Hsp5B在骨肉瘤中的表达及对其恶性生物学行为调控的实验研究

Osteosarcoma is prone to locally invasive growth and metastasis. Therefore，investigating the metastasis-related factors and mechanisms is important for patients. Recently, many studies found that αB-Crystallin (Hsp5B) overexpressed in a variety of tumors and negatively correlated with prognosis. Our previous studies showed: ① Hsp5B was highly expressed in osteosarcoma tissues and associated with the lymph node metastasis; ②The interference of Hsp5B expression inhibited the osteosarcoma cells invasion;③ The interference of Hsp5B down-regulate the activation of ERK1/2 and the secretion of MMP9, while up-regulate the E-cadherin expression. On this basis, we will detect the mRNA and proteases expression of Hsp5B, E-cadherin, MMP9 and uPA, and analyze their relativity in osteosarcoma tissues. We will also analyse the correlation between the Hsp5B expression and patients prognosis by follow-up studied; The relationship of Hsp5B expression with the cell proliferation, migration, metastasis, E-cadherin expression, MMP9 expression and Upa expression will be studied by gene interference. We will establish the signaling molecules and pathway, and then confirm the key signaling molecules affected by Hsp5B. lastly, the vivo study will be performed to validate the above results. The research will provide the theoretical basis for Hsp5B as the new indicator of clinically judging the osteosarcoma invasion and metastasis ability, and as the molecular target of controlling osteosarcoma recurrence and metastasis.

骨肉瘤在局部呈侵袭性生长且易转移,故探索其侵袭转移的因素及机制对患者有重要意义。近年许多研究发现Hsp5B在多种肿瘤中过表达，且高表达患者预后差。我们前期研究示：①在骨肉瘤组织中Hsp5B呈高表达且与淋巴结转移相关；②干扰Hsp5B 表达明显抑制骨肉瘤细胞的侵袭；③下调Hsp5B，细胞中ERK1/2的激活与MMP9 的分泌下调，而E-钙黏素表达升高。我们拟在此基础上，检测骨肉瘤组织中Hsp5B与E-钙黏素、MMPs 及uPA的mRNA与蛋白表达并分析它们的相关性，结合随访研究Hsp5B与患者预后关系；通过基因干扰，于细胞水平研究Hsp5B表达与骨肉瘤细胞增殖、移动、侵袭及E-钙黏素、MMPs、uPA表达的关系；建立信号分子和通路，确立受Hsp5B影响的关键信号分子；最后裸鼠体内实验证明。本研究将为Hsp5B作为临床判断骨肉瘤侵袭转移能力的新指标、控制骨肉瘤复发转移的分子靶点提供理论依据。

骨肉瘤（Osteosarcoma）是临床骨科最为常见的原发性骨恶性肿瘤，约占所有骨肿瘤20%，也是青少年中最常见的恶性骨肿瘤，其特点是产生骨样组织和不成熟骨基质的肿瘤细胞大量增殖，恶性程度高且易出现早期肺转移。.HSP5B(αB-Crystallin,Cryab)是一种sHSP，最近有研究提示其在肿瘤细胞中呈高表达，有可能为一潜在的癌基因。我们在mRNA及蛋白骨检测人骨肉瘤细胞株、肉瘤肿瘤组织及相应癌旁组织中Hsp5B、MMPs及E-cadherin表达；在MG-63细胞干扰Hsp5B表达，分析Hsp5B表达与骨肉瘤生物学特性、MMP9及E-cadherin的表达的关系；最后检测Hsp5B表达差异的骨肉瘤细胞信号转导变化。我们发现Hsp5B在骨肉瘤组织中表达明显高于相应癌旁组织（p<0.01）,且高表达Hsp5B组织有MMP9表达上调及E-cadherin表达下调。我们建立Hsp5B的慢病毒干扰质粒，并感染MG-63细胞，建立MG-63-vshRNA-Hsp5B稳定细胞株，干扰效果满意。进一步在MG-63-vshRNA-Hsp5B与MG-63-Mock细胞中检测发现，Hsp5B对细胞增殖没有影响，而其表达与细胞的移动及侵袭影响呈正相关；与MG-63-Mock比较发现，E-cadherin在MG-63-vshRNA-Hsp5B表达上调，而MMP9相反。在骨肉瘤细胞中，Hsp5B高表达诱导ERK1/2过激活，而对Akt几乎没有影响，且抑制ERK1/2信号可以逆转Hsp5B诱导的E-cadherin表达下调及MMP9表达上调。结论：我们认为Hsp5B高表达可以作为骨肉瘤恶性表型的一新指标，其可作为骨肉瘤患者预后预测及治疗靶点。

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