PGM1基因在肝细胞癌中的抑癌功能及分子机制

Recently, by using iTRAQ-2DLC-ESI-MS/MS technique, we identified 147 proteins, including 52 proteins up-regulated and 95 proteins down-regulated in small Hepatocellular Carcinoma (sHCC), and found that Phosphoglucomutase-1 (PGM1) can be served as candidate immunohistochemistry markers for distinction of small HCC from DN. PGM1 belongs to the phosphohexose mutase family and participates in both the breakdown and synthesis of glucose. PGM1 mainly expressed in cytoplasm as monomer and binds magnesium ion as cofactor. In addition, defects in PGM1 is the cause of glycogen storage disease type 14 (GSD14). GSD14 related metabolic disorder resulting in a myopathy characterized by exercise-induced intolerance with episodes of rhabdomyolysis, normal elevation of lactate, and hyperammonemia on a forearm-exercise test. .It was a first proposal that PGM1 protein can be used as immunohistochemistry markers. However, how the PGM1 related and participate to the biological behavior of HCC cell and hepatocarcinogenesis has not yet been studied. .Moreover, in the preliminary experiment, we found that PGM1 highly expressed in MHCC-97L and lowly expressed in HCC-LM3. In addition, PGM1 expression in HCC tissue showed significant prognostic value from our preliminary experiment. From these results, we hypothesize that PGM1 may serve as a potential tumor suppressor gene in HCC，and may be involved in carcinogenesis. The aim of this project is to identify diagnostic value of PGM1 in HCC prospectively，and to approach that change of PGM1 expressions and its effect on HCC cell line, including cell growth, invasion, apoptosis and tumor growth in mice model. The project will bring to light the involvement PGM1 in hepatocarcinogenesis.

前期工作中我们利用蛋白组学方法，发现磷酸葡萄糖变位酶1（PGM1）基因在肝细胞癌（HCC）癌前病变中高表达，HCC组织中低表达，并与HCC患者的预后显著相关。据此，我们认为PGM1是HCC的一种重要的抑癌基因，目前对该基因的功能研究尚未见报道。本研究首先在细胞学水平上利用RNAi和过表达技术，改变PGM1的表达量，探讨对HCC细胞的生长、侵袭、凋亡等细胞学功能的影响，并采用裸鼠种植瘤模型进行体内功能验证；在分子水平上采用PCR-SSCP技术检测PGM1编码区外显子基因突变位点，采用MSP-PCR法检测PGM1 DNA启动子区的甲基化，探讨PGM1在HCC中被下调的分子机制。同时将采用常规免疫组化方法进行前瞻性研究，对500例HCC组织进行PGM1检测，确定PGM1的HCC诊断效率。通过本项目阐述PGM1在HCC发生发展中的作用机制，为提高HCC的诊断效率和评估预后提供新的分子标志物。

第一部分：常规免疫组化实验.完成PGM1作为标志物的723例常规免疫组化实验。结果提示692例HCC的肿瘤病灶中PGM1表达阴性比率27.5%，+为29.9%，++为28.3%，+++为14.3%例, 对应癌旁中PGM1表达阴性为1.2%，+为7.8%，++为12.1%，+++为78.8%。另外，PGM1在局灶性增生结节，肝细胞腺瘤，异型增生结节中阴性率为0-12.5%。结果，提示HCC肿瘤组织中PGM1低表达（阴性和+）的比率较癌旁组织、良性结节（局灶性增生结节，肝细胞腺瘤，异型增生结节）高，提示恶性程度高的肿瘤组织PGM1表达水平低。.第二部分：PGM1 DNA启动子区CpG甲基化分析.完成HCC，DN，慢性肝炎/肝硬化样本的PGM1 DNA启动子区CpG甲基化分析。检测的15例HCC及对应癌旁样本10例结果提示，HCC组织及癌旁皆未发生甲基化。.第三部分 PGM1干扰（shRNA）及PGM1过表达（OE）对细胞功能的影响.首先，在L02，Hep3B，Hep1，HepG2，SMMC-7721，Huh7，PLC，MHCC-97L，MHCC-97H，LM3细胞中进行了PGM1蛋白检测。利用慢病毒颗粒感染细胞，进行PGM1干扰及PGM1过表达后，观察对细胞生长（MTT或计数），细胞周期，克隆形成，侵袭转移的影响。.结果，PGM1过表达能抑制细胞生长，而PGM1干扰在2株细胞进行实验后未能得到相同的结果。细胞周期检测结果提示，PGM1过表达引起肝癌细胞S和G2期的增加，而G1期减少。克隆形成实验提示，PGM1过表达引起肝癌细胞克隆形成能力的降低。侵袭迁移实验提示，PGM1过表达显著提高Hep1细胞的侵袭迁移能力，而LM3细胞未发生变化。当在Huh7细胞及SMMC-7721细胞中干扰PGM1表达后检测侵袭迁移能力，未能得到相同的结果。.第四部分 裸鼠皮下种植瘤实验.PGM1过表达（OE）Hep1细胞和Flag慢病毒感染Hep1细胞后，打入裸鼠皮下，观察其种植瘤的生长情况。结果显示，PGM1过表达（OE）能抑制Hep1细胞的裸鼠皮下生长。即，将细胞打入皮下第5周开始出现PGM1过表达抑制肿瘤生长（体积）的效果。当实验结束后取出肿瘤测量其重量的结果表明PGM1过表达显著抑制肿瘤生长（重量），并具有统计学意义。.另外，PGM1干扰病毒感染肝癌细胞株的皮下种植瘤实验正在准备中。