亚致死照射后大鼠肝癌细胞基因表达谱的变化及其生物信息学分析

发布时间：2018-05-02 03:55

本文选题：肝肿瘤 + 放射疗法　；参考：《肿瘤》2017年07期

【摘要】：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(P0.001)。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(P0.001)。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。
[Abstract]:Aim: to investigate the changes of gene expression profile and cell metastasis ability in hepatocellular carcinoma cells after sublethal irradiation and its possible molecular mechanism. Methods: Mc A-RH7777 cells of hepatocellular carcinoma were irradiated for 6 Gy at one time, and the remaining cells were selected as McA-RH7777-6Gy cell lines. The changes of mRNA expression in MC A-RH7777 and McA-RH7777-6Gy cells were detected by gene chip technique. Then, bioinformatics analysis of genes with different expression levels was carried out by using DAVID database, including go biofunctional enrichment analysis and KEGG signaling pathway enrichment analysis. Real-time fluorescence quantitative PCR assay was used to verify the difference of the expression of 2(SMAD family member 2 SMAD2 and MET proto-oncogene m RNA between the three tissue inhibitor of matrix metalloproteinases (2(tissue inhibitor of metalloproteinase 2) and TIMP2SMAD family member (2(SMAD family member 2 SMAD2) and MET proto-oncogene (m RNA) in the two groups. Furthermore, the difference of migration and invasion ability between the two groups was detected by scratch healing test and Transwell chamber assay. Results: compared with wild type Mc A-RH7777-6Gy cells, a series of tumor-related gene expressions were significantly changed in irradiated Mc A-RH7777-6Gy cells. The results of real-time fluorescence quantitative PCR analysis showed that the expression levels of TIMP2SMAD2 and MET mRNA in Mc A-RH7777-6Gy cells were up-regulated by 13.000,14.516 and 6.384 times, respectively, compared with those of McA-RH7777 cells. Bioinformatics analysis showed that these genes were mostly enriched in the biological processes associated with tumor metastasis. For example, signal transduction of Ras protein, cell cycle arrest, cell migration, cell adhesion, negative regulation of apoptosis, positive regulation of cell proliferation, positive regulation of epithelial-mesenchymal transformation and positive regulation of activity of MAP kinase, etc. These genes are mostly enriched in proteoglycan signaling pathway, phosphoinositide-3 kinase- phosphoinosititide-3 kinase- PI3Ka-PKB pathway, virogenic signal pathway Rap1 signal pathway, Ras signal pathway and so on. The results of scratch healing test showed that the rate of scratch healing in McA-RH7777-6Gy group was significantly higher than that in McA-RH7777 group (P0.001N. Transwell chamber method). The results showed that the average number of cells in McA-RH7777-6Gy group was significantly higher than that in Mc A-RH7777 group (P0.001). Conclusion: the migration and invasiveness of residual HCC cells after sublethal irradiation were significantly enhanced, which may be related to the change of gene expression in tumor metastasis related signaling pathway.
【作者单位】：江南大学附属医院肿瘤研究所;江南大学附属医院肿瘤放疗科;
【基金】：国家自然科学基金资助项目(编号:81301920、81301784、81672328) 江苏省自然科学基金项目(编号:BK20151108、BK20150004)~~
【分类号】：R735.7

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