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目的 :初步探讨亚致死照射后残存的肝癌细胞中基因表达谱和细胞转移能力的变化,以及其可能的分子作用机制。方法:对肝癌Mc A-RH7777细胞进行一次性照射(6 Gy),筛选残存细胞即为McA-RH7777-6Gy细胞系。采用基因芯片技术检测Mc A-RH7777和McA-RH7777-6Gy细胞中mRNA表达的变化。然后,利用DAVID数据库对表达水平有明显差异的基因进行生物信息学分析,包括GO生物功能富集分析和KEGG信号通路富集分析。采用实时荧光定量PCR法验证2组细胞中上述分析差异较大的3个基因基质金属蛋白酶组织抑制因子2(tissue inhibitor of metalloproteinase 2,TIMP2)、SMAD家族成员2(SMAD family member 2,SMAD2)和MET原癌基因m RNA表达差异。进一步采用划痕愈合实验和Transwell小室法检测2组细胞的迁移和侵袭能力的差异。结果:基因芯片检测结果显示,照射后残存Mc A-RH7777-6Gy细胞与野生型Mc A-RH7777细胞相比,一系列与肿瘤相关的基因表达都发生了明显变化。实时荧光定量PCR检测结果显示,与McA-RH7777细胞相比,Mc A-RH7777-6Gy细胞中TIMP2、SMAD2和MET mRNA的表达水平分别上调13.000、14.516和6.384倍。GO生物信息学分析发现,这些基因多富集于肿瘤转移相关的生物学过程,如Ras蛋白信号转导、细胞周期阻滞、细胞迁移、细胞黏附、凋亡负调控、细胞增殖正调控、上皮-间质转化正调控和MAP激酶活性正调控等。KEGG信号通路分析发现,这些基因多富集在蛋白多糖信号通路、磷脂酰肌醇-3激酶(phosphoinositide-3 kinase,PI3K)-蛋白激酶B(protein kinase B,PKB,又称Akt)信号通路、病毒致瘤信号通路、FoxO信号通路、Rap1信号通路、Hippo信号通路和Ras信号通路等。划痕愈合实验结果表明,McA-RH7777-6Gy组划痕愈合率明显高于McA-RH7777组划痕愈合率(P<0.001)。Transwell小室法检测结果显示,McA-RH7777-6Gy组细胞侵袭至小室滤膜下的平均细胞数明显多于Mc A-RH7777组(P<0.001)。结论:亚致死照射后残存的肝癌细胞的迁移和侵袭能力明显增强,这可能与肿瘤转移相关信号通路基因的表达发生改变有关。
OBJECTIVE: To investigate the changes of gene expression and cell metastasis in surviving hepatocarcinoma cells after sublethal irradiation as well as the possible molecular mechanisms. Methods: A single irradiation (6 Gy) was performed on the hepatoma Mc A-RH7777 cells and the remaining cells were screened for McA-RH7777-6 Gy line. The changes of mRNA expression in Mc A-RH7777 and McA-RH7777-6 Gy cells were detected by gene chip technique. Then, using the DAVID database, the genes with significant differences in expression level were analyzed by bioinformatics, including enrichment analysis of GO biological function and enrichment analysis of KEGG signaling pathway. Real-time fluorescent quantitative PCR was used to examine the expression of three genes, tissue inhibitor of metalloproteinase 2 (TIMP2), SMAD family member 2 (SMAD2) MET proto-oncogene m RNA expression differences. Scratch healing experiments and Transwell chamber assay were further used to detect the differences in migration and invasion between the two groups of cells. Results: The results of microarray assay showed that a series of tumor-related gene expressions were significantly changed in the surviving Mc A-RH7777-6Gy cells compared with the wild-type Mc A-RH7777 cells. The results of real-time quantitative PCR showed that the expression of TIMP2, SMAD2 and MET mRNA in McA-RH7777-6Gy cells were up-regulated by 13.000, 14.516 and 6.384 times compared with McA-RH7777 cells respectively. GO bioinformatics analysis found that these genes are mostly involved in the biological processes involved in tumor metastasis, such as Ras protein signal transduction, cell cycle arrest, cell migration, cell adhesion, negative regulation of apoptosis, positive regulation of cell proliferation, epithelial - Positive regulation of mesenchymal transition and positive regulation of MAP kinase activity. KEGG signaling pathway analysis found that these genes are mostly enriched in the proteoglycan signaling pathway, the phosphoinositide-3 kinase (PI3K) -kinase B (PKB) pathway, Virus oncogenic signaling pathway, FoxO signaling pathway, Rap1 signaling pathway, Hippo signaling pathway and Ras signaling pathway. Scratch healing experiments showed that the wound healing rate in McA-RH7777-6Gy group was significantly higher than that in McA-RH7777 group (P <0.001). The results of Transwell chamber assay showed that the average number of cells in McA-RH7777-6Gy group infiltrating the cell membrane was significantly more than that of Mc A-RH7777 group (P <0.001). CONCLUSION: The migration and invasion ability of surviving hepatocarcinoma cells after sublethal irradiation are significantly increased, which may be related to the alteration of the expression of signal transduction pathways involved in tumor metastasis.