TIP30抑制胰腺癌转移和EMT的作用与机制研究

Tip30, also called CC3 or HTIP2, is a putative tumor suppressor gene located on human chromosome 11p15.1. It was originally identified by a differential display analysis of messenger RNA from the highly metastatic human variant small cell lung cancer (SCLC) versus less metastatic classic SCLC cell line. Decreased TIP30 expression has been detected in some tumor cells such as melanoma, breast cancer, neuroblastoma, glioblastoma, colon cancer and hepatocellular carcinoma. The underlying molecular mechanism of TIP30 inhibits tumor metastasis as well as the expression and function of TIP30 in pancreatic cancer remains unknown. In current study, we found TIP30 were decreased in 5 pancreatic cancer cell lines compared with the normal pancreatic cells and over-expression of TIP30 may promote cell proliferation and migration. Further, TIP30 was methylated and decreased during TGFb induced EMT. The results indicate that TIP30 may play an important role in tumor metastasis and EMT in pancreatic cancer. We will detected the expression of TIP30 in human pancreatic cancer and analysis the correlations between TIP30 expression and the clinical characteristic, and then evaluate the potential role of TIP30 as a prognostic factor in pancreatic cancer. Further, we try to investigate the underlying molecular mechanism of TIP30 down-regulated by TGFb. The expression of TIP30 will be deceased by RNA interference or increased by transected with over expression plasmid, then tumorigenicity and metastasis potentials of tumor cells will studied in vitro and in nude mice. Apoptosis will be examined by TUNEL assay and Annexin V-FITC kit. Wound-healing and Matrigel invasion assays will used to determine migration and invasion potentials of tumor cells. RT-PCR, Western blot and Immunofluorscence will be used to analysis EMT. We also try to investigated the molecular mechanism of TIP30 regulated EMT and tumor metastasis by ChIP-on-chip and expression microarray analysis. Our findings will present functional and mechanistic insight into the critical role of TIP30 in tumor metastasis of cancer.

TIP30是转移相关的经典的抑癌基因，在多种肿瘤中存在表达下调，但其抑制转移的分子机制及其在胰腺癌中的表达水平和作用尚无研究。我们前期研究表明，TIP30在多种胰腺癌细胞系中表达量下降，过表达TIP30可促进细胞增殖和转移能力。同时，TGFb诱导胰腺癌细胞EMT过程中伴随TIP30的启动子区甲基化及表达下调。基于此，本研究将通过临床资料分析，揭示TIP30表达与胰腺癌转移复发以及病人生存时间的关系，评价TIP30作为预后指标的价值；通过细胞增殖、迁移和侵袭等体外实验和胰腺癌转移裸鼠动物模型，深入探讨TIP30对胰腺癌转移的体内外生物学效应以及作用环节；阐明TGFb诱导TIP30表达下调的分子机制；结合ChIP-on-chip技术及全基因组表达谱芯片技术，从全基因组的水平系统性地研究TIP30调节的靶基因及其相关的下游功能性通路，充分阐明TIP30抑制肿瘤转移及EMT的分子机制。

胰腺癌是我国常见的消化道恶性肿瘤之一，发病隐匿，容易转移，预后极差，因此，深入研究胰腺癌发生、进展和转移中所涉及的关键分子机制尤为重要。TIP30 是恶性肿瘤转移相关的经典抑癌基因，已经被证实在多种恶性肿瘤中存在表达下调。然而TIP30在胰腺癌中的表达水平以及TIP30对胰腺癌的发生、进展和转移的作用尚无研究。本课题通过（1）免疫组化及甲基化特异性PCR等方法，检测胰腺癌临床标本中TIP30蛋白表达及DNA甲基化状态，分析其与临床病理学指标的关系；（2）慢病毒转染胰腺癌细胞，对TIP30的表达进行干扰或过表达调节，从分子和细胞水平以及实验动物体内水平探讨TIP30与胰腺癌侵袭、转移及EMT的关系及机制；（3）基因芯片检测抑制TIP30表达后胰腺癌细胞基因表达谱的变化及其下游通路；（4）探索了新的小鼠原位胰腺癌模型的建模方法，首次阐明了TIP30在胰腺癌中的表达情况，并从表观遗传学的角度研究了其在胰腺癌组织中表达水平降低的原因，证实了TIP30对胰腺癌增殖、侵袭、转移的抑制作用，并揭示了TGF-β通过促进甲基化抑制TIP30表达和促进胰腺癌细胞EMT的机制。今后通过研制特异性调节TIP30的靶向药物，对部分胰腺癌病人进行生物治疗，很可能具有重要的临床应用价值和应用前景。