靶向Stathmin的siRNA与紫杉醇联合作用于鼻咽癌细胞的效应及机制的研究

发布时间：2018-01-24 16:46

本文关键词： Stathmin siRNA 紫杉醇潜伏膜蛋白1 微管鼻咽癌凋亡增殖侵袭转移　出处：《中南大学》2011年博士论文　论文类型：学位论文

【摘要】：目的：微管是构成细胞骨架的主要组成成分之一,其对于细胞的各种主动运动至关重要,这包括细胞形态的改变、细胞的移动、染色体的分离和细胞的分裂等。细胞中各种微管结构和功能的差异决定于其亚细胞定位及其与之结合的微管结合蛋白。Stathmin就是一个已经明确的使微管失稳定的微管结合蛋白,它通过促使微管解聚和隔离组成微管的Tubulin亚基而使微管失稳定,多种激酶可以通过磷酸化和去磷酸化而使Stathmin蛋白的微管失稳定活性失活和活化。鼻咽癌是中国人群特有的高发肿瘤,EB病毒的感染与鼻咽癌的发生发展密切相关,EB病毒编码的潜伏膜蛋白LMP1是一个已经确认的瘤致蛋白质。前期的研究发现Stathmin是LMP1调控网络中的一个下游分子,LMP1可以通过多条通路调节Stathmin的磷酸化,使微管失稳定而促使细胞永生化、增强肿瘤细胞增殖、转移和侵袭。 siRNA是人工RNAi技术中一个重要小分子,其可以激发与之互补的目标mRNA的沉默。具有高特异性、高效率、可遗传等重要特性。本研究以期通过构建的靶向Stathmin的siRNA质粒沉默其蛋白质的表达,降低微管的解聚,增强微管的聚合,从而促使肿瘤细胞凋亡、抑制其增殖、转移和侵袭。紫杉醇是作用于细胞微管的主要抗肿瘤药物之一,它通过促进微管聚合,抑制其解聚,保持微管稳定,抑制细胞有丝分裂,具有显著的放射增敏作用,有着与靶向Stathmin的siRNA类似的作用。因此,本研究拟通过二者联合应用以期放大它们对肿瘤细胞的生物学效应。方法：本文以鼻咽癌细胞CNE1-LMP1为研究模型,用本实验室构建的靶向Stathmin的siRNA质粒为策略,探讨靶向Stathmin的siRNA抑制其蛋白质的表达而对鼻咽癌细胞的凋亡和增殖、侵袭和迁移的影响,并评价Stathmin的siRNA与抗微管化疗药物联合应用的效应。本研究首先以RT-PCR和Western blot证实本研究所应用的靶向Stathmin的siRNA质粒可否抑制鼻咽癌细胞CNE1-LMP1的Stathmin的mRNA和蛋白质的表达水平。以MTT实验证实靶向siRNA对鼻咽癌细胞生长增殖的抑制作用。利用流式细胞术确定靶向Stathmin的siRNA诱导鼻咽癌细胞的凋亡、细胞周期和线粒体膜电位的改变,以AO/EB染色和TUNEL实验证实其对鼻咽癌细胞凋亡的诱导作用,以Western blot检测siRNA转染后细胞凋亡标志性蛋白质Caspase的变化,以期确认鼻咽癌细胞的凋亡及凋亡途径。其次,以Western blot检测转染靶向Stathmin的siRNA的鼻咽癌细胞中可溶性微管和聚合性微管比例的变化。以间接荧光染色检测该siRNA质粒对微管多聚化的调节作用。以划痕实验检测该siRNA对鼻咽癌细胞在二维平面上迁移能力的影响。以Transwell检测该siRNA对鼻咽癌细胞在三维基质中迁移和侵袭能力的调节。再次,以MTT实验检测转染靶向Stathmin的siRNA和紫杉醇联合应用对鼻咽癌细胞增殖作用的影响。以流式细胞术检测二者联合应用对细胞的促凋亡作用和细胞周期的改变。以间接荧光法和Western blot检测转染该siRNA和紫杉醇联合应用对鼻咽癌细胞微管产生影响。最后,以RT-PCR和Western blot检测紫杉醇对鼻咽癌细胞以及其它多种高表达Stathmin细胞A375、MGC和Hela中Stathmin表达在mRNA和蛋白水平的调节作用。结果：首先,RT-PCR和Western blot实验证实了本研究所用的靶向Stathmin的siRNA质粒可以明显抑制鼻咽癌细胞Stathmin的mRNA和蛋白质表达水平。MTT实验检测细胞的生长曲线显示该siRNA的转染对鼻咽癌细胞的增殖也有明显的抑制作用。流式细胞术分析显示该siRNA可明显促进鼻咽癌细胞的凋亡(达到30.9%),使肿瘤细胞阻滞于G2/M期(16.3%)。同时,AO/EB染色、TUNEL实验以及Western blot检测凋亡标志性蛋白Caspase-3、8和9也进一步证实该siRNA对细胞凋亡的促进作用。并且,Caspase和线粒体膜电位的检测也明确该siRNA是通过线粒体凋亡途径诱导细胞凋亡的。其次,Western blot证实向鼻咽癌细胞转染靶向Stathmin的siRNA可明显增加细胞的聚合性微管的量而减少可溶性微管,使得聚合性微管／可溶性微管的比值明显提高(P/S=3.43)；间接免疫荧光也显示转染siRNA的细胞微管长而成束,荧光强度明显提高。虽然二维的划痕实验并没有显示转染了该siRNA的鼻咽癌细胞运动能力有明显的改变,但Transwell实验显示转染该siRNA的细胞迁移和侵袭能力都受到明显抑制。再次,将靶向Stathmin的siRNA质粒和紫杉醇联合应用于鼻咽癌细胞CNE1-LMP1。MTT实验显示联合应用对细胞增殖的抑制效果明显高于其中任何一种方式的单独应用。而且,转染了该siRNA的细胞,其细胞增殖的受抑制的程度与紫杉醇在一定的浓度范围内有剂量关系。间接免疫荧光实验同样显示虽然二者单独应用都可以一定程度上使被处理的细胞微管变粗和变长,荧光强度有所增强,但二者联合应用的结果显示出微管变得更粗和更长,荧光强度更强。同样,Western blot检测细胞的可溶性和聚合性微管的量也显示虽然二者单独应用都可以一定程度上使被处理细胞的聚合性微管增加,可溶性微管减少,聚合性微管／可溶性微管的比值有所增加。但二者联合使用,细胞中的聚合性微管增加和可溶性微管减少的程度要明显的多,聚合性微管／可溶性微管的比值显著提高(P/S=2.05)。最后,研究还发现在鼻咽癌细胞以及其它多种高表达Stathmin的肿瘤细胞中,紫杉醇对细胞中的Stathmin表达有明显的抑制作用,而且,在鼻咽癌细胞中,这种抑制作用与紫杉醇的浓度有一定的剂量效应。结论：靶向Stathmin的siRNA通过抑制其蛋白质的表达而抑制鼻咽癌细胞的增殖,迁移和侵袭,并通过线粒体途径促进细胞凋亡。这种对鼻咽癌细胞凋亡和增殖,迁移和侵袭的调控是通过增强微管的聚合而减少其降解,使微管多聚化、使微管稳定而实现的。紫杉醇可下调Stathmin的表达,靶向Stathmin的siRNA与化疗药物紫杉醇对鼻咽癌细胞有联合治疗效应。这将为鼻咽癌临床的化学治疗提供了一条新的启示。
[Abstract]:Objective: the microtubule is one of the major components of cytoskeleton, it is important for cell active movement, including the changes in cell morphology, cell migration, cell division and chromosome separation. The differences of structure and function of all cells in the microtubule depend on its subcellular localization and binding of microtubule associated protein.Stathmin is already a clear microtubule instability of microtubule binding protein, it makes microtubule instability by Tubulin subunit to microtubule depolymerization and isolation composed of microtubules, multiple kinases through phosphorylation and dephosphorylation of the Stathmin protein and microtubule instability activity inactivation and activation of nasopharyngeal carcinoma is Chinese people. The high incidence of tumors, closely related to the occurrence and development of nasopharyngeal carcinoma and infection of EB virus, EB virus encoding latent membrane protein LMP1 is a confirmed tumor Protein discovery. Previous studies found that Stathmin is a downstream molecule in LMP1 regulatory network. LMP1 can regulate phosphorylation of Stathmin through multiple pathways, causing microtubule instability, promoting cell immortalization and enhancing tumor cell proliferation, metastasis and invasion.
SiRNA is a small molecule artificial RNAi technology, which can stimulate and complementary target mRNA silencing. With high specificity, high efficiency, genetic and other important features. In this study, in order to silence the expression of siRNA protein of plasmid Stathmin by constructing the target, reduce the microtubule depolymerization of microtubules enhanced the polymerization, thereby inducing tumor cell apoptosis, inhibit the proliferation, metastasis and invasion.
Taxol is one of the main antitumor drugs to cells, it is by promoting microtubule polymerization, inhibit the depolymerization of microtubules remain stable, inhibition of cell mitosis, and has remarkable radiosensitizing effect, with Stathmin targeting siRNA similar role. Therefore, this study proposed by the combination of the two methods in order to enlarge their biological effect on tumor cells.
Methods: the nasopharyngeal carcinoma cell line CNE1-LMP1 as a model constructed by our laboratory siRNA plasmid targeting Stathmin strategy, to investigate the expression of Stathmin targeting siRNA inhibits the protein on nasopharyngeal carcinoma cell apoptosis and proliferation, invasion and migration in vitro, and assess the effect of Stathmin and siRNA price antimicrotubule chemotherapy application.
This study firstly by RT-PCR and Western blot confirmed that the expression level of mRNA protein and the research on Application of the siRNA plasmid targeting Stathmin could inhibit CNE1-LMP1 nasopharyngeal carcinoma cell line Stathmin. MTT experiment confirmed that targeting inhibition of siRNA on proliferation of nasopharyngeal carcinoma cells. Using flow cytometry to determine apoptosis of nasopharyngeal carcinoma cell targeting Stathmin siRNA, cell cycle and mitochondrial membrane potential changes with AO/EB staining and TUNEL experiments showed that the induction of apoptosis of nasopharyngeal carcinoma cells, to change Western blot detection siRNA transfected cells apoptosis marker protein Caspase, in order to confirm the apoptosis and apoptosis of nasopharyngeal carcinoma cells.
Secondly, the changes of soluble microtubule with Western detected by blot targeting Stathmin siRNA in nasopharyngeal carcinoma cells and the proportion of polymeric microtubules by indirect fluorescent staining. The siRNA plasmid multimerization of microtubule regulation. In order to influence the scratch assay siRNA on nasopharyngeal carcinoma cell migration in the two-dimensional plane. Detected by Transwell the regulation of siRNA on nasopharyngeal carcinoma cells in three-dimensional matrix in migration and invasion.
Again, to transfect the target detection of MTT experiment to Stathmin siRNA and paclitaxel on the proliferation of nasopharyngeal carcinoma cells. The change was measured by flow cytometry two combined application of cell apoptosis and cell cycle. By indirect immunofluorescence and Western blot detection combined with the transfection of siRNA and paclitaxel the influence on NPC cell microtubules.
Finally, we used RT-PCR and Western blot to detect paclitaxel regulating the expression of Stathmin in A375, MGC and Hela of nasopharyngeal carcinoma cells and many other high expression Stathmin cells at mRNA and protein level.
Results: first, RT-PCR and Western blot experiments confirmed the growth curve of mRNA.MTT and protein expression assay were used in this study targeted siRNA plasmid Stathmin could inhibit the proliferation of nasopharyngeal carcinoma cell line Stathmin showed that transfection of the siRNA in nasopharyngeal carcinoma cells also significantly inhibited. The analysis shows that the siRNA significantly to promote the apoptosis of nasopharyngeal carcinoma cells by flow cytometry (up to 30.9%), the tumor cells in G2/M phase (16.3%). At the same time, AO/EB staining, TUNEL assay and Western blot apoptosis protein marker Caspase-3,8 and 9 confirmed the role of siRNA on cell apoptosis. And the detection of Caspase and mitochondrial membrane potential the siRNA is also clearly induced apoptosis through the mitochondrial apoptotic pathway.
Secondly, Western blot confirmed to nasopharyngeal carcinoma cells transfected with Stathmin targeting siRNA could significantly increase the amount of polymerization of microtubule cells and reduce the ratio of soluble tubulin, microtubule polymerization / soluble microtubules increased significantly (P/S=3.43); indirect immunofluorescence also showed that transfection of siRNA cells with long microtubule bundles, the fluorescence intensity was obviously improved. Although the two-dimensional scratch test did not show the movement ability of the transfected nasopharyngeal carcinoma cell siRNA has obvious change, but Transwell test showed that cell migration and invasion of the transfected siRNA were inhibited.
Again, the siRNA plasmid targeting Stathmin and paclitaxel in nasopharyngeal carcinoma cell CNE1-LMP1.MTT assay showed that the inhibitory effect of combined application of cell proliferation was significantly higher than that of the single application in any way. Moreover, the transfected siRNA cells, the cell proliferation was inhibited and the degree of paclitaxel dose-dependent in concentration within a certain range. Indirect immunofluorescence assay also showed that although the two separate applications can make the processed microtubule thicker and longer to a certain extent, the fluorescence intensity increased, but the combination of the two results show that microtubules become thicker and longer, higher fluorescence intensity. Similarly, soluble Western blot detection cells and aggregation of microtubules also show that although the two separate applications, can to some extent make polymerization of MTS treatment increased soluble microtubule The ratio of aggregated microtubules / soluble microtubules increased. However, the combination of two components increased the number of aggregated microtubules and the decrease of soluble microtubules in cells. The ratio of polymerized microtubules / soluble microtubules increased significantly (P/S=2.05).
Finally, the study also found in NPC cells and other higher expression of Stathmin in tumor cells, and paclitaxel inhibited significantly in the cells Stathmin expression in nasopharyngeal carcinoma cells, and the inhibitory effect of paclitaxel concentration and the dose effect.
Conclusion: the Stathmin targeting siRNA can inhibit the expression of the protein and the inhibition of nasopharyngeal carcinoma cell proliferation, migration and invasion, and promote cell apoptosis through the mitochondrial pathway. The apoptosis and proliferation of nasopharyngeal carcinoma cells, the regulation of migration and invasion and reduce its degradation by enhancing the polymerization of microtubules, microtubule polymerization,. Microtubule stabilizing and implementation. The expression of paclitaxel can downregulate Stathmin, Stathmin targeting siRNA and paclitaxel in nasopharyngeal carcinoma cells with combined treatment effect. This will provide a new inspiration for the chemical treatment of nasopharyngeal carcinoma.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.63

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