中文摘要：

目的探讨乙型肝炎病毒剪接特异性蛋白（Hepatitis B spliced protein,HBSP）与转化生长因子1诱导蛋白1（transforming growth factor-β1-induced transcript 1,TGFβ1I1）相互作用对TGFβ1诱导的肝癌细胞上皮间质转化（epithelialmesenchymal transition,EMT）的影响。方法构建HBSP慢病毒表达载体,利用293T细胞包装慢病毒颗粒,感染Huh7肝癌细胞株。以5ng/mLTGFβ1分别诱导稳定表达HBSP的慢病毒细胞株及其对照细胞株,观察细胞形态的变化,并提取细胞蛋白,Westernblot检测上皮间质转化标志物E-钙黏素（E-cadherin,E-cad）、紧密连接蛋白（Claudin-1）、β-链蛋白（β-catenin）、N-钙黏素（N-cadherin,N-cad）的变化。进而以TGFβ1I1特异性siRNA转染上述细胞,Westernblot观察以上指标变化情况。最后以侵袭小室实验和划痕实验分别检测TGFβ1诱导的细胞侵袭与迁移能力的变化。结果筛选获得稳定表达HBSP的慢病毒细胞株Huh7-HBSP-flag-HIV及其对照细胞株Huh7-flag-HIV。以TGFβ1诱导后在显微镜下观察到细胞形态由紧密的上皮形态变为松散的间质形态;特异性抗体检测表明上皮标志物E-cad、Claudin-1、β-catenin表达量下降,而间质标志物N-cad表达上升。侵袭小室实验和划痕实验表明TGFβ1诱导的HBSP表达株侵袭及迁移能力增强。而转染TGFβ1I1特异性siRNA可逆转以上现象。结论 HBSP与TGFβ1I1相互作用可促进TGFβ1诱导的肝癌细胞上皮间质转化并增强其侵袭迁移能力,提示HBSP在HBV相关性肝细胞肝癌发生发展中具有重要的致病意义。

英文摘要：

To investigate the TGFβ1-induced epithelial-mesenchymal transition （EMT） of Huh7 hepatoma cells caused by interaction of hepatitis B spliced protein （HBSP） with transforming growth factor beta-l-induced transcript 1 protein （TGFβ1I1）, coding region of HBSP was cloned into lentiviral expression vector. Huh7 hepatoma cells were infected by recom-binant lentivirus packaged in 293T cells. Stable cell lines expressing HBSP or control cells were selected by puromycin. Cells were incubated with 5 ng/mL TGFβ1 for 24 h, and observed under contrast-phase microspeope. Then the whole cell lysates were collected for western blot analysis using specific antibodies against EMT markers including E-cadherin, N-eadherin, Claudin-1 and β-catenin. To evaluate the effects of HBSP-TGFβ1I1 interaction on EMT, TGFβ1-induced EMT marker transition, as well as cell invasion and migra- tion were explored after knocking down of TGFβ1I1 by siR-NA. Results showed that Huh7 cell lines expressing HBSP （Huh7-HBSP-flag-HIV） and control cell lines （Huh7-flag-HIV） were successfully established. Huh7-HBSP-flag-HIV cells lost their pebble-like shape and tight cell-cell adhesion and transformed into the mesenchymal-like cells in the presence of TGFβ1. Decreased expression level of epithelial marker of E-cadherin, Claudin-1, β-catenin, increased expression level of mesenchymal marker of N-cadherin, and enhanced migration and invasion abilities were observed in Huh7-HBSP-flag-HIV cells as compared to the control cells. Moreover, the changes of EMT markers and metastasis abilities of Huh7-HBSP-flag HIV ceils could he reversed when TGFβ1I1 was knocked down by siRNA. In conclusion, HBSP could promote hepatoma cell migration and invasion by triggering EMT via interaction with TGFβ1I1. Our findings highlight new insights for HBSP-induced HCC progression.