwhile,the activation of TGF-β/Smad3 signaling pathway was also detected by immunoblot.After giving signaling pathway inhibitor,the changes of invasion ability of MKN-45 cells were observed by Transwell invasion experiment.The role of TGF-βautocrine and related signaling pathway activation in CDH17-regulated invasion of gastric cancer cells was evaluated.Results After transfecting siRNA-CDH17 for silencing CDH17 expression in MKN-45 cells,the expression of TGF-βwas significantly decreased compared with non-transfection group,its concentration in supernatants was also significantly reduced[（510±55）pg/mL vs.（115±20）pg/mL,P〈0.01].The immunoblots revealed that phosphorylation level of Smad3 after CDH17silence was also significantly diminished.However,giving the TGF-β/Smad3 signaling inhibitor SIS3（10μmol/L）could also suppress the phosphorylation level of Smad3 when CDH17was highly expressed,meanwhile silencing CDH17 and inhibiting Smad3 phosphorylation could significantly decrease the invasion of MKN-45 gastric cancer cells（P〈0.05）.Conclusion CDH17 could participate in the invasion of gastric cancer cells by promoting TGF-βautocrine to activate TGF-β/Smad3 signaling pathway.