转化生长因子-β1诱导人肺腺癌细胞侵袭迁移的机制

Effect of transforming growth factor-β1 on the invasion and migration of human lung adenocarcinoma cells and epithelial mesenchymal transformation and autophagy

目的探讨转化生长因子-β1（TGF-β1）影响肺腺癌细胞侵袭迁移，以及对上皮-间充质转化（EMT）和自噬的影响。 方法Transwell检测体外TGF-β1对肺腺癌细胞A549和SK-LU-1迁移与侵袭能力的影响；免疫荧光检测TGF-β1引起的A549细胞自噬水平变化；Western blot实验检测在A549细胞中TGF-β1对EMT标志分子及自噬调控基因表达的影响。 结果在A549和SK-LU-1细胞中，sh-TGF-β1干扰效率约为60%，GV358-TGF-β1过表达组过表达效率均在4倍以上；迁移实验结果显示，A549细胞中，随机干扰对照组（shNC组）迁移率为1.02 ± 0.06，TGF-β1过表达组为5.14 ± 0.43（t＝16.440，P＝0.004）；SK-LU-1细胞中，shNC组迁移率为1.04 ± 0.08，TGF-β1过表达组为4.92 ± 0.48（t＝13.810，P＝0.005），差异均有统计学意义；侵袭实验结果显示，A549细胞中，随机干扰对照组（shNC组）侵袭率为0.96±0.07，TGF-β1过表达组为4.28±0.51（t＝11.170，P＝0.007）；SK-LU-1细胞中，shNC组侵袭率为1.03±0.05，TGF-β1过表达组为4.11±0.36（t＝10.410，P＝0.009），差异均有统计学意义；免疫荧光实验结果可见，与对照组比较，TGF-β1过表达组点状分布的自噬小体数目明显增多（t＝22.340，P＝0.000），荧光强度显著增强；Western blot实验结果显示，TGF-β1过表达组E-钙黏蛋白（E-cadherin）的表达量明显减少，波形蛋白（Vimentin）、Twist、Snail的表达显著增加；自噬相关蛋白Beclin1表达上调，磷酸化的雷帕霉素靶蛋白（p-mTOR）表达上调；而TGF-β1干扰组的结果则与过表达组相反。 结论在肺腺癌细胞中，TGF-β1阳性表达诱导的自噬和EMT可能与其增强肺腺癌细胞侵袭迁移能力相关。

ObjectiveTo investigate the effect of transforming growth factor-β1 （TGF-β1） on the invasion and migration of lung adenocarcinoma cells and the epithelial-mesenchymal transition （EMT） and autophagy of epithelial cells. MethodsTranswell assay was used to detect the effect of TGF-β1 on the migration and invasion of lung adenocarcinoma A549 and SK-LU-1 cells. The changes of autophagy in A549 cells induced by TGF-β1 were examined by immunofluorescence. The effect of TGF-β1 on the expression of EMT marker and autophagy regulatory genes in A549 cells was detected by Western blotting. ResultsThe interference efficiency of sh-TGF-β1 was about 60% in A549 and SK-LU-1 cells, and the overexpression efficiency in GV358-TGF-β1 overexpression group was more than 4 times. The migration assay showed that in A549 cells, the migration was 1.02±0.06 in shNC group, and 5.14±0.43 in TGF-β1 overexpression group （t=16.440, P=0.004）; In SK-LU-1 cells, the migration was 1.04±0.08 in shNC group, and 4.92±0.48 in TGF-β1 overexpression group （t=13.810, P=0.005）. The invasion assay showed that in A549 cells, the invasion rate was 0.96±0.07 in the shNC group, and 4.28±0.51 in the TGF-β1 overexpression group （t=11.170, P=0.007）; In SK-LU-1 cells, the invasion rate was 1.03±0.05 in the shNC group, and 4.11±0.36 in the TGF-β1 overexpression group （t=10.410, P=0.009）. The results of immunofluorescence showed that as compared with the control group, the number of autophagic bodies punctured in TGF-β1 overexpression group was significantly increased （t=22.340, P=0.000）, and the fluorescence intensity was significantly increased. The results of Western blotting revealed the expression of E-cadherin in TGF-β1 overexpression group was significantly decreased, and that of Vimentin, Twist and Snail significantly increased as compared with the control group. The autophagy-related protein Beclin1 and phosphorylated mammalian target of rapamycin （p-mTOR） were significantly upregulated. The results in the TGF-β1 interference group showed the opposite trend to the TGF-β1 over-expression group. ConclusionIn lung adenocarcinoma cells, autophagy and EMT induced by TGF-β1 positive expression may be related to their ability to enhance invasion and migration of lung adenocarcinoma cells.