本文選題：胰腺癌 + microRNA��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：背景胰腺癌早期診斷困難、惡性程度高,是預(yù)后最差的惡性腫瘤之一,5年生存率低于7%。MiRNAs因可同時(shí)參與腫瘤的增殖、凋亡、耐藥及侵襲等多個(gè)功能通路的調(diào)控而受到廣泛關(guān)注。miR-1285屬于miR-612/1285/3187-5p簇中的一員,目前只有在肝癌和腎細(xì)胞腎癌中有所報(bào)道,但其在胰腺癌中的調(diào)控機(jī)制仍不明確,因此我們將miR-1285作為本研究的研究對象,深入研究其對胰腺癌增殖、周期、化療敏感性、侵襲、遷移、凋亡等惡性生物學(xué)行為的影響,并尋找其靶基因,探索其調(diào)控機(jī)制。YAP1蛋白是Hippo信號通路的主要效應(yīng)因子之一,近兩年來大量研究證實(shí)其在多種惡性腫瘤中均發(fā)揮促癌作用。目的1.明確miR-1285對胰腺癌細(xì)胞株增殖、周期、化療敏感性、侵襲、遷移、凋亡等表型的調(diào)控作用；2.明確miR-1285的靶基因；3.初步探索miR-1285及靶基因的下游信號通路。方法本研究首先通過實(shí)時(shí)定量PCR(探針法)檢測檢測miR-1285在各細(xì)胞系中的表達(dá)量,并選取細(xì)胞株作為研究對象。隨后,通過脂質(zhì)體瞬時(shí)轉(zhuǎn)染法改變細(xì)胞內(nèi)源性miR-1285的表達(dá)后,采用CCK-8細(xì)胞增殖實(shí)驗(yàn)檢測miR-1285對胰腺癌細(xì)胞增殖能力和化療敏感性的影響,采用PI單標(biāo)記流式細(xì)胞術(shù)檢測細(xì)胞周期,采用transwell法檢測細(xì)胞遷移和侵襲能力,采用Annexin V/PI雙標(biāo)記流式細(xì)胞術(shù)檢測細(xì)胞凋亡,驗(yàn)證miR-1285對胰腺癌細(xì)胞系惡性生物學(xué)行為的影響。隨后,通過生物信息學(xué)數(shù)據(jù)庫預(yù)測miR-1285的靶基因,將多個(gè)數(shù)據(jù)庫預(yù)測到的結(jié)果進(jìn)行比對,并實(shí)時(shí)定量PCR(染料法)和western blot法分別檢測靶基因mRNA和蛋白的表達(dá),同時(shí)進(jìn)一步驗(yàn)證miR-1285-YAP1的下游效應(yīng)蛋白的表達(dá)水平。結(jié)果1.MiR-1285在胰腺癌耐藥株中較親本株低表達(dá)；2.體外試驗(yàn)中,miR-1285可顯著抑制胰腺癌細(xì)胞增殖,抑制胰腺癌細(xì)胞G1→S期轉(zhuǎn)化,增加吉西他濱化療敏感性,抑制細(xì)胞遷移和侵襲能力,促進(jìn)胰腺癌細(xì)胞凋亡；3.MiR-1285的靶基因?yàn)閅AP1,可能通過抑制YAP1mRNA翻譯的途徑抑制YAP1蛋白表達(dá)；4. MiR-1285-YAP1下游通路蛋白包括,EGFR、β-catenin、Bim、PTEN等。結(jié)論1.MiR-1285通過下調(diào)YAP1蛋白表達(dá),在胰腺癌細(xì)胞株中發(fā)揮抑癌作用；2. EGFR、β-catenin、Bim、PTEN等蛋白參與miR-1285-YAP1對胰腺癌細(xì)胞惡性表型的調(diào)控。
[Abstract]:Background the early diagnosis of pancreatic cancer is difficult, the malignant degree is high, and the prognosis is the worst. The 5-year survival rate is lower than that of 7%.MiRNAs because it can participate in the proliferation and apoptosis of the tumor at the same time. The regulation of multifunctional pathways, such as drug resistance and invasion, has attracted wide attention. MiR-1285 is a member of the miR-612/1285/3187-5p cluster. At present, it has been reported only in liver cancer and renal cell renal cell carcinoma, but its regulatory mechanism in pancreatic cancer is still unclear. Therefore, we take miR-1285 as the object of this study, study the effects of miR-1285 on the proliferation, cycle, chemosensitivity, invasion, migration, apoptosis and other malignant biological behaviors of pancreatic cancer, and search for its target gene. To explore its regulatory mechanism. YAP1 protein is one of the main effector factors of Hippo signaling pathway. In recent two years, a large number of studies have confirmed that YAP1 protein plays a role in promoting cancer in many kinds of malignant tumors. Objective 1. To elucidate the regulatory effects of miR-1285 on cell proliferation, cell cycle, chemosensitivity, invasion, migration, apoptosis and other phenotypes of pancreatic cancer cells. The target gene of miR-1285 was identified. To explore the downstream signaling pathway of miR-1285 and target gene. Methods in this study, the expression of miR-1285 in various cell lines was detected by real-time quantitative PCR (probe method), and the cell line was selected as the research object. Then, after the expression of endogenous miR-1285 was changed by liposome transient transfection, the effects of miR-1285 on the proliferation and chemosensitivity of pancreatic cancer cells were detected by CCK-8 cell proliferation assay, and the cell cycle was detected by Pi single-labeled flow cytometry. Cell migration and invasion were detected by transwell assay, apoptosis was detected by Annexin V/PI double labeling flow cytometry, and the effect of miR-1285 on malignant biological behavior of pancreatic cancer cell line was tested. Then, the target genes of miR-1285 were predicted by bioinformatics database, and the results of multiple databases were compared, and the expression of mRNA and protein were detected by real-time quantification of mRNA and western blot, respectively. At the same time, the expression level of downstream effector protein of miR-1285-YAP1 was further verified. Results the expression of 1.MiR-1285 was lower in resistant strains of pancreatic cancer than that in parent strains. In vitro, miR-1285 could significantly inhibit the proliferation of pancreatic cancer cells, inhibit the transformation of pancreatic cancer cells in G1 and S phase, increase the chemosensitivity of gemcitabine, and inhibit the migration and invasion of pancreatic cancer cells. 3. The target gene of MiR-1285 may inhibit the expression of YAP1 protein by inhibiting YAP1mRNA translation. The downstream pathway proteins of MiR-1285-YAP1 include EGFR, 尾 -catenin, and so on. Conclusion 1.MiR-1285 plays an inhibitory role in pancreatic cancer cells by down-regulating the expression of YAP1 protein. EGFR, 尾 -cateninine miR-1285-YAP1 and other proteins are involved in the regulation of malignant phenotype of pancreatic cancer cells.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.9

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