胃癌耐药相关microRNA表达谱及miR-17-5p逆转胃癌细胞耐药作用的研究

发布时间：2018-06-25 15:59

本文选题：MicroRNA + miR-17-5p　；参考：《浙江大学》2016年博士论文

【摘要】：背景：胃癌是全球肿瘤致死主要原因之一,在我国是仅次于肺癌死亡率排名第二的恶性肿瘤。化疗是胃癌治疗的重要手段之一。胃癌细胞对化疗药物产生耐药性导致胃癌化疗失败的主要原因之一。胃癌患者如果对化疗出现耐药情况,预后较差,所以明确胃癌细胞产生耐药的分子机制十分重要。microRNA (miRNA)是内源性非编码小RNA分子,它们通过调节靶基因mRNA的翻译或降解而起到调节基因的作用。miRNA在肿瘤中通过对下游靶基因的调节,对其发生与发展起到至关重要的作用。近年来的研究证实了miRNA在抗肿瘤药物的敏感性和耐药性中起了重要作用。以特定的miRNA为治疗靶点,通过比较miRNA表达谱,与肿瘤细胞耐药性密切相关的特定miRNA将被识别,这可能为新的靶向治疗方案开辟途径,从而改善治疗效果。然而miRNA影响胃癌细胞耐药性的作用机制并不明了。因此准确寻找对胃癌耐药细胞影响较大的miRNA并探讨其作用机制,成为亟待解决的重要问题。本研究拟在两种耐药胃癌细胞SGC7901/DDP和BGC823/5-FU中筛选出与亲本细胞表达差异较大的miRNA,并深入探讨其对细胞耐药性的影响作用及其作用机制。目的：在两种耐药胃癌细胞SGC7901/DDP和BGC823/5-FU中分析miRNA表达谱,筛选耐药相关特异性miRNA;研究耐药相关miRNA对胃癌细胞株耐药性的影响；寻找潜在靶点,探讨其作用分子机制,以期为新的靶向治疗方案开辟途径。方法：采用基因芯片技术分别检测胃癌细胞SGC7901及其耐药细胞SGC7901/DDP、胃癌细胞BGC823及其耐药细胞BGC823/5-FU的miRNA的表达差异情况,寻找在两株胃癌耐药细胞中与亲本细胞之间寻找表达差异较大的miRNA,并采用实时定量PCR方法进行验证。通过体外转染抑制序列anti-miR-17-5p序列下调细胞中的miR-17-5p表达,CCK-8法分析转染后细胞对长春新碱、阿霉素、5-氟尿嘧啶和顺铂的药物敏感性变化。运用生物信息学工具PicTar和Miranda algorithms预测miR-17-5p的靶基因。构建荧光素酶报告质粒pGL3-p21-3'-UTR验证miR-17-5p与预测靶基因p21的相互作用。通过WesternBlot方法检测胃癌耐药细胞株SGC7901/DDP和BGC823/5-FU中miR-17-5p对p21的调控作用。流式细胞术检测miR-17-5p对胃癌耐药细胞株SGC7901/DDP和BGC823/5-FU细胞凋亡和细胞周期的影响。细胞计数的方法检测miR-17-5p对胃癌耐药细胞株SGC7901/DDP和BGC823/5-FU细胞生长的影响。结果：基因芯片分析结果显示：与SGC7901细胞相比,SGC7901/DDP细胞中miR-17-5p的表达明显上调(P0.01); BGC823/5-FU细胞中miR-17-5p的表达较BGC823细胞明显上调(P0.01)。实时定量荧光PCR分析结果显示：耐药SGC7901/DDP与BGC823/5-FU细胞中miR-17-5p表达水平均显著上调(P0.01)。抑制SGC7901/DDP细胞和BGC823/5-FU细胞中miR-17-5p表达可有效提高细胞对顺铂、阿霉素、长春新碱和5-FU的药物敏感性(P0.05)。根据生物信息学工具的预测miR-17-5p的靶基因可能为p21。对p21基因3’非编码区报告质粒荧光素酶活性测定显示miR-17-5p可显著降低pGL3-p21-3'-UTR相对荧光酶活性(P0.05)。在胃癌耐药细胞SGC7901/DDP和BGC823/5-FU中抑制miR-17-5p表达可明显增加p21蛋白的表达(P0.05)。抑制miR-17-5p表达还可抑制胃癌耐药细胞SGC7901/DDP和BGC823/5-FU的生长(P0.05),并诱导细胞凋亡(P0.05)。在胃癌耐药细胞SGC7901/DDP和BGC823/5-FU中抑制miR-17-5p表达可使G1期细胞明显减少,而S期细胞明显增多,引起细胞周期G1-S期阻滞(P0.05)。结论：与亲代非耐药胃癌细胞相比,miR-17-5p在耐药细胞SGC7901/DDP和BGC823/5-FU细胞中表达显著增高。抑制miR-17-5p表达可明显改善胃癌细胞耐药性,同时抑制胃癌耐药细胞的生长活力、诱导细胞凋亡病引起细胞周期G1-S期阻滞。究其分子机制,miR-17-5p可能是通过调控其靶基因p21的表达影响胃癌耐药细胞的凋亡和增殖。本研究发现miR-17-5p的表达可明显降低胃癌细胞的耐药性,为临床上克服胃癌化疗时细胞耐药性难题提供理论支持和新的分子靶标。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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