Chen Dong, Wang Yan, Zhang Kejun, Jiao Xuelong, Yan Bomin, Liang Jun
Department of General Surgery, the Affiliated Hospital of Qingdao Medical College, Qingdao University, Qingdao 266003, China.
Department of Endodontics, School of Stomatology, Shandong University, Jinan 250012, China.
Int J Mol Sci. 2012;13(8):10594-10607. doi: 10.3390/ijms130810594. Epub 2012 Aug 23.
Secreted clusterin (sCLU) has been shown to be overexpressed in metastatic hepatocellular carcinoma (HCC) tissue, and its overexpression in HCC cells increases cell migration and the formation of liver metastatic tumor nodules in vivo. In this study, we tested the hypothesis that sCLU plays a role in the invasiveness of human HCC and may be associated with its metastatic spread. HCCLM3, a human hepatocellular carcinoma cell line, was transiently transfected with an antisense oligonucleotide (ASO) against sCLU (OGX-011). HepG2 liver hepatocellular cells were transiently transfected with the pc.DNA3.1-sCLU plasmid to overexpress sCLU, and subsequently evaluated for effects on invasion and the expression of molecules involved in invasion. We observed that suppression of the sCLU gene significantly reduced the invasive capability of the highly invasive HCCLM3 cells, and vice versa in the low invasive HepG2 cell line. The results revealed that knockdown of sCLU by OGX-011 resulted in a significant increase in the expression of E-cadherin and a decrease in matrix metalloproteinase-2 (MMP-2) gene transcription. Overexpression of sCLU by transfection with pc.DNA3.1-sCLU significantly decreased the expression of E-cadherin and increased MMP-2 gene transcription. These data were further verified by reverse transcription-PCR and Western blot analysis. A significant reduction in MMP-2 expression and an increase in E-cadherin expression in sCLU-knockdown HCCLM3 cells were observed, as well as a significant increase in MMP-2 expression and a decrease in E-cadherin expression in HepG2 cells overexpressing sCLU. These data indicate a role for sCLU in augmenting MMP-2 transcription and decreasing E-cadherin expression. Our data show the involvement of sCLU in human HCC invasion, and demonstrate that silencing sCLU gene expression inhibits the invasion of human HCC cells by inhibiting MMP-2 expression and promoting E-cadherin expression. Thus, OGX-011 could be an effective therapeutic agent for HCC.
分泌型簇集素（sCLU）已被证明在转移性肝细胞癌（HCC）组织中过表达，其在肝癌细胞中的过表达会增加细胞迁移以及体内肝转移瘤结节的形成。在本研究中，我们验证了以下假设：sCLU在人类肝癌的侵袭性中起作用，并且可能与其转移扩散有关。人肝癌细胞系HCCLM3用针对sCLU的反义寡核苷酸（ASO，即OGX - 011）进行瞬时转染。人肝癌HepG2细胞用pc.DNA3.1 - sCLU质粒进行瞬时转染以过表达sCLU，随后评估其对侵袭以及侵袭相关分子表达的影响。我们观察到，sCLU基因的抑制显著降低了高侵袭性HCCLM3细胞的侵袭能力，而在低侵袭性的HepG2细胞系中情况则相反。结果显示，OGX - 011敲低sCLU导致E - 钙黏蛋白表达显著增加，基质金属蛋白酶 - 2（MMP - 2）基因转录减少。用pc.DNA3.1 - sCLU转染过表达sCLU则显著降低E - 钙黏蛋白表达并增加MMP - 2基因转录。这些数据通过逆转录 - PCR和蛋白质印迹分析得到进一步验证。在敲低sCLU的HCCLM3细胞中观察到MMP - 2表达显著降低以及E - 钙黏蛋白表达增加，而在过表达sCLU的HepG2细胞中观察到MMP - 2表达显著增加以及E - 钙黏蛋白表达减少。这些数据表明sCLU在增强MMP - 2转录和降低E - 钙黏蛋白表达中起作用。我们的数据表明sCLU参与人类肝癌侵袭，并证明沉默sCLU基因表达通过抑制MMP - 2表达和促进E - 钙黏蛋白表达来抑制人类肝癌细胞的侵袭。因此，OGX - 011可能是一种有效的肝癌治疗药物。
