沉默DDX49基因对骨肉瘤细胞增殖的影响

发布时间：2018-03-06 08:00

本文选题：骨肉瘤　切入点：DDX49基因　出处：《兰州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景骨肉瘤是最常见的骨原发性恶性肿瘤,其恶性程度高,容易发生远处转移。与其它的骨肿瘤相比,骨肉瘤患者的预后较差。然而,骨肉瘤患者的生存率在过去30年里没有得到明显的提高。很多学者认为通过研究骨肉瘤的生物学特性将有助于找到治疗骨肉瘤的有效方法。DEAD-box RNA解旋酶不仅参与几乎所有与RNA代谢有关的过程,还参与调控转录和翻译,并且与细胞增殖及肿瘤转化有关。DDX49是DEAD-box RNA解旋酶家族中的一个成员,目前尚无具体阐述DDX49在肿瘤细胞中的表达及其生物学功能的研究报道。目的本研究旨在研究DDX49基因在骨肉瘤细胞增殖及凋亡中的作用,为骨肉瘤的诊断和治疗提供新的分子靶点。方法用实时定量聚合酶链反应(Quantitative real-time polymerase chain reaction,qRT-PCR)检测DDX49基因在四种骨肉瘤细胞中的表达,用慢病毒介导的RNA干扰技术沉默DDX49在U-2OS细胞中的表达,然后用qRT-PCR检测DDX49基因的敲减效率,用Celigo细胞扫描分析仪动态观察细胞增殖情况及细胞个数的变化,用噻唑兰(methylthiazoletetrazolium,MTT)实验检测细胞活力,用流式细胞仪检测细胞凋亡。结果qRT-PCR结果DDX49基因在四种骨肉瘤细胞中有表达;经RNA干扰慢病毒感染U-2OS细胞3天后,qRT-PCR结果显示RNA干扰慢病毒能有效抑制U-2OS细胞中DDX49基因在mRNA水平的表达;经慢病毒感染U-2OS细胞后用Celigo细胞扫描分析仪连续检测5天,结果显示,抑制DDX49基因的表达能显著抑制U-2OS细胞的增殖;经慢病毒感染U-2OS细胞3天后,MTT实验连续检测5天,结果显示,与DDX49基因非沉默组相比,沉默组在波长490 nm处的吸光率随时间增加逐渐降低,表明抑制DDX49基因的表达能明显降低U-2OS细胞的活力;经慢病毒感染5天后,DDX49沉默组发生凋亡的U-2OS细胞显著增加,提示DDX49基因与U-2OS细胞的凋亡显著相关。结论DDX49基因在人骨肉瘤细胞U-2OS细胞的增殖和凋亡中起重要作用,可能是潜在的治疗骨肉瘤的分子靶点。
[Abstract]:Background Osteosarcoma is the most common primary malignant tumor of bone, which is highly malignant and prone to distant metastasis. The survival rate of osteosarcoma patients has not improved significantly in the past 30 years. Many scholars believe that studying the biological characteristics of osteosarcoma will help to find an effective way to treat osteosarcoma. DEAD-box RNA helicase is not only involved in several aspects of osteosarcoma. All processes associated with RNA metabolism, DDX49 is also involved in the regulation of transcription and translation, and is a member of the DEAD-box RNA helicase family associated with cell proliferation and tumor transformation. At present, there are no reports on the expression and biological function of DDX49 in tumor cells. Objective to study the role of DDX49 gene in the proliferation and apoptosis of osteosarcoma cells. Methods the expression of DDX49 gene in four kinds of osteosarcoma cells was detected by real-time quantitative real-time polymerase chain reactionation qRT-PCR, which provided a new molecular target for the diagnosis and treatment of osteosarcoma. Lentivirus mediated RNA interference technique was used to silence the expression of DDX49 in U-2OS cells, then qRT-PCR was used to detect the knockdown efficiency of DDX49 gene, and Celigo cell scanning analyzer was used to dynamically observe the cell proliferation and cell number. Cell viability was detected by methylthiazole tetrazolium MTT assay and apoptosis was detected by flow cytometry. Results qRT-PCR results showed that DDX49 gene was expressed in four osteosarcoma cells. After 3 days of lentivirus infection with RNA, the results of qRT-PCR showed that RNA interfering lentivirus could effectively inhibit the expression of DDX49 gene in U-2OS cells at the mRNA level, and detected by Celigo cell scanning analyzer for 5 days after lentivirus infection. Inhibiting the expression of DDX49 gene could significantly inhibit the proliferation of U-2OS cells, and after 3 days of lentivirus infection, the proliferation of U-2OS cells was detected continuously for 5 days. The results showed that, compared with the non-silencing group of DDX49 gene, the proliferation of U-2OS cells was significantly inhibited after lentivirus infection. The absorptivity of the silencing group at 490nm decreased gradually with the increase of time, which indicated that inhibiting the expression of DDX49 gene could significantly reduce the activity of U-2OS cells, and that the apoptosis of U-2OS cells in the silencing group after 5 days of lentivirus infection was significantly increased. Conclusion DDX49 gene plays an important role in the proliferation and apoptosis of human osteosarcoma cell line U-2OS and may be a potential molecular target for the treatment of osteosarcoma.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738.1

【相似文献】