S100A4 通过 NF-κB 依赖性 MMP-9 信号调控肝癌 HepG2 细胞的迁移和侵袭。

S100A4 regulates migration and invasion in hepatocellular carcinoma HepG2 cells via NF-κB-dependent MMP-9 signal.

机构信息

Department of Surgery, the Affiliated Hospital of Medical College, Qingdao University, Qingdao, China.

出版信息

Eur Rev Med Pharmacol Sci. 2013 Sep;17(17):2372-82.

Abstract

BACKGROUND AND OBJECTIVES

We previously showed that the calcium-binding protein S100A4 is overexpressed and related to metastasis in hepatocellular carcinoma (HCC). However, whether S100A4 participates in the regulation of metastasis and its mechanisms in HCC is mostly unknown. Given the associations of S100A4, nuclear factor-kB (NF-kB/RelA) and MMP-9 with metastasis in a variety of malignancies, we explored a potential role of S100A4 in HCC metastasis and its mechanism.

METHODS

20 patients with HCC invasion (Lymph node metastasis, microvascular invasion, major portal vein invasion and intrahepatic metastasis) and 20 patients without HCC invasion were included. These tissues were detected for the expression of S100A4, NF-kB/RelA and MMP-9 by immunohistochemistry and quantitative real time polymerase chain reaction (Q-PCR). Correlation between the expressions of S100A4, NF-kB/RelA and MMP-9 with the invasion was analysed. The expressions of S100A4, nuclear factor-kB and MMP-9 was evaluated in HepG2 cells by western blot and immunohistochemistry. HepG2 cells were stably transfected with S100A4-specific small interfering RNA (S100A4 siRNA) to knockdown of S100A4, then transiently transfected with S100A4 cDNA to rescure the S100A4 level and evaluated for effects on invasion and expression analysis for molecules involved in invasion. After the HepG2 cells recurred the S100A4 levels, the HepG2 cells was treated with 5 µM Pyrrolidine Dithiocarbamate (PDTC) (a selective NF κ B inhibitor) to inhibit the NF-kB activity, or treated with Batimast (BB94: a MMPs inhibitor) to inhibit the MMP-9 activity. The expression analysis for molecules involved in invasion was analyzed.

RESULTS

A significant increase of S100A4, NF-kB/RelA and MMP-9 expression in HCC tissues with invasion than that of without invasion. A positive correlation was observed between S100A4, NF-kB/RelA, MMP-9 and invasion, respectively. In addition, S100A4 was positively correlated with NF-kB and MMP-9. S100A4 siRNA mediated knockdown of S100A4 in HepG2 cells resulted in significant reduction in the NF-kB activity and MMP-9 expression, and dramatically decreased its invasion. Moreover, the HepG2 cell metastatic potential was rescued by overexpression of S100A4 completely, at the same time, the NF-kB activity and MMP-9 expression was also increased. Pretreatment with PDTC or BB94 was observed to significantly reduce NF-kB activity and MMP-9 expression and dramatically decreased S100A4 -induced invasion.

CONCLUSIONS

Our findings indicate that S100A4 contributes to HCC metastasis by activation of NF-kB dependent MMP-9 expression, suggesting S100A4 as a novel diagnostic biomarker and therapeutic target in HCC.

摘要

背景与目的

我们之前的研究表明，钙结合蛋白 S100A4 在肝癌（HCC）中过度表达并与转移相关。然而，S100A4 是否参与 HCC 转移的调节及其机制在很大程度上尚不清楚。鉴于 S100A4、核因子-kB（NF-kB/RelA）和 MMP-9 与多种恶性肿瘤转移的相关性，我们探讨了 S100A4 在 HCC 转移中的潜在作用及其机制。

方法

纳入 20 例 HCC 侵袭（淋巴结转移、微血管侵犯、主门静脉侵犯和肝内转移）患者和 20 例无 HCC 侵袭患者。采用免疫组织化学和实时定量聚合酶链反应（Q-PCR）检测这些组织中 S100A4、NF-kB/RelA 和 MMP-9 的表达。分析 S100A4、NF-kB/RelA 和 MMP-9 的表达与侵袭的相关性。采用 Western blot 和免疫组织化学检测 HepG2 细胞中 S100A4、核因子-kB 和 MMP-9 的表达。用 S100A4 特异性小干扰 RNA（S100A4 siRNA）稳定转染 HepG2 细胞以敲低 S100A4，然后瞬时转染 S100A4 cDNA 以恢复 S100A4 水平，并评估其对侵袭的影响和对侵袭相关分子的表达分析。当 HepG2 细胞恢复 S100A4 水平后，用 5μM 吡咯烷二硫代氨基甲酸盐（PDTC）（一种选择性 NF-κB 抑制剂）处理 HepG2 细胞以抑制 NF-kB 活性，或用巴替马（BB94：一种 MMPs 抑制剂）处理以抑制 MMP-9 活性。分析侵袭相关分子的表达分析。

结果

侵袭性 HCC 组织中 S100A4、NF-kB/RelA 和 MMP-9 的表达明显高于无侵袭性 HCC 组织。S100A4、NF-kB/RelA、MMP-9 与侵袭性之间呈正相关。此外，S100A4 与 NF-kB 和 MMP-9 呈正相关。S100A4 siRNA 介导的 HepG2 细胞 S100A4 敲低导致 NF-kB 活性和 MMP-9 表达显著降低，侵袭性显著降低。此外，S100A4 的过表达完全挽救了 HepG2 细胞的转移潜能，同时 NF-kB 活性和 MMP-9 表达也增加。用 PDTC 或 BB94 预处理可显著降低 NF-kB 活性和 MMP-9 表达，并显著降低 S100A4 诱导的侵袭。