缺氧诱导肝癌细胞EMT转化及耐药的初步实验研究

Preliminary Study on HCC EMT Transformation and Drug Resistance Induced by Hypoxia

【目的】观察缺氧对肝癌细胞侵袭转移能力的影响,并进一步探讨缺氧环境下肝癌细胞耐药机制。【方法】利用HepG2、SMMC-7721细胞建立肝癌细胞缺氧模型,利用倒置显微镜观察细胞形态学变化;Transwell迁移与侵袭实验检测肿瘤细胞体外侵袭转移能力;MTT法检测肿瘤细胞活性;Western blot检测EMT相关蛋白E-cadherin、Vinmentin、N-cadherin和肿瘤耐药基因ABCG2的表达;采用流式细胞术检测凋亡和细胞周期变化。【结果】缺氧培养48 h后的肝癌细胞从上皮细胞样表型转化为间质细胞样表型;缺氧条件下肝癌细胞E-cadherin表达显著下降,Vimentin和N-cadherin表达显著升高（P〈0.05）。同时,缺氧条件下肝癌细胞迁移和侵袭能力显著增强（P〈0.05）。缺氧可导致HepG2、SMMC-7721停滞于静止期（G0/G1）的细胞数显著增多,缺氧条件下肝癌细胞对cisplatin耐药性显著增加（P〈0.05）; 缺氧条件下ABCG2蛋白表达增加（P〈0.05）。【结论】缺氧能诱导肝癌细胞EMT转化而致侵袭转移,同时可导致对cisplatin耐药。

【Objective】To observe the effect of hypoxia on invasion and metastasis of hepatocellular carcinoma （HCC） cells and to investigate the mechanism of drug resistance in HCC cells induced by hypoxia.【Methods】HCC hypoxia model was established by using HepG2 and SMMC-7721 cells. The morphological changes were observed by inverted microscope; invasion and metastasis of tumor cells in vitro were detected by Transwell migration and invasion assays; the cell activity was tested by MTT assay. Epithelial mesenchymal transition （EMT） related protein including E-cadherin, Vinmentin, N-cadherin and the expression of tumor drug resistance gene ABCG2 were detected by Western blotting. Apoptosis and cell cycle changes were detected by flow cytometry.【Results】HCC cells transformed from epithelial like phenotype to mesenchymal cell like phenotype after 48 h of hypoxia culture; the expression of E-cadherin decreased significantly and the expression of Vimentin and N-cadherin increased significantly under hypoxia （P〈0.05）; at the same time, the migration and invasion ability of HCC cells increased significantly under hypoxia （P〈0.05）; hypoxia can lead to a significant increase in the number of HepG2 and SMMC-7721 arrested in quiescent phase （G0/G1）; under hypoxia, the resistance of HCC cells to cisplatin was significantly increased （P〈0.05）; the expression of ABCG2 protein was increased under hypoxic conditions （P〈0.05）.【Conclusion】Hypoxia induces HCC EMT transformation and causes invasion and metastasis. At the same time, hypoxia may also induce resistance to cisplatin.