乙型肝炎病毒 X 蛋白在调节 LIM 和 SH3 蛋白 1（LASP-1）表达以介导肝癌细胞增殖和迁移中的作用。
Role of hepatitis B virus X protein in regulating LIM and SH3 protein 1 (LASP-1) expression to mediate proliferation and migration of hepatoma cells.
机构信息
Department of Pathogenic biology and Laboratory of Infection and Immunology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China.
出版信息
Virol J. 2012 Aug 16;9:163. doi: 10.1186/1743-422X-9-163.
BACKGROUND
Hepatitis B virus X protein (HBx) has been shown to be responsible for the development of hepatocellular carcinoma (HCC) caused by Hepatitis B virus infection. However, its potential effect on the progression of hepatocellular carcinoma remains yet unclear. LIM and SH3 protein 1 (LASP-1), a focal adhesion protein, is expressed in an up-regulation manner in the HCC tissues. LASP-1 plays an important role in the regulation of proliferation and migration of HCC. In this study, we investigated the effect of LASP-1 involved in HBx-related tumor progression.
METHODS
LASP-1 levels in the HBx stable transfected HepG2 and Huh-7 cells were detected by RT-PCR and western blot analysis. The cellular localization of LASP-1 was assessed by immunofluorescence analysis. The activity of phosphatidylinositol 3-kinase (PI3-K) pathway was demonstrated by western blot assay. The HBx-expressing cells were transfected with specific small interference RNA (siRNA) against LASP-1. The proliferation and migration ability of cells were evaluated by cell viability assay and plate clone formation assay. The migration ability of cells was detected by transwell assay and wound healing assay.
RESULTS
RT-PCR and western blot analysis indicated the expression of LASP-1 was increased in the stable HBx-expressing cells compared with the control cells. Immunofluorescence study revealed that the distributions of LASP-1 in HepG2-HBX cells were mainly in pseudopods and the cytoplasm while they were mainly localized in the cytoplasm of HepG2-Mock cells. The cellular localizations of LASP-1 in Huh-7-HBX cells were in the perinuclear fractions while they were mainly localized in the cytoplasm of Huh-7-Mock cells. The upregulation of LASP-1 was inhibited after treatment with LY294002, PI3-K pathway inhibitor. Overexpression of LASP-1 in the stable HBx-expressing cells enhanced the proliferation and migration ability of hepatocellular cells. siRNA-mediated LASP-1 knowdown in the stable HBx-expressing cells significantly suppressed hepatocellular cells proliferation and migration.
CONCLUSIONS
These results demonstrated that HBx could upregulate LASP-1 through PI3-K pathway to promote the proliferation and migration of hepatoma cells.
背景
乙型肝炎病毒 X 蛋白（HBx）已被证明是导致乙型肝炎病毒感染引起的肝细胞癌（HCC）的罪魁祸首。然而，其在肝细胞癌进展中的潜在作用尚不清楚。LIM 和 SH3 蛋白 1（LASP-1）是一种黏着斑蛋白，在 HCC 组织中呈上调表达。LASP-1 在调节 HCC 的增殖和迁移中发挥重要作用。在这项研究中，我们研究了 LASP-1 在 HBx 相关肿瘤进展中的作用。
方法
通过 RT-PCR 和 Western blot 分析检测 HBx 稳定转染 HepG2 和 Huh-7 细胞中的 LASP-1 水平。通过免疫荧光分析评估 LASP-1 的细胞定位。通过 Western blot 分析检测磷酸肌醇 3-激酶（PI3-K）途径的活性。用针对 LASP-1 的特异性小干扰 RNA（siRNA）转染表达 HBx 的细胞。通过细胞活力测定和平板克隆形成测定评估细胞的增殖和迁移能力。通过 Transwell 测定和划痕愈合测定检测细胞的迁移能力。
结果
RT-PCR 和 Western blot 分析表明，与对照细胞相比，稳定表达 HBx 的细胞中 LASP-1 的表达增加。免疫荧光研究表明，HepG2-HBX 细胞中 LASP-1 的分布主要在伪足和细胞质中，而 HepG2-Mock 细胞中主要定位于细胞质中。Huh-7-HBX 细胞中 LASP-1 的细胞定位在核周区，而 Huh-7-Mock 细胞中主要定位于细胞质中。PI3-K 途径抑制剂 LY294002 处理后，LASP-1 的上调受到抑制。在稳定表达 HBx 的细胞中过表达 LASP-1 增强了肝癌细胞的增殖和迁移能力。在稳定表达 HBx 的细胞中，siRNA 介导的 LASP-1 下调显著抑制了肝癌细胞的增殖和迁移。
结论
这些结果表明，HBx 可以通过 PI3-K 途径上调 LASP-1 来促进肝癌细胞的增殖和迁移。
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