目的：本研究旨在探讨lncRNA PCBP1-AS1在肝细胞癌EMT转化中的调控作用及其机制,为肝细胞癌的防治提供新靶点。方法：收集了21例肝细胞癌患者的肝癌组织和相应的癌旁正常组织作为细胞检测标本。采用Real-Time PCR检测和蛋白免疫印迹法检测细胞中lncRNA PCBP1-AS1,miR-199,XBP1等基因的表达;使用皮尔逊相关系数法分析lncRNA PCBP1-AS1表达水平与肝细胞癌TNM分期的相关性;并利用生物信息学预测及荧光素酶报告基因实验验证lncRNA PCBP1-AS1与miR-199间的相互作用;通过转染miR-199 mimc或inhibitor构建miR-199 mimc或inhibitor的Hep3B细胞模型;Transwell实验检测细胞的侵袭能力;划痕实验检测细胞的迁移能力;CCK-8法检测细胞活力;LDH释放实验检测细胞毒性。结果：与癌旁组织相比,肝癌组织中LncRNA PCBP1-AS1表达明显增加,其水平与肝细胞癌的TNM分期呈正相关;信息学预测lncRNA PCBP1-AS1与miR-199存在相互作用;miR-199 mimic可以显著减少转染lncRNA PCBP1-AS1-wt报告基因质粒的Hep3B细胞荧光素酶活性;转染lncRNA PCBP1-AS1质粒可明显上调Hep3B细胞中lncRNA PCBP1-AS1、 XBP1、Snai1、Vimentin mRNA及蛋白的表达,下调miR-199、E-cadherin mRNA及蛋白的表达,增加Hep3B细胞的活力、迁移、侵袭能力,减少Hep3B细胞LDH的释放,而上述作用可被miR-199 mimic抑制或被miR-199 inhibitor促进。结论：lncRNA PCBP1-AS1通过结合miR-199并减少其表达水平,可上调肝癌细胞中XBP1Snai1、Vimentin的表达,同时下调E-cadherin基因的表达,从而促进了上皮-间充质转化（EMT）,是肝细胞癌的潜在靶点。
Abstract
Objective This study aims to investigate the regulatory role of lncRNA PCBP1-AS1 in EMT transformation of hepatocellular carcinoma and its possible mechanism, so as to provide a new target for the prevention and treatment of hepatocellular carcinoma. Methods Real-Time PCR was used to detect the expression of lncRNA PCBP1-AS1, miR-199, XBP1 and other genes in liver tissue of HCC patients or HCC cells. Pearson correlation coefficient was used to analyze the correlation between lncRNA PCBP1-AS1 expression and TNM stage of hepatocellular carcinoma. Bioinformatics was used for predicting the interaction between lncRNA PCBP1-AS1 and miR-199, which was verified via luciferase reporter gene experiment. Lipo2000 was used for transfering PCBP1-AS1 plasmid or miR-199 mimc (inhibitor) into Hep3B cells. Transwell assay was used for detecting the invasive ability of cells. Scratch test was used for detecting the migration ability of cells. CCK-8 kit was used for detecting the cell activity. LDH release assay was used for detecting the cytotoxicity. Western blot was used for detecting the expression of XBP1 and other genes. Results Compared with the normal liver tissues, the expression level of LncRNA PCBP1-AS1 in liver cancer tissues was significantlyincreased. The expression level of LncRNA PCBP1-AS1 was positively correlated with the TNM stage of hepatocellular carcinoma. It was predicted that lncRNA PCBP1-AS1 interacted with miR-199 mimic, and miR-199 mimic could significantly reduce the luciferase activity of Hep3B cells transfected with lncRNA PCBP1-AS1-wt luciferase reporter gene. LncRNA PCBP1-AS1 overexpression of can significantly increase the expression of lncRNA PCBP1-AS1, XBP1, Snai1 and Vimentin in Hep3B cells, significantly decrease the expression of miR-199 and E-cadherin, significantly increase the viability of H cells, and significantly reduce LDH release, significantly increase the migration and invasion ability of Hep3B cells, and these effects could be inhibited by miR-199 mimic or promoted by miR-199 inhibitor. Conclusion LncRNA PCBP1-AS1 can upregulatethe expression of Snai1 and Vimentin, downregulate the expression of E-cadherin in liver cancer cells and promote its epithelial-mesenchymal transformation (EMT) via sponging the expression of miR-199. LncRNA PCBP1-AS1 is a potential target for hepatocellular carcinoma.
关键词
肝细胞癌 /
lncRNA PCBP1-AS1 /
miR-199 /
XBP1 /
上皮间充质转化
Key words
hepatocellular carcinoma /
lncRNA PCBP1-AS1 /
miR-199 /
XBP1 /
epithelial mesenchymal transformation
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基金
长沙市自然科学基金“缺氧导致miRNA-199低表达促进肝癌转移的分子机制”（kq2004154）
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