MiR-1285对胰腺癌细胞恶性生物学行为的影响及作用机制

发布时间：2018-04-21 00:16

本文选题：胰腺癌 + microRNA　；参考：《北京协和医学院》2015年博士论文

【摘要】：背景胰腺癌早期诊断困难、恶性程度高,是预后最差的恶性肿瘤之一,5年生存率低于7%。MiRNAs因可同时参与肿瘤的增殖、凋亡、耐药及侵袭等多个功能通路的调控而受到广泛关注。miR-1285属于miR-612/1285/3187-5p簇中的一员,目前只有在肝癌和肾细胞肾癌中有所报道,但其在胰腺癌中的调控机制仍不明确,因此我们将miR-1285作为本研究的研究对象,深入研究其对胰腺癌增殖、周期、化疗敏感性、侵袭、迁移、凋亡等恶性生物学行为的影响,并寻找其靶基因,探索其调控机制。YAP1蛋白是Hippo信号通路的主要效应因子之一,近两年来大量研究证实其在多种恶性肿瘤中均发挥促癌作用。目的1.明确miR-1285对胰腺癌细胞株增殖、周期、化疗敏感性、侵袭、迁移、凋亡等表型的调控作用；2.明确miR-1285的靶基因；3.初步探索miR-1285及靶基因的下游信号通路。方法本研究首先通过实时定量PCR(探针法)检测检测miR-1285在各细胞系中的表达量,并选取细胞株作为研究对象。随后,通过脂质体瞬时转染法改变细胞内源性miR-1285的表达后,采用CCK-8细胞增殖实验检测miR-1285对胰腺癌细胞增殖能力和化疗敏感性的影响,采用PI单标记流式细胞术检测细胞周期,采用transwell法检测细胞迁移和侵袭能力,采用Annexin V/PI双标记流式细胞术检测细胞凋亡,验证miR-1285对胰腺癌细胞系恶性生物学行为的影响。随后,通过生物信息学数据库预测miR-1285的靶基因,将多个数据库预测到的结果进行比对,并实时定量PCR(染料法)和western blot法分别检测靶基因mRNA和蛋白的表达,同时进一步验证miR-1285-YAP1的下游效应蛋白的表达水平。结果1.MiR-1285在胰腺癌耐药株中较亲本株低表达；2.体外试验中,miR-1285可显著抑制胰腺癌细胞增殖,抑制胰腺癌细胞G1→S期转化,增加吉西他滨化疗敏感性,抑制细胞迁移和侵袭能力,促进胰腺癌细胞凋亡；3.MiR-1285的靶基因为YAP1,可能通过抑制YAP1mRNA翻译的途径抑制YAP1蛋白表达；4. MiR-1285-YAP1下游通路蛋白包括,EGFR、β-catenin、Bim、PTEN等。结论1.MiR-1285通过下调YAP1蛋白表达,在胰腺癌细胞株中发挥抑癌作用；2. EGFR、β-catenin、Bim、PTEN等蛋白参与miR-1285-YAP1对胰腺癌细胞恶性表型的调控。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.9

【共引文献】