肝星状细胞条件培养基激活ERK1/2通路诱导肝癌细胞增殖及上皮间质转化

Hepatic stellate cell conditioned medium induces proliferation and epithelial-mesenchymal transition via activating ERK1/2 signaling pathway in hepatoma cells

目的探讨肝星状细胞(HSC)对肝癌细胞(HCC)恶性生物学行为的影响及其相关机制。方法分别培养SMMC-7721肝癌细胞、Hep G2肝癌细胞和LX-2 HSC,采用LX-2 HSC条件培养基(LX2-CM)、丝裂原激活蛋白激酶(MAPK)特异性抑制剂U0126处理肝癌细胞,TranswellTM小室检测肝癌细胞的侵袭和迁移能力,CCK-8法检测细胞的增殖情况,实时定量PCR和Western blot法分别检测两种肝癌细胞磷酸化的胞外信号调节激酶1/2(p-ERK1/2)、ERK1/2、c-Myc、波形蛋白(vimentin)、上皮钙黏素(E-cadherin)的mRNA和蛋白水平。结果 LX2-CM能促进SMMC-7721细胞和Hep G2细胞的增殖、侵袭和迁移,其作用可被U0126阻断。LX2-CM可以上调p-ERK1/2、c-Myc、vimentin的水平,下调E-cadherin的水平;U0126处理细胞后,p-ERK1/2、c-Myc、vimentin的水平显著降低,E-cadherin水平明显升高。结论 LX2-CM能通过ERK1/2通路激活c-Myc,促进肝癌细胞的增殖、侵袭和迁移,并诱导上皮间质转化。

Objective To investigate the influence of hepatic stellate cells（ HSCs） on malignant biological behavior of hepatoma cells and related mechanisms. Methods Human hepatoma cell lines SMMC-7721 and Hep G2,and hepatic stellate cell line LX-2 were cultured separately. HSC conditioned medium（ LX2-CM）,MAPK specific inhibitor U0126 were used to treat hepatoma cells,separately or together. The invasion and migration abilities of hepatoma cells were detected by TranswellTMassay,and cell proliferation was analyzed by CCK-8 assay. The mRNA and protein expression levels of p-ERK1 /2,ERK1 /2,c-Myc,vimentin and E-cadherin were determined by real-time PCR and Western blot analysis,respectively.Results LX2-CM promoted the proliferation,invasion and migration of SMMC-7721 and Hep G2 cells,and these effects were inhibited by U0126. LX2-CM up-regulated the expression levels of p-ERK1 /2,c-Myc,vimentin and down-regulated the expression level of E-cadherin. Conversely,after U0126 treatment,the expression levels of p-ERK1 /2,c-Myc and vimentin decreased significantly,while E-cadherin expression level increased. Conclusion LX2-CM could activate c-Myc via ERK1 /2signaling pathway in hepatoma cells,and consequently promote cell proliferation,invasion and migration,and also induce epithelial-mesenchymal transition.