| 摘要: |
| 目的探讨索拉菲尼耐药肝癌细胞发生上皮间质变(EMT)及侵袭、转移能力增强的机制。方法通过药物连续诱导人肝癌细胞株Huh7建立索拉菲尼耐药肝癌细胞株Huh7R，显微镜下观察细胞形态学变化；CCK-8细胞增殖试验检测Huh7R细胞增殖能力；荧光定量PCR检测ABC家族耐药基因ABCB1、ABCC1、ABCG2及EMT相关调控锌指蛋白转录因子Snail、Slug基因表达水平；Westernblot检测耐药蛋白ABCG2，EMT标记分子E-cadherin、Vimentin及N-cadherin，转录因子Snail、Slug蛋白表达水平；Transwell小室侵袭试验检测Huh7R细胞迁移、侵袭能力。结果Huh7R细胞呈细长形，伴纤维状突起，形态学上符合EMT改变；CCK-8细胞增殖试验显示在索拉菲尼作用下，Huh7R细胞生存率高于Huh7细胞；荧光定量PCR结果显示，Huh7R细胞ABCB1、ABCC1、ABCG2及Snail、Slug基因表达水平均高于Huh7细胞（均P＜0.05）；Westernblot显示，Huh7R细胞上皮标记蛋白E-cadherin表达水平低于Huh7细胞（P＜0.05），而间质标记蛋白Vimenti、N-cadherin及Snail、Slug蛋白表达水平均高于Huh7细胞（均P＜0.05）；Transwell小室侵袭试验显示Huh7R细胞迁移、侵袭能力均较Huh7细胞增强。结论长期低剂量索拉菲尼暴露会促进肝癌细胞Snail和Slug表达，诱导肿瘤细胞发生EMT，增强其侵袭、转移能力。 |
| 关键词: 肝癌 索拉菲尼 转录因子 上皮间质变 耐药 |
| DOI：10.12056/j.issn.1006-2785.2017.39.04.2016-953 |
| 分类号: |
| 基金项目:浙江省“十二五”基层卫生适宜技术成果转化工程重大项目（2013T301-12、15）；嘉兴市科技计划项目（2013AY21042-5）；嘉兴市重点科技创新团队项目（[2013]3号） |
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| Epithelial-mesenchymal transition and migrant/invasion ability in human sorafenib-resistant hepatocellular carcinoma Huh7 cells |
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LIU Jun, WANG Qingqing, CAO Haoqiang, ZHANG Hao
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Bengbu Medical College
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| Abstract: |
| Objective To investigate epithelial-mesenchymal transition (EMT) and migrant/invasion ability in sorafenib- resistant human hepatocellular carcinoma cells (Huh7R). Methods The Huh7R was induced by long-term exposure to low dose sorafenib. The morphological changes of Huh7R was observed with phase contrast microscopy, the drug sensitivity was evaluated with cytotoxicity cell counting kit-8 (CCK-8) assay; the expression of ABC family resistance genes A BCB1, ABCC1, A BCG2, and transcription factor genes Snail, Slug was detected with real-time PCR (RT-PCR), the resistance related protein ABCG2, EMT marker E-cadherin, Vimentin and N-cadherin, transcription factors Snail and Slug was detected with Western blot. Migration and invasion properties of Huh7R cells were assessed using Transwell assays. Results Phase contrast microscopy showed that the Huh7R cells were elongated with fibrous projections and the morphological changes were consistent with typical EMT changes. CCK-8 results showed that the Huh7R cells were less sensitive to sorafenib than parental Huh7 cells. Fluorescence quantitative PCR results showed that compared to the parental Huh7 cells, the expression of resistance gene A BCB1, ABCC1 and transcription factor Snail, Slug in Huh7R cells was up-regulated significantly, and Western blot results showed that the expression of ABCG2 protein was significantly increased, the expression of epithelial marker E-cadherin protein was decreased, and the expression of mesenchymal marker protein Vimentin and N-cadherin increased, the expression levels of snail and slug were up-regulated. Transwell invasion assay indicated that Huh7R migration and invasion ability was increased compared with parental cells. Conclusion Theresultsindicatethatlong term exposure to low dosesorafenibpromotes theexpression of snail and Slug in human heparocellular carcinoma Huh7R cells, which mediates EMT and enhances the migrant and invasion ability of Huh7Rcells. |
| Key words: Hepatocellular carcinoma Sorafenib Transcription factor Epithelial mesenchymal transition Drug
resistance |
