NOP16低表达增强前列腺癌化疗敏感性的初步研究

发布时间：2018-08-31 14:03

【摘要】：目的:研究NOP16表达水平改变对前列腺癌细胞体外、体内Docetaxel化疗敏感性的影响,并初步探讨其分子机制。方法:运用RT-q PCR方法检测NOP16在PCa和BPH组织中的表达量;利用RT-q PCR及Western Blot检测NOP16在正常前列腺细胞系RWPE-1以及PCa细胞系LNCa P和PC3中的表达量;构建慢病毒载体,将其与PC3细胞一起培养转染PC3细胞,使NOP16短发卡RNA(sh RNA)在PC3细胞内进行表达,经药物筛选及进行稳定转染获得稳定NOP16低水平的PC3细胞株,并使用RT-q PCR、Western Blot验证转染后细胞中NOP16的表达量;使用cellcount法和CCK-8法检测NOP16-KDPC3细胞增殖能力的变化,使用细胞迁移迁徙实验技术(Transwell)检测NOP16-KDPC3细胞迁徙能力,磷脂酰丝氨酸外翻分析(Annexin V)检测NOP16-KDPC3细胞的凋亡的变化。使用不同浓度Docetaxel处理PC3细胞24小时、48小时后CCK-8法检测细胞活性;使用亚致死量Docetaxel处理PC3-Vector、PC3-NOP16-KD细胞,使用CCK-8检测细胞活性。使用PC3-Vector和PC3-NOP16-KD细胞做单克隆形成实验并且应用Docetaxel处理,检测细胞增殖能力;培养PC3-Vector和PC3-NOP16-KD细胞并且应用Docetaxel处理后做凋亡实验,检测细胞凋亡情况;培养PC3-Vector和PC3-NOP16-KD细胞并且应用Docetaxel处理后做细胞周期实验,检测细胞增殖情况;培养PC3-Vector和PC3-NOP16-KD细胞并且应用Docetaxel处理后做Transwell实验,检测细胞迁徙情况。使用Western Blot检测PC3、PC3-Vector和PC3-NOP16-KD三种细胞分别被0nmol/L、5nmol/L、10nmol/L三种浓度的Docetaxel处理24小时后的Caspase3含量,检测细胞凋亡。结果:NOP16在Pca组织中表达高于BPH组织;NOP16在前列腺癌细胞系LNCa P及PC3中表达高于正常前列腺细胞系RWPE-1,而在上述两种不同的前列腺癌细胞系比较中没有观察到明显的变化;使用生物工程技术,利用慢病毒作为载体将sh RNA转染进入PC3细胞,将NOP16表达下调。可以通过PC3细胞建立PC3-NOP16-KD稳定表达的细胞;通过特异性sh RNA的慢病毒载体可以下调内源性NOP16水平,借此可出现细胞体外增殖能力下降的现象;通过PC3细胞的NOP16表达降低可以抑制细胞迁徙能力;观察PC3-NOP16-KD细胞可以发现与正常PC3细胞相比其明显易发生凋亡;观察发现PC3-NOP16-KD细胞内蛋白合成水平较低。PC3细胞经过不同浓度Docetaxel处理后,细胞的活性降低,计算其半数致死量发现其中IC50约在30nmol/L(24h)、20nmol/L(48h)之间;使用亚致死量浓度的Docetaxel,分别暴露PC3-Vector、PC3-NOP16-KD细胞,结果显示:随Docetaxel浓度逐渐加大后细胞活性呈明显下降,NOP16-KD细胞对Docetaxel的浓度更加敏感,提示抑制NOP16可以强化Docetaxel对前列腺癌细胞的抑制作用;NOP16-KD组细胞克隆形成数目显著少于对照组,与对照组相比Docetaxel处理后NOP16-KD组细胞单克隆数明显降低,提示NOP16可能通过参与细胞增殖能力抵抗Docetaxel对细胞的损伤作用;与对照组相比NOP16-KD组细胞凋亡比率明显较高,与对照组相比Docetaxel处理后NOP16-KD组细胞凋亡同样明显升高,提示抑制NOP16表达可能提高PC3细胞的Docetaxel化疗敏感性;细胞周期实验显示NOP16-KD组细胞增殖能力低于对照组;此外,Docetaxel处理后NOP16-KD组细胞增值能力同样显著低于对照组。提示NOP16可能通过参与细胞增殖能力抵抗Docetaxel对细胞的抑制作用;细胞迁徙实验结果显示NOP16-KD组细胞迁徙能力低于对照组,Docetaxel处理后NOP16-KD组细胞迁徙能力同样显著低于对照组,提示NOP16可能通过参与细胞迁徙能力抵抗Docetaxel对细胞的抑制作用;PC3-NOP16-KD组Caspase3含量大于对照组,此外经过Docetaxel处理后Caspase3含量呈现Docetaxel剂量依赖性增加明显大于对照组,说明NOP16表达异常与Docetaxel化疗可以协同作用诱发凋亡。裸鼠成瘤实验证实NOP16-KD细胞成瘤体积小于对照组,Docetaxel化疗后NOP16-KD组小鼠肿瘤体积进一步缩小,体内实验显示NOP16能够显著提高Docetaxel化疗敏感性。结论:NOP16异常表达可改变前列腺癌细胞的体外生物学行为,体外、体内实验显示NOP16-KD可以提高Docetaxel化疗敏感性。NOP16具有成为基因治疗前列腺癌的潜在靶点。
[Abstract]:Objective: To study the effect of NOP16 expression on the sensitivity of prostate cancer cells to Docetaxel chemotherapy in vitro and in vivo, and to explore its molecular mechanism.Methods: The expression of NOP16 in PCa and BPH tissues was detected by RT-q PCR, and the expression of NOP16 in normal prostate cell line RWPE-1 and PCa cell line L was detected by RT-q PCR and Western Blot. The expression of NOP16 short hairpin RNA (sh RNA) in PC3 cells was obtained by drug screening and stable transfection, and the expression of NOP16 in PC3 cells was verified by RT-q PCR and Western Blot. Cell count assay and CCK-8 assay were used to detect the proliferation of NOP16-KDPC3 cells, cell migration assay (Transwell) was used to detect the migration ability of NOP16-KDPC3 cells, and phosphatidylserine valgus assay (Annexin V) was used to detect the apoptosis of NOP16-KDPC3 cells. Cell viability was measured by CCK-8 assay hours later; PC3-Vector and PC3-NOP16-KD cells were treated with sublethal dose of Docetaxel, and the cell viability was detected by CCK-8 assay. Monoclonal formation experiments were performed with PC3-Vector and PC3-NOP16-KD cells, and cell proliferation was detected by Docetaxel treatment; PC3-Vector and PC3-NOP16-KD cells were cultured with Docetax. Cell proliferation was detected by cell cycle assay after treatment with Docetaxel; PC3-Vector and PC3-NOP16-KD cells were cultured with Docetaxel; PC3-Vector and PC3-NOP16-KD cells were cultured with Docetaxel and Transwell assay was performed after treatment with Docetaxel. The expression of NOP16 in PC3, PC3-Vector and PC3-NOP16-KD cell lines LNCa P and PC3-NOP16-KD was higher than that in normal prostate cell lines RWPE-1 and NOP16 was higher than that in Pca and PC3-NOP16-KD cell lines RWPE-1. No significant changes were observed in the two different prostate cancer cell lines mentioned above; sh RNA was transfected into PC3 cells using lentiviruses as vectors by biotechnology and NOP16 expression was down-regulated. PC3-NOP16-KD stably expressed cells could be established by PC3 cells; and sh RNA-specific lentiviral vectors could be down-regulated. The level of endogenous NOP16 could induce the decrease of cell proliferation in vitro; the decrease of NOP16 expression in PC3 cells could inhibit cell migration; the apoptosis of PC3-NOP16-KD cells could be observed in comparison with normal PC3 cells; the protein synthesis level of PC3-NOP16-KD cells was lower than that of normal PC3 cells. The activity of PC3-Vector and PC3-NOP16-KD cells decreased after treatment with different concentrations of Docetaxel, and the half lethal dose of IC50 was found to be between 30 nmol/L (24 h) and 20 nmol/L (48 h). PC3-Vector and PC3-NOP16-KD cells were exposed to sublethal concentration of Docetaxel, respectively. Cells were more sensitive to the concentration of Docetaxel, suggesting that inhibiting NOP16 could enhance the inhibitory effect of Docetaxel on prostate cancer cells; the number of cell clones in NOP16-KD group was significantly less than that in control group, and the number of monoclonal cells in NOP16-KD group was significantly lower than that in control group, suggesting that NOP16 might participate in cell proliferation by inhibiting the concentration of Docetaxel. Compared with the control group, the apoptosis rate of NOP16-KD group was significantly higher, and the apoptosis rate of NOP16-KD group was also significantly higher, suggesting that inhibiting the expression of NOP16 might increase the sensitivity of PC3 cells to Docetaxel chemotherapy. In addition, the proliferation of NOP16-KD cells after Docetaxel treatment was also significantly lower than that of the control group. NOP16 may be involved in the ability of cell proliferation to resist the inhibition of Docetaxel on cells. Cell migration test showed that NOP16-KD cells migration ability was lower than that of the control group, and NOP16-KD cells were fine after Docetaxel treatment. Cell migration ability was also significantly lower than that of the control group, suggesting that NOP16 might be able to resist the inhibition of Docetaxel by participating in cell migration ability; the content of Caspase 3 in PC3-NOP16-KD group was higher than that of the control group; moreover, the content of Caspase 3 in Docetaxel-treated group was significantly higher than that in the control group, suggesting that the expression of NOP16 was different. The tumor-forming volume of NOP16-KD cells in nude mice was smaller than that of the control group. The tumor volume of NOP16-KD group was further reduced after Docetaxel chemotherapy. In vivo experiments showed that NOP16 could significantly increase the sensitivity of Docetaxel chemotherapy. Conclusion: The abnormal expression of NOP16 could change prostate cancer cells. Biological behavior in vitro and in vivo experiments have shown that NOP16-KD can increase the sensitivity of Docetaxel to chemotherapy. NOP16 may be a potential target for gene therapy of prostate cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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