本文選題：miR-194-1 + HIF-1α��； 參考：《中山大學》2017年碩士論文

【摘要】：本研究以雄激素非依賴型前列腺癌細胞系DU-145為研究背景,探討缺氧對miR-194-1啟動子活性的影響,對DU-145細胞遷移和侵襲能力的影響,以及缺氧誘導(dǎo)因子HIF-1α與mi R-194-1的關(guān)系。揭示HIF-1α及miR-194在前列腺癌的發(fā)生發(fā)展中的作用和意義,為DU-145發(fā)病機理研究、病理診斷及預(yù)后判斷等提供新的依據(jù),同時將HIF-1α或miRNA作為靶基因的基因治療可能成為日后治療前列腺癌的一種有效途徑。首先基于表觀遺傳學數(shù)據(jù)庫預(yù)測了miRNA-194-1上游啟動子序列和缺氧反應(yīng)元件(HRE)序列,構(gòu)建含啟動子序列的熒光素酶報告載體,通過截短表達報告載體和瞬時轉(zhuǎn)染檢測螢火蟲熒光素酶強度的方法確定其啟動子序列。為了驗證缺氧對miR-194-1轉(zhuǎn)錄的影響,我們構(gòu)建了含有HRE缺失突變的miR-194-1啟動子載體,并檢測啟動子的熒光活性。然后用氯化鈷模擬缺氧,研究缺氧處理對前列腺上皮細胞和前列腺癌細胞的HIF-1α與miR-194-1表達的影響。構(gòu)建HIF-1α過表達載體以及合成HIF-1αsiRNA序列并轉(zhuǎn)染細胞,通過RT-qPCR和Western Blot實驗檢測HIF-1α和miR-194-1的表達水平,進一步驗證HIF-1α與miR-194-1的關(guān)系,之后在細胞水平通過劃痕實驗和transwell實驗探討缺氧對前列腺癌細胞DU-145遷移和侵襲的影響。結(jié)果:(1)成功構(gòu)建pGL3-Basic-194-1和缺失HRE序列的pGL3-Basic-194-1-△HRE熒光素酶載體,轉(zhuǎn)染后pGL3-Basic-194-1的啟動子熒光活性顯著升高,這表明啟動子序列是正確的;pGL-3-Basic-194-1-△HRE比空載體的熒光活性還要低,且在常氧和缺氧條件下,熒光活性都沒發(fā)生變化,表明缺失HRE序列后,啟動子活性顯著降低,且不再受缺氧調(diào)控,驗證了miR-194-1核心啟動子序列中包含HRE序列。(2)miR-194-1在前列腺癌細胞中的表達量低于前列腺上皮細胞系,提示了其作為日后前列腺癌診斷治療的可能性。(3)成功構(gòu)建HIF-1α過表達載體和合成siRNA序列,轉(zhuǎn)染細胞后,檢測到HIF-1α過表達會引起miR-194-1表達的下調(diào);抑制HIF-1α會導(dǎo)致miR-194-1表達的上調(diào)。(4)HIF-1α過表達或缺氧會使DU-145細胞的遷移和侵襲能力增強;siRNA抑制HIF-1α表達,則細胞遷移和侵襲能力下降。結(jié)論:實驗成功構(gòu)建了miR-194-1的啟動子序列,且驗證了缺氧會調(diào)控miR-194-1啟動子的轉(zhuǎn)錄調(diào)控。并且缺氧或HIF-1α過表達會促進DU-145細胞的遷移和侵襲;轉(zhuǎn)染siRNA會抑制DU-145的遷移和侵襲。
[Abstract]:In this study, androgen independent prostate cancer cell line DU-145 was used to investigate the effects of hypoxia on the activity of miR-194-1 promoter, migration and invasion of DU-145 cells, and the relationship between hypoxia inducible factor HIF-1 偽 and miR-194-1. To reveal the role and significance of HIF-1 偽 and miR-194 in the occurrence and development of prostate cancer, and to provide a new basis for the study of pathogenesis, pathological diagnosis and prognosis of DU-145. At the same time, gene therapy with HIF-1 偽 or miRNA as target gene may be an effective way to treat prostate cancer in the future. Firstly, the upstream promoter sequence and hypoxia response element (HRE) sequence of miRNA-194-1 were predicted based on epigenetics database, and luciferase report vector containing promoter sequence was constructed. The promoter sequence was determined by truncated expression report vector and transient transfection to detect the luciferase intensity of firefly. In order to verify the effect of hypoxia on the transcription of miR-194-1, we constructed the promoter vector containing HRE deletion mutation and detected the fluorescence activity of the promoter. Then the effects of hypoxia on the expression of HIF-1 偽 and miR-194-1 in prostate epithelial cells and prostate cancer cells were studied. HIF-1 偽 overexpression vector was constructed and HIF-1 偽 siRNA sequence was synthesized and transfected into cells. The expression levels of HIF-1 偽 and miR-194-1 were detected by RT-qPCR and Western Blot to further verify the relationship between HIF-1 偽 and miR-194-1. Then the effects of hypoxia on the migration and invasion of prostate cancer cell DU-145 were investigated at cell level by scratch test and transwell assay. Results: (1) pGL3-Basic-194-1 and pGL3-Basic-194-1-HRE luciferase vector were constructed successfully. The fluorescence activity of pGL3-Basic-194-1 promoter was significantly increased after transfection, which indicated that the promoter sequence was correct and the fluorescence activity of pGL3-Basic-194-1-HRE was lower than that of empty vector. The fluorescence activity did not change in normoxic and hypoxic conditions, indicating that the promoter activity decreased significantly after HRE deletion, and was no longer regulated by hypoxia. It was verified that the core promoter sequence of miR-194-1 contained HRE sequence. (2) the expression of miR-194-1 in prostate cancer cells was lower than that in prostate epithelial cells. The results suggested that HIF-1 偽 overexpression vector and siRNA sequence were successfully constructed. After transfection, the overexpression of HIF-1 偽 resulted in down-regulation of miR-194-1 expression. Inhibition of HIF-1 偽 resulted in upregulation of miR-194-1 expression. (4) overexpression of HIF-1 偽 or hypoxia increased migration and invasion ability of DU-145 cells. SiRNA inhibited HIF-1 偽 expression, then cell migration and invasion decreased. Conclusion: the promoter sequence of miR-194-1 was successfully constructed, and it was verified that hypoxia could regulate the transcription of miR-194-1 promoter. Hypoxia or overexpression of HIF-1 偽 promoted migration and invasion of DU-145 cells, and transfection of siRNA inhibited migration and invasion of DU-145 cells.
【學位授予單位】：中山大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.25

【參考文獻】

相關(guān)期刊論文 前10條

1 秦承東;任正剛;湯釗猷;;缺氧微環(huán)境在腫瘤進展中的作用[J];腫瘤;2016年01期

2 牛黛醇;李效宇;;MicroRNA在魚類胚胎發(fā)育中的調(diào)控作用[J];四川動物;2015年06期

3 郭德正;周宗和;羅興成;;HIF-1α與胃腸道腫瘤的關(guān)系研究進展[J];中國民康醫(yī)學;2015年08期

4 張哲;孫立;袁勝濤;;拓撲替康聯(lián)合用藥抗腫瘤研究進展[J];中國臨床藥理學與治療學;2015年01期

5 趙文鋒;;正確認識前列腺癌[J];保健醫(yī)苑;2015年01期

6 歐陽偉煒;羅松;蘇勝發(fā);盧冰;;前列腺特異性膜抗原PSMA的研究現(xiàn)狀[J];實用腫瘤雜志;2014年05期

7 王婧;李冰;劉翠翠;朱陣;張繼瑜;;啟動子結(jié)構(gòu)和功能研究進展[J];生物技術(shù)通報;2014年08期

8 蔡秀紅;黃貽濤;張子平;王藝磊;;缺氧誘導(dǎo)因子-1(HIF-1)及其在水生動物中的研究進展[J];農(nóng)業(yè)生物技術(shù)學報;2014年01期

9 黃寶星;宿恒川;曹萬里;孫�？�;;前列腺癌內(nèi)分泌治療方法研究及預(yù)后分析[J];中華男科學雜志;2013年09期

10 林艷端;申鍔;胡兵;;三種常見的前列腺癌細胞系LNCap、PC3和DU145的生物學特性[J];中華臨床醫(yī)師雜志(電子版);2013年11期

相關(guān)博士學位論文 前4條

1 韓康;microRNA-194在骨肉瘤中作用及機制的研究[D];第四軍醫(yī)大學;2015年

2 於雷;MicroRNA在肝癌早期診斷中的價值及其影響肝癌預(yù)后的機理初探[D];復(fù)旦大學;2012年

3 余嫻;缺氧通過HIF-1α誘導(dǎo)心肌細胞分泌TNF-α的機制研究[D];華中科技大學;2010年

4 任加強;前列腺特異性膜抗原（PSMA）在前列腺癌的病理診斷及免疫治療的基礎(chǔ)研究[D];復(fù)旦大學;2005年

，

本文編號：2103786