臺灣博碩士論文加值系統

胃癌是全世界常見癌症死亡原因，尤其好發於亞洲地區。根據衛生署2008年統計資料顯示，胃癌在台灣男性及女性十大癌症中排名皆第五位，是所有癌症的5.9%。手術切除腫瘤是目前主要治療的方法。早期胃癌五年存活率可達九成，但一旦侵襲至晚期胃癌，五年存活率降到20-30%，其中轉移是造成癌症病人死亡的主要原因。因此了解侵襲的機轉對控制胃癌的轉移是非常關鍵的。
肌動素調節蛋白 (gelsolin) 位在人類的第九對染色體，主要是藉由肌動蛋白的動態調節，進而調控細胞形態維持、細胞移動、細胞分化及細胞凋零。在過去研究中已知在許多人類癌症中，gelsolin蛋白質表現下降，且在一些癌組織之大規模基因體掃描實驗中也証明它是表現量最顯著受到抑制的蛋白質之一，然而胃癌發展與gelsolin 之間的關係尚未被完整研究。本研究主要目的在探討gelsolin與胃癌侵襲過程的關係。在免疫組織化學染色分析中發現，與正常胃黏膜組織比較，癌組織其gelsolin表現相對減少。而且隨著侵襲的過程中，gelsolin表現更為明顯減少。在病人的預後因子方面，gelsolin表現量的減少與較差的預後相關，在統計上為一獨立的胃癌預後因子。在胃癌細胞株gelsolin的過度表現會抑制癌細胞的移動、侵襲及增生的能力。在gelsolin之遺傳分析方面， 我們選擇四個單一核甘酸多型性來分析正常胃黏膜細胞，胃癌細胞及胃癌細胞株的對偶基因頻率，實驗發現這三者並沒有統計上的差別，也就是胃癌在gelsolin的遺傳分析上是沒有差異性的.另外, 在gelsolin的表觀遺傳表現調節方面，實驗中以胃癌細胞株加上去氧核糖核酸甲基化抑制劑及去組織蛋白乙醯化抑制劑治療，發現gelsolin表現明顯上升。同時在胃癌病患的腫瘤組織中明顯有甲基特異聚合酵素連鎖反應結果。顯示胃癌中gelsolin表現可能是經由去氧核糖核酸甲基化及組織蛋白乙醯化所調控。
綜觀之，gelsolin的表現在胃癌臨床上具有預後之指標意義尤其於初期癌症並且與癌細胞侵犯行為等有關。以胃癌細胞株模型及基因遺傳分析探討的後續研究中進一步發現gelsolin在促進癌侵襲力之結果與基因去氧核糖核酸甲基化及組織蛋白乙醯化有關。

Gastric cancer is one of the most common causes of cancer death in the world, with a particularly high incidence in Asia. In Taiwan, it accounts for an estimated 5.9% of all malignancies in 2008. Surgical resection is the only potential curative method to treat patients with gastric cancer. The prognosis at stage I and II gastric cancer is promising (5-year survival about 90%), but it deteriorates rapidly at stage III and IV (5-year survival about 20~30%). Therefore, to understand the pathophysiology of metastasis is demanded for future treatment of gastric cancer. It is important to understand the molecular genetic events involved in the tumor progression and metastases of gastric cancer.
Discovered in 1971, gelsolin is located at chromosome 9q33, an actin-binding protein involving in dynamic change of the actin cytoskeleton. It has been known to regulate cell morphology maintenance, motility, cellular differentiation, and apoptosis. It is one of the most commonly down regulated proteins in many types of human malignancies. However, the role of gelsolin in gastric carcinogenesis has not been well evaluated. In this study, we demonstrated that gelsolin expression was decreased in gastric cancer tissue, as compared with normal gastric mucosal epithelium. Nevertheless, a significant negative association between gelsolin expression and the cancer progression was observed. Clinicopathological evaluation revealed that gelsolin expression was negatively correlated with the serum CEA level, lymph node invasion and TMN stage. Significant higher survival rate was associated with increasing gelsolin expression in gastric cancer patients, especially at the early stage of disease, implying the potential role of gelsolin as an independent prognostic biomarker in gastric cancer.
The molecular mechanisms of gelsolin expression in gastric cancer cell motility and invasion, as well as cellular proliferation were further investigated in gastric cells. Overexpression of gelsolin in gastric cancer cells could suppress cell motility, cell invasion and cell proliferation. Four selected single nucleotide polymorphisms (SNPs) in coding region have been genotyped using the TaqMan SNP genotyping assay. The genotype frequency of four SNPs in gelsolin gene was not significantly different between gastric cancer cells, gastric cancer tissue and gastric epithelium in gastric cancer patients. In addition, treatment of gastric cells with 5-azacytidine (a demethylation agent) and trichostatin A (a histone deacetylation agent) also enhanced gelsolin expression. In the methylation-specific PCR study, the result showed a tumor-specific hypermethylation of the gelsolin promoter. These genetic and epigenetic analyses suggested that an epigenetic regulatory mechanism of DNA methylation and histone acetylation could be one of the mechanisms for gelsolin expression modulation in gastric cancer cells.
In summary, loss of gelsolin expression is a significantly positive biomarker during gastric cancer progression and participates in gastric carcinogenesis through the modulation of cell motility, cell invasion, and cell proliferation. Gelsolin play a role as a tumor suppressor in gastric cancer and it’s down-regulated may be regulated by changes in DNA hypermethylation and histone acetylation.

Contents
Abstract…………………………………………………………i
English Abstract……………………………………………i
Chinese Abstract……………………………………………iii
Acknowledgement…………………………………………………v
Contents…………………………………………………………vi
Table Contents…………………………………………………viii
Figure Contents………………………………………………ix
Abbreviation …………………………………………………x
Medication ……………………………………………………xi
Introduction…………………………………………………1
Gastric cancer……………………………………………1
Genetic alteration in humancancer…………………5
Epigenetic alteration in human cancer………………6
Gelsolin………………………………………………………8
Materials and Methods…………………………………………12
Human subject and tissue collection……………………12
Gastric cancer cell lines…………………………………12
Immunohistochemistry (IHC) and grading score………13
Transfection of gelsolin overexpression constructs……13
Western blot analysis………………………………………14
Cell proliferation assay…………………………………15
Cell Migration assay………………………………………15
Immunofluorescence of cytoskeleton……………………16
Long-term activity of gastric carcinoma xenografts in vivo…………16
Genotyping of gelsolin gene single nucleotide polymorphisms…16
Epigenetic regulation of gelsolin expression……………17
Methylation-specific PCR………………………………………17
Statistics………………………………………………………18
Results……………………………………………………………19
Discussion…………………………………………………………25
Summary……………………………………………………………28
References…………………………………………………………29
Tables………………………………………………………………36
Figures……………………………………………………………42

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