Zhu Mingyue, Lu Yan, Li Wei, Guo Junli, Dong Xu, Lin Bo, Chen Yi, Xie Xieju, Li Mengsen
1. Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Haikou 571199, Hainan Province, PR. China; 2. Key Laboratory of Molecular Biology, Hainan Medical College, Haikou 571199, PR. China.
1. Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Haikou 571199, Hainan Province, PR. China; 3. Department of Pathophysiology, Hainan Medical College, Haikou 571199, Hainan Province, PR. China.
J Cancer. 2016 May 12;7(8):935-46. doi: 10.7150/jca.13628. eCollection 2016.
The infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear.
Fifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting.
we found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while L-02-X and HLE-X cells(stably transfected with pcDNA3.1-HBx) were silenced AFP expression by AFP-siRNA.
HBx through stimulating expression of AFP to promote malignant behaviors of human normal liver cells and HCC cells; AFP maybe used as a novel biotarget for therapeutics of HCC patients.
乙型肝炎病毒（HBV）感染与肝细胞癌（HCC）的发生密切相关，HBV-X蛋白（HBx）能够在正常肝细胞中诱导甲胎蛋白（AFP）的表达，且AFP具有促进正常肝细胞恶性转化的功能，但AFP在HCC细胞恶性行为中所起的作用仍不清楚。
通过肝切除术从临床患者中收集56份肝组织样本（包括正常肝组织、HBV相关性肝炎肝组织和HBV相关性HCC组织），经病理切片诊断这些组织，采用免疫组织化学染色和蛋白质免疫印迹法检测AFP、Ras和CXCR4的表达；采用MTT法检测人正常肝细胞系L-02细胞和人肝癌细胞系HLE细胞（不产生AFP）的增殖情况；采用划痕愈合试验比较L-02和HLE细胞的修复能力；采用Transwell小室试验分析这些细胞的迁移和侵袭能力；构建HBx表达载体（pcDNA3.1-HBx）并转染至L-02和HLE细胞，也通过MTT法、Transwell小室试验检测pcDNA3.1-HBx对恶性行为的影响，采用蛋白质免疫印迹法检测AFP、Ras和CXCR4的表达。
我们发现，与HBV相关性HCC非转移组织和HBV相关性肝炎组织相比，AFP、Ras和CXCR4在HBV相关性HCC及淋巴结转移组织中的表达显著升高；与正常肝组织相比，AFP、Ras和CXCR4在HBV相关性肝炎组织中的表达显著增强；当用pcDNA3.1-HBx载体转染L-02和HLE细胞时，细胞的生长率、迁移和侵袭能力以及AFP、Ras和CXCR4的表达均受到显著促进。当用AFP-siRNA使L-02-X和HLE-X细胞（稳定转染pcDNA3.1-HBx）沉默AFP表达时，其增殖率、迁移和侵袭能力以及Ras和CXCR4的表达均受到显著抑制。
HBx通过刺激AFP的表达来促进人正常肝细胞和HCC细胞的恶性行为；AFP可能作为HCC患者治疗的新型生物靶点。
