BIRC基因为靶点治疗去势抵抗性前列腺癌的初步研究

发布时间：2018-03-09 05:36

本文选题：前列腺癌　切入点：IAP　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究BIRC6基因在前列腺癌中的表达水平及对前列腺癌细胞体内、外生物学行为的影响,并观察联合降低以BIRC6为基础的IAP基因家族表达对前列腺癌细胞生物学行为的影响。方法:运用Western Blot与RT-qPCR检测BIRC6在正常前列腺细胞RWPE-1与前列腺癌细胞系LNCaP、PC-3、C4-2和DU145中蛋白及mRNA的表达水平;构建BIRC6低表达质粒,扩增后提取BIRC6低表达质粒,转染至PC-3细胞,分别应用Western Blot与RT-qPCR验证BIRC6在PC-3细胞系中蛋白和mRNA表达水平;使用MTT、CCK-8方法检测低表达BIRC6的PC-3的细胞增殖能力;应用单克隆形成实验观察BIRC6低表达后对PC-3细胞生长状况的影响;用Transwell方法检测BIRC6低表达对前列腺癌细胞侵袭能力的变化;采取磷脂酰丝氨酸外翻法(Annexin V)检测BIRC6低表达后对前列腺癌凋亡的改变;使用流式细胞术分析BIRC-6低表达后对细胞生长周期的变化;将PC-3细胞种植于NOD-SCID小鼠皮下,使用BIRC6特异反义寡核酸腹腔注射观察对肿瘤生长的影响。同时设计除BIRC6外的IAP家族基因BIRC2低表达质粒,与BIRC6低表达质粒共转染PC-3细胞,观察其对上述生物学行为的影响。记录、整理实验数据及图像资料,需使用统计分析的实验结果均运用SPSS软件进行统计学分析。结果:前列腺癌细胞系中BIRC-6的蛋白及mRNA表达情况普遍高于正常前列腺细胞,并且在LNCaP及PC-3细胞系表达量显著升高;我们发现BIRC6基因在CRPC患者组织中表达明显升高,故选用了雄激素非依赖性PC-3细胞作为研究对象;构建BIRC6低表达质粒转染PC-3细胞后发现,BIRC6低表达可显著抑制PC-3细胞生长,降低其侵袭能力,促进细胞的凋亡,影响细胞生长周期,使停滞于G2/M期的细胞明显增多。在同时转染BIRC6与BIRC2低表达质粒的PC-3细胞中,我们同样观察到抑制前列腺癌细胞增殖和促进凋亡的现象,并且其抑制作用更加强烈。在小鼠皮下成瘤实验中,同时抑制两种IAP基因的反义寡核苷酸可更有效的抑制肿瘤的生长。结论:BIRC6表达改变可影响前列腺癌细胞生物学行为,并且同时降低以BIRC6为基础的联合IAP家族成员BIRC2可更有效的抑制前列腺癌在体内、外生长。
[Abstract]:Objective: to study the expression level of BIRC6 gene in prostate cancer and its effect on the biological behavior of prostate cancer cells in vivo and in vitro. To observe the effect of combined reduction of IAP gene family expression based on BIRC6 on the biological behavior of prostate cancer cells. Methods: Western Blot and RT-qPCR were used to detect the expression of BIRC6 in normal prostatic cells RWPE-1 and prostate cancer cell lines LNCaPnPC-3C4-2 and DU145. The expression level of white and mRNA; The low expression plasmid of BIRC6 was constructed, the low expression plasmid of BIRC6 was extracted and transfected into PC-3 cells, the expression level of BIRC6 and mRNA in PC-3 cell line were verified by Western Blot and RT-qPCR, and the proliferation ability of PC-3 with low expression of BIRC6 was detected by MTT- CCK-8 method. The effect of low expression of BIRC6 on the growth of PC-3 cells was observed by monoclonal formation assay, and the change of invasion ability of prostate cancer cells by low expression of BIRC6 was detected by Transwell method. The changes of apoptosis in prostate cancer were detected by Annexin V) after low expression of BIRC6. Flow cytometry was used to analyze the changes of cell cycle after low expression of BIRC-6. PC-3 cells were implanted subcutaneously in NOD-SCID mice. The effect of BIRC6 specific antisense oligodeoxynucleic acid (BIRC6) on tumor growth was observed by intraperitoneal injection. The low expression plasmid of IAP family gene BIRC2 except BIRC6 was designed and co-transfected with BIRC6 low expression plasmid to observe its effect on the above biological behavior. The experimental data and image data were analyzed by SPSS software. Results: the expression of BIRC-6 protein and mRNA in prostate cancer cell line was generally higher than that in normal prostate cell line. The expression of BIRC6 gene was significantly increased in the tissues of CRPC patients, so androgen independent PC-3 cells were selected as the research objects. After transfection of PC-3 cells with BIRC6 low expression plasmid, it was found that low expression of BIRC6 could significantly inhibit the growth of PC-3 cells, reduce its invasion ability, promote cell apoptosis and affect cell growth cycle. In PC-3 cells transfected with both BIRC6 and BIRC2 low expression plasmids, we also observed the inhibition of prostate cancer cell proliferation and the promotion of apoptosis. In murine subcutaneous tumorigenesis, antisense oligonucleotides of two IAP genes can inhibit tumor growth more effectively. Conclusion the change of the expression of BIRC6 may affect the biological behavior of prostate cancer cells. At the same time, the reduction of BIRC2, a member of the IAP family based on BIRC6, was more effective in inhibiting prostate cancer growth in vivo and in vitro.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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