辐射诱导线粒体功能障碍激活转化生长因子β1通路促进胰腺癌细胞上皮间质转化

Mitochondrial dysfunction-induced expression of TGF-β1 pathway promoting epithelial-mesenchymal transition in pancreatic cancer cells after X-ray exposure

目的研究X射线照射后胰腺癌细胞上皮间质转化的发生特点,探究线粒体功能障碍及其诱导的转化生长因子β1(TGF-β1)表达增加在上皮间质转化中的作用。方法采用6 MV X射线多次(2 Gy×20次或4 Gy×10次)照射胰腺癌细胞株PATU1988 t,利用transwell小室检测细胞迁移性,采用实时荧光定量方法检测上皮间质转化(EMT)相关因子E-cadherin、Vimentin、N-cadherin和MMPs家族(MMP2、MMP9)及线粒体复合体I的关键亚基及TGF-β1的转录水平变化。应用线粒体氧化磷酸化解偶联剂碳酰氰基-对-氯苯腙(CCCP)破坏线粒体膜电位,检测复合体亚基、TGF-β1含量及EMT相关分子的变化。应用小干扰RNA抑制TGF-β1的表达后,检测上皮间质转化及迁移变化。结果多次照射后,存活肿瘤细胞迁移数增加(t=21.90、35.64,P<0.05),上皮间质转化增强,表现为上皮间质转化分子E-cadherin表达下降(t=8.37、6.77,P<0.05),N-cadherin(t=4.42、4.77,P<0.05)、Vimentin(t=4.57、3.02,P<0.05)、MMP2(t=7.27、26.08,P<0.05)和MMP9(t=13.26、7.29,P<0.05)表达均增加,TGF-β1(t=90.49、35.17,P<0.05)的含量也增加。沉默TGF-β1后细胞上皮间质转化及迁移性下降(t=38.66、11.54,P<0.05)。细胞发生线粒体功能障碍,具体表现为线粒体膜电位(t=6.94、29.71,P<0.05)和复合体相关亚基的表达量下降;加入CCCP破坏线粒体膜电位后(t=16.51,P<0.05),复合体亚基含量下降,TGF-β1的表达量增加(t=47.93,P<0.05),上皮间质转化增强。结论辐射破坏了线粒体功能,进而激活了TGF-β1的表达,诱导胰腺癌细胞发生上皮间质转化,迁移性增强。

Objective To investigate epithelial-mesenchymal transition and to explore the effects of mitochondrial dysfunction and increased expression of TGF-β1 pathway on epithelial-mesenchymal transition(EMT)in pancreatic adenocarcinoma after X-ray irradiation.Methods Split-dose irradiations of total 40 Gy(2 Gy×20 and 4 Gy×10)of 6 MV X-rays were performed on PATU1988 t cells.The migration of the cells were examined through transwell filter chambers.Real-time PCR was adopted to detect the expression of EMT-related factors E-cadherin,Vimentin,N-cadherin,and MMPs(MMP2 and MMP9),critical subunits of mitochondrial complex I,and TGF-β1.The expression of EMT-related factors and content of TGF-β1 was detected after carbonylcyanide-m-chlorophenylhydrazone(CCCP)treatment.Meanwhile,the migration potential of pancreatic cells was detected after small interfering RNA(siRNA)knockdown of the expression of TGF-β1.Results After irradiation,the migration capacities of the cancer cells increased(t=21.90,35.64,P<0.05).The expression of N-cadherin(t=4.42,4.77,P<0.05),Vimentin(t=4.57,3.02,P<0.05),MMP2(t=7.27,26.08,P<0.05),and MMP9(t=13.26,7.29,P<0.05)all increased,while the expression of E-cadherin deceased(t=8.37,6.77,P<0.05).The expression of TGF-β1(t=90.49,35.17,P<0.05)increased.The expression of TGF-β1 decreased with small interfering RNA,which paralleled the inhibition of the epithelial-mesenchymal transition and migration(t=38.66,11.54,P<0.05).Mitochondrial dysfunction was reflected by the decline in the membrane potential(t=6.94,29.71,P<0.05)and complex-related subunits.The expression of TGF-β1(t=47.93,P<0.05)and EMT-related factors further increased after mitochondrial function was destroyed(t=16.51,P<0.05).Conclusions Radiation-induced mitochondrial dysfunction can increase the expression of TGF-β1,which promotes epithelial-mesenchymal transition,and result in the migration of pancreatic cancer cell line.