The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
Mol Cancer. 2010 Apr 19;9:81. doi: 10.1186/1476-4598-9-81.
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. There is an urgent need to develop novel biomarkers for early diagnosis, as well as to identify new drug targets for therapeutic interventions.
54 paired HCC samples and 21 normal liver tissues were obtained from West China Hospital of Sichuan University. Informed consent was obtained from all the patients or their relatives prior to analysis, and the project was approved by the Institutional Ethics Committee of Sichuan University. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics was employed to profile the differentially expressed proteins between a HepG2 human hepatoma cell line and an immortal hepatic cell line L02. Validation of PGAM1 expression was performed by semi-quantitative RT-PCR, immunoblot and immunohistochemistry using clinical samples. shRNA expressing plasmids specifically targeting PGAM1 were designed and constructed by GenePharma Corporation (Shanghai, China), and were utilized to silence expression of PGAM1 in vitro and in vivo. Cell proliferation was measured by a combination of colony formation assay and Ki67 staining. Apoptosis was examined by flow cytometry and TUNEL assay.
A total of 63 dysregulated proteins were identified, including 51 up-regulated proteins, and 12 down-regulated proteins (over 2-fold, p < 0.01). Phosphoglycerate mutase 1 (PGAM1) was found markedly upregulated. Clinico-pathological analysis indicated that overexpression of PGAM1 was associated with 66.7% HCC, and strongly correlated with poor differentiation and decreased survival rates (p < 0.01). shRNAs-mediated repression of PGAM1 expression resulted in significant inhibition in liver cancer cell growth both in vitro and in vivo.
Our studies suggested that PGAM1 plays an important role in hepatocarcinogenesis, and should be a potential diagnostic biomarker, as well as an attractive therapeutic target for hepatocellular carcinoma.
肝细胞癌（HCC）是全球最常见的恶性肿瘤之一，由于对常规化疗的耐药性和放疗效果有限，预后较差。因此，迫切需要开发新的生物标志物进行早期诊断，并确定新的药物靶点用于治疗干预。
从四川大学华西医院获得 54 对 HCC 样本和 21 个正常肝组织。在分析前获得了所有患者或其亲属的知情同意，该项目得到了四川大学机构伦理委员会的批准。采用基于稳定同位素标记与氨基酸在细胞培养中的应用（SILAC）的蛋白质组学方法来分析 HepG2 人肝癌细胞系和永生化肝细胞系 L02 之间差异表达的蛋白质。使用临床样本通过半定量 RT-PCR、免疫印迹和免疫组化验证 PGAM1 的表达。由 GenePharma 公司（中国上海）设计并构建了特异性靶向 PGAM1 的 shRNA 表达质粒，并用于体外和体内沉默 PGAM1 的表达。通过集落形成试验和 Ki67 染色相结合来测量细胞增殖。通过流式细胞术和 TUNEL 测定来检查细胞凋亡。
共鉴定出 63 个失调蛋白，其中 51 个上调蛋白，12 个下调蛋白（超过 2 倍，p < 0.01）。发现磷酸甘油酸变位酶 1（PGAM1）明显上调。临床病理分析表明，PGAM1 的过表达与 66.7%的 HCC 相关，与分化不良和生存率降低密切相关（p < 0.01）。shRNA 介导的 PGAM1 表达抑制导致肝癌细胞在体外和体内的生长均显著受到抑制。
我们的研究表明，PGAM1 在肝癌发生中起重要作用，它可能是一种有潜力的诊断生物标志物，也是肝细胞癌的一个有吸引力的治疗靶点。
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