Elucidating Regulatory Pathway of Estrogen Receptors in Helicobacter pylori-infected Gastric Cancer

本研究為探討胃幽門螺旋桿菌(Helicobacter pylori, H. pylori)感染所導致的胃癌中，人類雌激素受器(estrogen receptors, ERs)相關的分子調控機制。我們首先分析8片胃癌病人胃部組織的互補去氧核醣核酸微陣列晶片(cDNA microarray)以得到幽門螺旋桿菌與胃癌發生相關資料。在諸多幽門螺旋桿菌感染相關表現不同的基因裡，共有82個基因表現達兩倍以上。經Ingenuity Pathway Analysis (IPA) 軟體分析後共有41個基因與4組癌症調控網路高度相關，其中雌激素受器扮演調控網路中重要的中心角色(crucial hub)，並調控20個癌症相關基因。以即時定量反轉錄聚合酶連鎖反應驗證幽門螺旋桿菌感染的胃癌細胞株AGS的基因表現，發現共有14個與雌激素受器相關的基因表現量達1.5倍以上。此外我們與Human Genome-wide High-affinity ERE資料庫進行比對後，建立了新的幽門螺旋桿菌感染所引發的胃癌之分子調控路徑。更進一步的實驗分析包括細胞存活試驗(MTT assay)、細胞週期分析(cell cycle analysis)、抗氧化測試 (oxidative stress resistance test)、細胞侵犯實驗(invasion assay)、西方墨點轉漬法(western blot)、以及即時定量反轉錄聚合酶連鎖反應。我們的結果顯示不論是1 nM 雌激素(estradiol)或10-5 M雌激素受體拮抗劑(estrogen receptor antagonist)法洛德(fulvestrant)均能有效減低AGS因幽門螺旋桿菌感染造成的增生、抗氧化力上升、和轉移能力。幽門螺旋桿菌會透過雌激素受器依賴(estrogen receptor dependent)的路徑使胃癌細胞生長、抗氧化、以及侵犯能力增加。以高生理濃度雌激素處理或用法洛德抑制胃癌細胞的雌激素受器能有效擾亂幽門螺旋桿菌感染所導致的各種不正常胃癌現象。我們的研究成果或許能為人類胃癌發生與進展調控機制帶來一線曙光。This study is to elucidate the regulatory mechanism of estrogen receptors in Helicobacter pylori (H. pylori)-infected Gastric cancer. The correlation between H. pylori infection and gastric cancer was first analyzed using the cDNA microarray data from 8 gastric cancer patients. Among the differentially expressed H. pylori-related genes, 82 were found to be up-regulated two-fold. Ingenuity Pathway Analysis (IPA) analysis showed these genes to be highly related to four pathways with cancer progression, where estrogen receptor (ER) was the crucial hub in the network, and regulating 20 cancer related genes. A total of 14 ER-related genes were confirmed by q-RT-PCR to be up-regulated 1.5-fold in H. pylori-infected human AGS cells. Moreover, we utilized a human genome-wide high-affinity ERE database and built a new regulatory pathway for H. pylori-infected gastric cancer. Further experimental analysis included MTT assay, cell cycle analysis, oxidative stress resistance analysis, invasion assay, western blot, and q-RT-PCR. Our results show that either 1 nM estradiol or 10-5 M fulvestrant can reduce AGS viability, oxidative resistance, and metastasis that were increased by H. pylori infection. H. pylori enhanced the growth, resistance, and metastasis abilities of gastric cancer cells through ER-dependent pathway. With high physiological concentration of estradiol or shutting down ERs by fulvestrant, it can disturb abnormal gastric cancer conditions caused by H. pylori infection. Our results may shed light on the intricate regulatory mechanism in human gastric cancer formation and progression