C/EBPβ在人正常肝细胞和肝癌细胞系中的差异表达及其与内质网应激相关细胞死亡的关系

Differential expression of C/EBP beta in human normal liver cells and hepatocellular carcinoma cell lines and its correlation with cell death related to endoplasmic reticulum stress

目的观察转录因子C/EBPβ在人永生化正常肝细胞和肝癌细胞系中的表达差异性,探讨其与肝癌细胞内质网应激介导的细胞死亡的相关性。方法培养人永生化正常肝细胞系HHL5、HL7702和肝癌SMMC7721、Bel7402、HepG2、Hep3B细胞系;Hep3B细胞用衣霉素诱导内质网应激细胞模型;倒置光学显微镜下观察细胞形态;MTT法检测细胞生长抑制率;Hoechst 33258染色后荧光显微镜下检测细胞凋亡;RT-PCR和Western blotting技术分别检测mRNA和蛋白的表达水平。结果 C/EBPβ的mRNA和蛋白亚型C/EBPβ-1在4种人肝癌细胞系和永生化正常肝细胞系中均有表达,但C/EBPβ-3仅在人永生化正常肝细胞系中表达,在肝癌细胞系中不表达;衣霉素能够上调人肝癌Hep3B细胞C/EBPβ的mRNA和蛋白表达水平,明显上调C/EBPβ-3的表达水平;衣霉素能够诱导人肝癌Hep3B细胞内质网应激介导的细胞死亡,表现为细胞凋亡和类凋亡两种方式。结论 C/EBPβ的蛋白亚型C/EBPβ-3在人永生化正常肝细胞中表达而在肝癌细胞中不表达;C/EBPβ参与内质网应激介导的人肝癌细胞死亡。

Objective To detect the expression profile of transcription factor C/EBPβ in human immortalized normal hepatic cell lines and hepatocellular carcinoma cell lines so as to determine the correlation between C/EBPβ with cell death mediated by endoplasmic reticulum stress in hepatocellular cells. Methods We cultured the human immortalized normal hepatic cells lines HHL5 and HL7702 and hepatocellular carcinoma cell lines SMMC7721; Be17402, HepG2 and Hep3B. Hep3B cells were used as the cell model in tunicamycin-induced endoplasmic reticulum stress. Cellular morphology was observed under an inverted optical microscope. MTT assay was used to assess the inhibition of cell growth. To detect cell apoptosis, the cells were dyed with Hoechst 33258 and observed using a fluorescence microscope. RT-PCR and Western blotting were used to detect the expression of at mRNA and protein levels, respectively. Results We found that normally the mRNA and protein isoform of C/EBPβ, C/EBPβ-1,were both expressed in all of the four hepatocellular cell lines and the two immortalized normal hepatic cell lines, while C/EBPβ protein isoform C/EBPβ-3 was only expressed in the two immortalized normal hepatic cell lines. Tunicamycin increased the expressions of both mRNA and protein of C/EBPβ in Hep3B cells and the increase of protein isoform C/EBPβ-3 was the most remarkable. In Hep3B ceils, cell death was induced by tunicamycin through endoplasmic reticulum stress activity. Apoptosis as well as paraptosis was observed in tunicamycin-induced cell death. Conclusion C/EBPβ-3, one of the protein isoforms of C/EBPβ, is only expressed in normal hepatic cell lines, but not in hepatocellular cell lines. C/EBPβ is involved in cell death mediated by endoplasrnic reticulum stress activity in hepatocellular carcinoma cells.