miR-15a和miR-16-1在骨肉瘤细胞凋亡机制中的作用

发布时间：2018-04-30 19:20

  本文选题：骨肉瘤 + miR-15a　； 参考：《第四军医大学》2015年博士论文

【摘要】：背景:骨肉瘤是最常见的原发性恶性骨肿瘤,临床上由于该肿瘤发病及进展迅速,早期易转移,治疗过程中容易产生耐药,致使许多患者失去生命。而导致骨肉瘤发生和发展的机制仍未被研究透彻。已有的实验成果认为,在癌细胞的基本代谢活动过程中,mi R-15a和mi R-16-1起到了重要调控作用。研究它们在骨肉瘤细胞内的具体功能有望进一步完善该肿瘤发生和发展的机制。目的:本实验旨在发现并验证mi R-15a和mi R-16-1在骨肉瘤细胞中的异常表达状态,研究它们对细胞生命程序的影响,揭示其在骨肉瘤细胞凋亡机制中的作用。方法:1.选取34例骨肉瘤病理蜡块样本进行实时定量PCR分析,并与前期组织芯片结果对比,分析差异表达情况。2.采用瞬时转染法构建高表达mi R-15a和mi R-16-1的骨肉瘤细胞系,观察转染效果并计算转染效率。3.搜集转染48小时后的存活细胞,使用TUNEL、流式细胞仪及MTT法研究高表达mi R-15a和mi R-16-1的SOSP-9607细胞在细胞周期、凋亡及增殖过程中的变化。4.利用基因信息学软件、文献回顾及RT-PCR实验方法确定靶基因研究对象。5.使用免疫组织化学、Western blot、RT-PCR及荧光素酶报告基因依次从组织细胞、蛋白、m RNA及DNA水平验证靶基因。结果:1.34例骨肉瘤蜡块标本中mi R-15a和mi R-16-1的相对表达定量与瘤旁标本相比有所降低,统计学分析有差异(P0.05)。2.转染4小时后,经过镜下观察并计算转染效率,结果表明转染成功,可用于后续实验。3.转染48小时后,流式细胞仪和TUNEL法凋亡检测结果表明高表达mi R-15a和mi R-16-1的SOSP-9607细胞凋亡率较抑制物组和对照组相比,显著增加(P0.05)。流式细胞仪周期分析结果显示转染后处于G0/G1期的细胞明显多于对照组,说明细胞周期减缓(P0.05)。以MTT法绘制的细胞增殖曲线显示转染后的肿瘤细胞增殖速度较其他组有所减缓(P0.05)。上述实验中,抑制物组和对照组的组间和组内结果对比均无差异(P0.05)。4.经过基因信息学软件筛选,文献回顾分析以及RT-PCR的初步筛查,最终确定待研究靶基因为CCND1。5.荧光素酶报告基因分析结果揭示出mi R-15a和mi R-16-1可以抑制CCND1-b组的荧光强度(P0.05),而CCND1-a组则不受影响(P0.05)。说明mi R-15a和mi R-16-1通过与CCND1基因3’UTR区域的CCND1-b段结合抑制CCND1基因的表达。结论:mi R-15a和mi R-16-1可以通过抑制CCND1基因表达促进SOSP-9607细胞凋亡、阻抑细胞周期,抑制细胞增殖。
[Abstract]:Background: osteosarcoma is the most common primary malignant bone tumor. Due to the rapid onset and progression of osteosarcoma early metastasis and drug resistance in the course of treatment many patients lost their lives. However, the mechanisms leading to the development of osteosarcoma have not been thoroughly studied. It has been suggested that mi R-15a and mi R-16-1 play an important role in the basic metabolic activity of cancer cells. The study of their specific function in osteosarcoma cells is expected to further improve the mechanism of tumorigenesis and development. Aim: to detect and verify the abnormal expression of mi R-15a and mi R-16-1 in osteosarcoma cells, to study their effects on cell life process and to reveal their role in the apoptosis mechanism of osteosarcoma cells. Method 1: 1. 34 cases of osteosarcoma were analyzed by real-time quantitative PCR and compared with the results of tissue microarray. Osteosarcoma cell lines with high expression of miR-15a and miR-16-1 were constructed by transient transfection method. The transfection effect was observed and the transfection efficiency was calculated. The survival cells were collected after 48 hours of transfection. Tunel, flow cytometry and MTT were used to study the changes in cell cycle, apoptosis and proliferation of SOSP-9607 cells with high expression of miR-15a and miR-16-1. Using gene informatics software, literature review and RT-PCR experiment method to determine the target gene research object. RT-PCR and luciferase reporter genes were used to identify target genes from tissue cells, protein RNA and DNA. Results the relative quantitative expression of miR-15a and miR-16-1 in paraffin specimens of 1. 34 cases of osteosarcoma was lower than that of the adjacent specimens, and there was a significant difference between them (P0.05. 2). After 4 hours of transfection, the transfection efficiency was observed and calculated under microscope. The results showed that the transfection was successful and could be used in the following experiment. 3. After 48 hours of transfection, the apoptotic rate of SOSP-9607 cells with high expression of miR-15a and miR-16-1 was significantly higher than that of the control group and the control group by flow cytometry and TUNEL assay, and the results showed that the apoptosis rate of SOSP-9607 cells with high expression of miR-15a and miR-16-1 was significantly higher than that of the control group. The results of flow cytometry showed that the number of cells in the G0/G1 phase after transfection was significantly higher than that in the control group, indicating that the cell cycle slowed down (P 0.05). The cell proliferation curve plotted by MTT method showed that the proliferation rate of tumor cells after transfection was slower than that of other groups. In the above experiments, there was no difference in the results between the inhibition group and the control group. After the screening of gene informatics software, literature review and preliminary screening of RT-PCR, the target gene was determined to be CCND 1.5. The results of luciferase reporter gene analysis showed that miR-15a and miR-16-1 could inhibit the fluorescence intensity of CCND1-b group (P0.05), but CCND1-a group was not affected. It is suggested that mi R-15a and mi R-16-1 inhibit the expression of CCND1 gene by binding to the CCND1-b segment of 3'UTR region of CCND1 gene. Conclusion the cell cycle and cell proliferation of SOSP-9607 cells can be inhibited by inhibiting the expression of CCND1 gene by inhibiting the apoptosis of SOSP-9607 cells.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R738.1
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本文编号：1825844