非小细胞肺癌中miR-181a-5p功能的研究.docVIP

miR-181a-5p在非小细胞肺癌中的功能研究 [摘 要] 目的 探索miR-181a-5p 的抑癌机制，并探讨它作为标志物用于检测肺癌细胞的可行性。方法：采用CCK8（Cell Counting Kit-8）方法和流式细胞术分别检测细胞增殖情况和细胞凋亡情况; 蛋白质印迹技术检测miR-181a-5p对靶蛋白表达的影响； 细胞划痕和Transwell实验检测miR-181a-5p对细胞株的运动抑制；利用双荧光报告基因实验对靶基因进行验证。结果：结果显示miR-181a-5p抑制可A549细胞株的增殖，并且促进细胞的早期凋亡；A549细胞的运动受到miR-181a-5p显著的抑制；miR-181a-5p 直接作用于靶基因的3′UTR行使调控。结论： miR-181a-5p可显著抑制NSCLC (non-small cell lung cancer) 细胞生物进程，发挥抑癌基因的作用。 [关键词] miR-181a-5p；增殖；迁移；凋亡；非小细胞肺癌 [中图分类号] [文献标识码] Analysis of miR-181a-5p function in Non- small cell lung cancer [Abstract] Objective The aims of this study were to explore the biologic functions in NSCLC of miR-181a-5p and to investigate whether it can be a marker for detecting NSCLC. Methods 1.To examine the cell growth and apoptosis, CCK8 and flow cytometry were applied ,respectively. 2. To determine the expression effects of miR-181a-5p, western blot blot was used.3. Transwell assay and wound healing assay. 4. Plasmid constructs and luciferase reporter assay.Results 1. miR-181a-5p inhibited cell proliferation and promoted cell apoptosis in vitro.2. Transwell assay and wound healing assay showed that miR-181-5p negatively regulated cell migration in vitro.3. miR-181a-5p down-regulated Kras expression by directly targeting its 3′-UTR 5. Kras is inversely correlated with miR-181a-5p expression in NSCLC. Conclusion miR-181a-5p might provide a potential treatment approach for NSCLC patients. [Keywords] miR-181a-5p;Proliferation；migration;?apoptosis;?non small?cell lung cancer 恶性肿瘤为全球较大的公共卫生问题之一，给人类的身心健康带来了极大的危害，并将在新世纪成为威胁人类生命的第一大杀手[1-2]。在肿瘤的发生发展过程中，miRNA通过调控基因表达影响细胞功能，起抑癌或促癌作用。起抑癌作用的miRNA主要通过调控功基因来抑制细胞的增殖、迁移或促进细胞的凋亡来抑制肿瘤的发生[3-4] 1 材料与方法 1.1 实验材料 非小细胞肺癌细胞株SPCA-1 购自ATCC；非小细胞肺癌细胞株A549 购自中科院生化细胞所细胞库。 1.3 实验方法 （1）细胞培养：非小细胞肺癌细胞株A549、SPCA-1分别培养在含有10%胎牛血清、青霉素105IU/L、链霉素100mg/L的1640和DMEM培养基中，培养基于37℃，CO2体积分数为5%的细胞培养箱内培养。细胞融合到85%左右时，用0.25%胰酶消化，传代[9]。。 （2）细胞增殖实验[5]： 取对数生长期的细胞，以1×104个/mL的密度接种于96孔培养板中，每孔接种100μL，培养待细胞贴壁后，再分别加入miR-181a-5p、anti-miR-181a-5p，每组设置6个复孔，同时设置不受处理因素的对照组（N