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研究生:施乃嘉
研究生(外文):Nai-Chia Shih
論文名稱:miR-31以及缺氧誘發因子在口腔鱗狀上皮癌細胞株中對HB-EGF的調控機制
論文名稱(外文):The regulation of miR-31 and Hypoxia-Inducible Factor-1 on Heparin-binding epidermal growth factor -like growth factor in OSCC cells
指導教授:林姝君林姝君引用關係
指導教授(外文):Shu-Chun Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:71
中文關鍵詞:微型RNA-31口腔癌
外文關鍵詞:miR-31Oral cancer
相關次數:
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  • 下載下載:15
  • 收藏至我的研究室書目清單書目收藏:0
臺灣十大死因之首為惡性腫瘤,而口腔癌在十大癌症死亡率排名第六,於男性癌症死亡率中排名第四,在過去十年罹患口腔癌的人數平均每年成長160%,故口腔癌對國人健康影響非常重大。微型RNA(MicroRNAs; miRNAs)是一群不會轉譯,約只有20~23核苷酸的小片段RNA,在近年來的研究中發現,miRNA在癌症扮演著重要的角色。本實驗室先前研究發現,miR-31在口腔鱗狀上皮細胞癌(OSCC)的組織以及OSCC細胞株中皆比正常的組織及細胞表現量高,因此推測在調控口腔鱗狀上皮細胞癌化過程當中,miR-31可能扮演著重要角色。缺氧誘發因子(Hypoxia-inducible factor -1 ; HIF-1)是細胞對缺氧環境的反應中很重要的蛋白,在缺氧的環境之下會被活化,幫助腫瘤細胞存活,使得癌症更加惡性。在先前實驗室的研究中發現,miR-31會藉由抑制缺氧誘導因子抑制因子(Factor Inhibiting HIF; FIH)來調控HIF-1,造成HIF-1表現量上升。HB-EGF (Heparin-Binding Epidermal Growth Factor -Like Growth Factor)是表皮生長因子家族(Epidermal Growth Factor; EGF) 之一,近年來研究指出,在很多癌症中,例如:胃癌,大腸癌…等,HB-EGF都有過度表現的情況,且在腫瘤的發展、增生、分化以及轉移上扮演著重要的角色。在先前實驗室微矩陣的實驗中發現miR-31大量表現的同時,HB-EGF mRNA也有一致的增幅現象,此外我們利用預測軟體找到在HB-EGF的啟動子上面有HIF-1的結合位置。因此本研究欲針對miR-31、HIF-1以及HB-EGF三者之間的關聯性做研究。研究結果顯示,在miR-31大量表現的口腔癌細胞株中,HB-EGF的mRNA以及蛋白質表現量均有上升。而處理模倣缺氧狀態之藥劑CoCl2後,HIF-1以及HB-EGF表現量均有上升。但在轉染HIF-1持續表現質體以及缺氧培養箱內,HIF-1表現量上升,但HB-EGF表現量卻下降。此外,抑制HB-EGF會造成口腔癌細胞株增生能力上升以及移行能力下降。由上述研究結果得知在口腔癌細胞中miR-31會調升HB-EGF的表現,然而HIF-1可能會調降HB-EGF表現,此外,HB-EGF在口腔癌細胞的增生能力中可能扮演抑制的角色,而在移行能力中則扮演促進的角色。
Malignant tumors are the top cause of death in Taiwan, oral squamous cell carcinoma (OSCC) is the sixth cause of death among ten most common cancers. In the past decade, the number of people suffering from oral cancer increased 160% each year, suggesting that oral cancer has a great impact on health in Taiwan. Micro-RNAs (miRNAs) are a group of fragmented RNA encompassing 20-23 nucleotides which do not undergo translation. In the recent years, miRNA was shown to play an important role in cancer. According to the previous results obtained from our laboratory, the level of miR-31 was demonstrated to be six-fold higher in OSCC cell lines or tissues with OSCC, as compared with normal tissues or cells, implicating that miR-31 may be involved in OSCC tumorigenesis. Hypoxia-induced factor (also called hypoxia-inducible factor -1; HIF-1) is an important factor in response to hypoxia. Under the hypoxic condition, HIF-1 is activated to aid the survival of tumor cells, thus providing the capacity for tumors to become more malignant. Previous reports from our laboratory have demonstrated that miR-31 up-regulates HIF-1 through the inhibition of factos inhibiting HIF (FIH). Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of epidermal growth factor (EGF) family, is overexpressed in various cancers, including gastric cancer, colorectal cancer, etc. Furthermore, HB-EGF is a critical factor involved in tumor development, proliferation, differentiation, metastasis. Our lab has also found that the overexpression of miR-31 accompanies with the augmentation of HB-EGF mRNA, as measured by microarray analysis. Besides, we found a HIF-1 binding site in HB-EGF promoter by a prediction softwar. Therefore, the purpose of this study is to examine the correlation of miR-31, HIF-1 and HB-EGF in OSCC cells line. The current result showed that overexpression of miR-31 increased the protein and mRNA levels of HB-EGF in oral carcinoma cell line. Cobalt chloride (CoCl2), a chemical which can be utilized to mimic hypoxia condition, upregulated the protein level of HIF-1 mRNA as well as protein and mRNA levels of HB-EGF. Hypoxia incubation or transfection with plasmid expressing constitutively activated HIF-1 up-regulated the expression of HIF in oral carcinoma cell line, however, the expression of HB-EGF was repressed. In addition, inhibition of HB-EGF expression increased proliferative capacity, but decreased the migration ability in oral carcinoma cells. Collectively, HB-EGF expression is up-regulated by miR-31 and modulated by HIF-1, where as HIF-1 may be indirect controlled by HB-EGF. Moreover, HB-EGF may inhibited proliferation and promoted migration.
中文摘要 i
英文摘要 iii
壹、 緒論 1
一、口腔癌 1
二、微型RNA (microRNA;miRNA) 1
三、miRNA與癌症 2
四、微型RNA-31 (miR-31) 3
五、缺氧誘發因子 (Hypoxia-Inducible Factor-1;HIF-1) 4
六、HB-EGF (Heparin-binding epidermal growth factor–like growth factor) 5
貳、 研究動機與目標 7
參、 材料與方法 9
一、細胞培養 (Cell culture) 9
二、萃取總RNA (Total RNA extraction) 9
三、反轉錄即時聚合酶連鎖反應 (Reverse transcription Real-time PCR) 10
四、西方墨點法 (Western blot) 12
五、病毒包裝(package virus) 15
六、病毒感染 (Infection) 18
七、染色質免疫沉澱技術(Chromatin Immunoprecipitation; ChIP) 18
八、MTT 21
九、細胞移行(Migration) 21
肆、 結果 22
一、製備可過度表現miR-31之細胞 22
二、在mirR-31過度表現的SAS細胞中HB-EGF的mRNA表現量上升 22
三、在miR-31過度表現的SAS細胞中HB-EGF的蛋白質表現量上升 23
四、CoCl2誘發HB-EGF mRNA表現量上升 23
五、CoCl2誘發HIF-1及HB-EGF 蛋白質表現上升 24
六、缺氧環境下HB-EGF mRNA表現量下降 24
七、缺氧環境下HB-EGF 蛋白質表現量下降 24
八、持續表現HIF-1造成HB-EGF mRNA及蛋白質的表現下降 25
九、HIF-1與HB-EGF啟動子的結合 25
十、建立HB-EGF刪減的穩定細胞株 26
十一、HB-EGF刪減能促進SAS細胞的增生 26
十二、HB-EGF刪減會抑制SAS細胞的移行 26
伍、 討論 28
陸、 圖列 31
圖一、偵測螢光確認慢病毒感染成功 31
圖二、SAS-miR-31細胞有過度表現miR-31 32
圖三、miR-31過度表現的SAS 穩定表現細胞株之HB-EGF mRNA表現量較高 33
圖四、miR-31過度表現的SAS 穩定表現細胞株之HB-EGF 蛋白質表現量較高 34
圖五、SAS細胞株處理CoCl2之後HB-EGF mRNA表現上升 35
圖六、SAS細胞株處理CoCl2之後HIF-1以及HB-EGF蛋白質表現量上升 36
圖七、SAS細胞株置於缺氧的培養箱之後HB-EGF mRNA表現下降 37
圖八、SAS細胞株置於缺氧的培養箱之後對HB-EGF蛋白質表現下降 38
圖九、持續表現HIF-1使HB-EGF的mRNA表現量下降 39
圖十、SAS細胞株處理CoCl2之後,HIF-1可與HB-EGF啟動子結合 41
圖十一、建立帶有HB-EGF shRNA的穩定細胞株 42
圖十二、HB-EGF shRNA能促進SAS細胞的增生 43
圖十三、HB-EGF shRNA會抑制SAS細胞的移行 44
柒、 表列 45
附表一:利用微矩陣分析偵測過量表現miR-31的SAS細胞 45
附表二:預測軟體PROMO預測可與HB-EGF促進子結合的因子 45
附表三:各項溶液配方 46
一、西方墨點法 46
1. 10x PBS 46
2. PMSF 46
3. Lysis buffer 46
4. 4x Lower buffer 46
5. 7.5%、10%、12.5% Lower gel 47
6. 4x Upper buffer 47
7. 4% Upper gel 47
8. SDS sample dye 47
9. 10x Running buffer (pH 8.3) 47
10.Transfer buffer 48
11. PBST 48
12. Blocking buffer 48
13. TAE溶液 48
二、TB培養液 48
附表四、初級抗體 49
附表五、二級抗體 49
附表六、引子序列 50
捌、 附圖列 51
附圖一、miR-31在人類口腔上皮細胞癌組織(A)及癌細胞株(B)中都有過度表現 51
附圖二、過度表現miR-31可抑制SAS細胞中FIH表現量(A),並進一步調升HIF-1活性(B) 52
附圖三、miR-31過度表現的口腔癌細胞株其增生能力、移行能力以及侵襲能力增加 53
附圖四、HIF-1α (ΔODD) map 圖 54
附圖五、pLV-EF1α-EGFP 55
玖、 參考文獻 56
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