本文選題：MicroRNA + miR-17-5p�。� 參考：《浙江大學》2016年博士論文

【摘要】：背景：胃癌是全球腫瘤致死主要原因之一,在我國是僅次于肺癌死亡率排名第二的惡性腫瘤�；熓俏赴┲委煹闹匾侄沃�。胃癌細胞對化療藥物產(chǎn)生耐藥性導致胃癌化療失敗的主要原因之一。胃癌患者如果對化療出現(xiàn)耐藥情況,預后較差,所以明確胃癌細胞產(chǎn)生耐藥的分子機制十分重要。microRNA (miRNA)是內源性非編碼小RNA分子,它們通過調節(jié)靶基因mRNA的翻譯或降解而起到調節(jié)基因的作用。miRNA在腫瘤中通過對下游靶基因的調節(jié),對其發(fā)生與發(fā)展起到至關重要的作用。近年來的研究證實了miRNA在抗腫瘤藥物的敏感性和耐藥性中起了重要作用。以特定的miRNA為治療靶點,通過比較miRNA表達譜,與腫瘤細胞耐藥性密切相關的特定miRNA將被識別,這可能為新的靶向治療方案開辟途徑,從而改善治療效果。然而miRNA影響胃癌細胞耐藥性的作用機制并不明了。因此準確尋找對胃癌耐藥細胞影響較大的miRNA并探討其作用機制,成為亟待解決的重要問題。本研究擬在兩種耐藥胃癌細胞SGC7901/DDP和BGC823/5-FU中篩選出與親本細胞表達差異較大的miRNA,并深入探討其對細胞耐藥性的影響作用及其作用機制。目的：在兩種耐藥胃癌細胞SGC7901/DDP和BGC823/5-FU中分析miRNA表達譜,篩選耐藥相關特異性miRNA;研究耐藥相關miRNA對胃癌細胞株耐藥性的影響；尋找潛在靶點,探討其作用分子機制,以期為新的靶向治療方案開辟途徑。方法：采用基因芯片技術分別檢測胃癌細胞SGC7901及其耐藥細胞SGC7901/DDP、胃癌細胞BGC823及其耐藥細胞BGC823/5-FU的miRNA的表達差異情況,尋找在兩株胃癌耐藥細胞中與親本細胞之間尋找表達差異較大的miRNA,并采用實時定量PCR方法進行驗證。通過體外轉染抑制序列anti-miR-17-5p序列下調細胞中的miR-17-5p表達,CCK-8法分析轉染后細胞對長春新堿、阿霉素、5-氟尿嘧啶和順鉑的藥物敏感性變化。運用生物信息學工具PicTar和Miranda algorithms預測miR-17-5p的靶基因。構建熒光素酶報告質粒pGL3-p21-3'-UTR驗證miR-17-5p與預測靶基因p21的相互作用。通過WesternBlot方法檢測胃癌耐藥細胞株SGC7901/DDP和BGC823/5-FU中miR-17-5p對p21的調控作用。流式細胞術檢測miR-17-5p對胃癌耐藥細胞株SGC7901/DDP和BGC823/5-FU細胞凋亡和細胞周期的影響。細胞計數(shù)的方法檢測miR-17-5p對胃癌耐藥細胞株SGC7901/DDP和BGC823/5-FU細胞生長的影響。結果：基因芯片分析結果顯示：與SGC7901細胞相比,SGC7901/DDP細胞中miR-17-5p的表達明顯上調(P0.01); BGC823/5-FU細胞中miR-17-5p的表達較BGC823細胞明顯上調(P0.01)。實時定量熒光PCR分析結果顯示：耐藥SGC7901/DDP與BGC823/5-FU細胞中miR-17-5p表達水平均顯著上調(P0.01)。抑制SGC7901/DDP細胞和BGC823/5-FU細胞中miR-17-5p表達可有效提高細胞對順鉑、阿霉素、長春新堿和5-FU的藥物敏感性(P0.05)。根據(jù)生物信息學工具的預測miR-17-5p的靶基因可能為p21。對p21基因3’非編碼區(qū)報告質粒熒光素酶活性測定顯示miR-17-5p可顯著降低pGL3-p21-3'-UTR相對熒光酶活性(P0.05)。在胃癌耐藥細胞SGC7901/DDP和BGC823/5-FU中抑制miR-17-5p表達可明顯增加p21蛋白的表達(P0.05)。抑制miR-17-5p表達還可抑制胃癌耐藥細胞SGC7901/DDP和BGC823/5-FU的生長(P0.05),并誘導細胞凋亡(P0.05)。在胃癌耐藥細胞SGC7901/DDP和BGC823/5-FU中抑制miR-17-5p表達可使G1期細胞明顯減少,而S期細胞明顯增多,引起細胞周期G1-S期阻滯(P0.05)。結論：與親代非耐藥胃癌細胞相比,miR-17-5p在耐藥細胞SGC7901/DDP和BGC823/5-FU細胞中表達顯著增高。抑制miR-17-5p表達可明顯改善胃癌細胞耐藥性,同時抑制胃癌耐藥細胞的生長活力、誘導細胞凋亡病引起細胞周期G1-S期阻滯。究其分子機制,miR-17-5p可能是通過調控其靶基因p21的表達影響胃癌耐藥細胞的凋亡和增殖。本研究發(fā)現(xiàn)miR-17-5p的表達可明顯降低胃癌細胞的耐藥性,為臨床上克服胃癌化療時細胞耐藥性難題提供理論支持和新的分子靶標。
[Abstract]:Background: gastric cancer is one of the leading causes of death in the world, and it is the second most malignant tumor next to lung cancer in our country. Chemotherapy is one of the most important methods for the treatment of gastric cancer. The drug resistance of gastric cancer cells to chemotherapy leads to one of the main reasons for the failure of chemotherapy for gastric cancer. .microRNA (miRNA) is an endogenous non coding small RNA molecule, which is an endogenous non coding small RNA molecule. They play the role of regulating gene by regulating the translation or degradation of the target gene mRNA, and.MiRNA plays an important role in the development and development of the tumor by regulating the target gene in the downstream. Recent studies have demonstrated that miRNA plays an important role in the sensitivity and resistance of antitumor drugs. Specific miRNA as the target of treatment, specific miRNA, which is closely related to the drug resistance of tumor cells, will be identified by comparing miRNA expression profiles. This may open up a way for the new target therapy and improve the therapeutic effect. However, the mechanism of the effect of miRNA on the drug resistance of gastric cancer cells is not clear. Therefore, it is an important problem to find out exactly how to find the miRNA and explore its mechanism of action to the drug resistant cells of gastric cancer. This study is to screen out the MI of two kinds of drug-resistant gastric cancer cells, SGC7901/DDP and BGC823/5-FU, which are different from those of the parent cells. RNA, and explore its effect on cell resistance and its mechanism of action. Objective: to analyze the miRNA expression profiles in two resistant gastric cancer cells, SGC7901/DDP and BGC823/5-FU, to screen resistance related specific miRNA, to study the response of drug resistance related miRNA to the drug resistance of gastric cancer cell lines, to search for potential targets and to explore the molecular mechanism of its action. System, in order to open up a new approach to target treatment. Methods: gene chip technology was used to detect the differential expression of miRNA in gastric cancer cell SGC7901 and its resistant cell SGC7901/DDP, gastric cancer cell BGC823 and its drug-resistant cells BGC823/5-FU, and to find the difference of expression between two gastric cancer cells and parental cells. Large miRNA, and verified by real-time quantitative PCR method. Down regulation of miR-17-5p expression in cells through in vitro transfection inhibition sequence anti-miR-17-5p sequence and CCK-8 assay for changes in drug sensitivity of transfected cells to vincristine, adriamycin, 5- fluorouracil and cisplatin. Use bioinformatics tool PicTar and Miranda algorithms preconditioning. The target gene of miR-17-5p was measured. A luciferase reporter plasmid pGL3-p21-3'-UTR was constructed to verify the interaction between miR-17-5p and the predicted target gene p21. The regulation of miR-17-5p on p21 in gastric cancer cell line SGC7901/DDP and BGC823/5-FU was detected by WesternBlot. Flow cytometry was used to detect miR-17-5p to the drug resistant cell line of gastric cancer. The effect of BGC823/5-FU cell apoptosis and cell cycle. Cell count method was used to detect the effect of miR-17-5p on the growth of SGC7901/DDP and BGC823/5-FU cells in gastric cancer cell lines. Results: gene chip analysis showed that the expression of miR-17-5p in SGC7901/DDP cells was obviously up regulated compared with SGC7901 cells (P0.01) and BGC823/5-FU cells in BGC823/5-FU cells. The expression of miR-17-5p was significantly higher than that of BGC823 cells (P0.01). Real-time quantitative fluorescence PCR analysis showed that the expression level of miR-17-5p in drug-resistant SGC7901/DDP and BGC823/5-FU cells increased significantly (P0.01). Inhibition of miR-17-5p expression in SGC7901/DDP and BGC823/5-FU cells could improve cells to cisplatin, adriamycin, vincristine and 5-F. U's drug sensitivity (P0.05). According to bioinformatics tools, the target gene for miR-17-5p may be p21. to the p21 gene 3 'non coding region report plasmid luciferase activity determination that miR-17-5p can significantly reduce the pGL3-p21-3'-UTR relative fluorescent enzyme activity (P0.05). Inhibition of miR-17-5p in gastric cancer resistant cells SGC7901/DDP and BGC823/5-FU is miR-17-5p. Expression can significantly increase the expression of p21 protein (P0.05). Inhibition of miR-17-5p expression also inhibits the growth of SGC7901/DDP and BGC823/5-FU in gastric cancer cells (P0.05) and induces apoptosis (P0.05). Inhibition of miR-17-5p expression in the SGC7901/DDP and BGC823/5-FU of gastric cancer cells can significantly reduce G1 phase cells, while S cells increase significantly. Cell cycle G1-S phase block (P0.05). Conclusion: the expression of miR-17-5p in the SGC7901/DDP and BGC823/5-FU cells of drug resistant cells is significantly higher than that of non drug resistant gastric cancer cells. Inhibition of miR-17-5p expression can obviously improve the drug resistance of gastric cancer cells, inhibit the growth of gastric cancer cells and induce cell apoptosis caused by cell cycle. Phase G1-S phase block. The molecular mechanism of miR-17-5p may be to influence the apoptosis and proliferation of gastric cancer cells by regulating the expression of its target gene p21. This study found that the expression of miR-17-5p can significantly reduce the drug resistance of gastric cancer cells and provide theoretical support and new molecular target for overcoming the problem of cell resistance in gastric cancer chemotherapy.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2

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