GRIM-19的亚细胞定位及在口腔鳞癌治疗中作用的研究

发布时间：2018-07-04 06:43

本文选题：干扰素和维甲酸联合应用诱导的细胞凋亡相关基因-19 + 亚细胞定位　；参考：《吉林大学》2017年博士论文

【摘要】：干扰素和维甲酸联合应用诱导的细胞凋亡相关基因-19(GRIM-19)被认为是一种新类型的抑癌基因,有研究显示GRIM-19基因敲除后,肿瘤细胞显示出对IFN-β/RA引发细胞凋亡的耐受能力。因此,研究GRIM-19在IFN-β/RA引发细胞凋亡中的作用机制,对基于该分子机制的肿瘤疗法的研究和开发具有重要意义。最初GRIM-19被认为是一种核内蛋白,后又被证实定位于线粒体,是线粒体复合物I的重要组成部分,这前后矛盾的研究结论值得注意。GRIM-19如在线粒体中可能更多地参与能量代谢相关过程;如在细胞核中则可能通过激活相关信号转导通路来参与凋亡调控。因此,应首先明确GRIM-19的亚细胞定位变化。鉴于过往成像技术的限制导致难以明确GRIM-19的胞内定位,本研究应用3D-SIM超高分辨率成像系统对GRIM-19在IFN-β/RA作用前后的亚细胞定位进行了观察。结果显示,正常状态下,GRIM-19主要分布于细胞质中,与线粒体具有明显的共定位现象,而在细胞凋亡过程中,GRIM-19的分布向细胞核内移动,细胞质中的GRIM-19明显减少。在明确凋亡过程中GRIM-19向细胞核移动后,接下来探讨GRIM-19对凋亡相关信号转导通路的影响。为保证后续实验的顺利进行,本研究首先成功构建了能表达GRIM-19的真核表达质粒——p GRIM-19,经RT-PCR和Western blotting等方法的检测,证明p GRIM-19能够显著上调肿瘤细胞中GRIM-19的表达。然后,本研究检测了GRIM-19的表达上调对相关信号通路的激活情况,研究发现,GRIM-19的上调能够抑制STAT3信号通路及VEGF、Bcl-2、cyclin D1的表达。综上,细胞凋亡过程中,GRIM-19的亚细胞定位发生从线粒体到细胞核的变化,并能够引起STAT3相关信号转导通路的抑制作用。上述结果为GRIM-19作为肿瘤治疗的靶点提供了可能,而已有报道显示GRIM-19能够抑制多种肿瘤的生长,但尚无其在口腔鳞癌中的作用研究。因此本研究试图揭示GRIM-19对口腔鳞癌的增殖、凋亡、转移及侵袭的影响。MTT法和克隆形成实验结果显示,GRIM-19的上调能够抑制口腔鳞癌细胞的增殖;吖啶橙染色法和Caspase活性法结果表明GRIM-19的上调能够促进口腔鳞癌细胞的增殖;细胞迁移实验证明GRIM-19的上调能够抑制口腔鳞癌的迁移;Transwell侵袭实验显示GRIM-19的上调能够抑制口腔鳞癌细胞的侵袭,进一步的侵袭机制证明GRIM-19的上调能够降低口腔鳞癌细胞中MMP-2和MMP-9的表达,从而抑制细胞侵袭。在上述体外实验的基础上,本研究检测了GRIM-19对小鼠体内口腔鳞癌肿瘤生长的影响。口腔鳞癌细胞接种于BALB裸鼠皮下,GRIM-19上调后肿瘤的重量和体积均明显下降,同时小鼠脾细胞的增殖也受到明显抑制。为进一步证明GRIM-19对口腔鳞癌肿瘤的抑制,将肿瘤组织取出后检测其中细胞的凋亡情况,TUNEL法结果显示,GRIM-19上调后裸鼠模型的肿瘤细胞凋亡明显得到增强。因此,GRIM-19确实具备成为口腔鳞癌治疗靶点的潜力。上面已证明GRIM-19有成为口腔鳞癌治疗靶点的可能,但以目前的药物研发水平,短时间内开发出GRIM-19的激活剂是十分困难的。因此首先寻求GRIM-19与现有抗肿瘤药物的联合治疗可能是当前GRIM-19在临床应用上更可行的策略。而现有口腔鳞癌治疗药物中,顺铂具有较好的治疗效果,但其毒性和耐药性等问题大大地限制了其进一步临床应用。已有报道称顺铂与抑癌基因的联合治疗能够克服其耐药性并增强治疗效果。因此,本研究试图探讨GRIM-19的表达上调对顺铂治疗口腔鳞癌效果的影响。首先确定了顺铂的合适使用浓度为10μM,而GRIM-19的上调能够增强顺铂对口腔鳞癌细胞增殖和细胞迁移的抑制、并能增强顺铂对细胞凋亡的促进。同时,体内实验结果也表明,相比于单独注射顺铂,GRIM-19的上调和顺铂联合治疗后口腔鳞癌肿瘤的重量和体积均明显减小,并能进一步抑制小鼠脾细胞的增殖。因此,GRIM-19的表达上调能够增强顺铂对口腔鳞癌的治疗效果。综上,在细胞凋亡过程中GRIM-19的亚细胞定位会发生变化,部分从线粒体向细胞核移动,并能够抑制核内STAT3相关信号转导通路的激活。同时,经验证GRIM-19具备成为口腔鳞癌治疗靶点的可能,其表达上调能够增强顺铂对口腔鳞癌的治疗效果。本研究揭示了GRIM-19的亚细胞定位变化及其对相关信号转导通路的影响,进一步阐述了GRIM-19在细胞凋亡中的作用机制。而关于GRIM-19在口腔鳞癌中治疗效果的研究,扩大了GRIM-19的潜在临床治疗范围,为GRIM-19相关的药物开发提供了初步的临床前实验数据。
[Abstract]:The combined use of interferon and retinoic acid to induce apoptosis related gene -19 (GRIM-19) is considered as a new type of tumor suppressor gene. Some studies have shown that after GRIM-19 knockout, the tumor cells show the tolerance to apoptosis induced by IFN- beta /RA. Therefore, the mechanism of GRIM-19 in the apoptosis induced by IFN- beta /RA is studied. The research and development of tumor therapy based on this molecular mechanism is of great significance. Initially, GRIM-19 was considered as an intranuclear protein, and then proved to be located in mitochondria, an important component of the mitochondrial complex I. This contradictory conclusion is worth noting that.GRIM-19 may be more involved in the energy generation of Xie Xiang in mitochondria. The process of closing the process, such as in the nucleus, may be involved in the regulation of apoptosis by activating the related signal transduction pathway. Therefore, the changes in the subcellular localization of GRIM-19 should be defined first. In view of the limitation of the past imaging techniques, the intracellular localization of GRIM-19 is difficult to be clearly defined. This study applied the 3D-SIM ultra high resolution imaging system to the effect of GRIM-19 in the IFN- beta /RA The subcellular localization was observed before and after. The results showed that in the normal state, GRIM-19 was mainly distributed in the cytoplasm, and there was a obvious co localization with the mitochondria. In the process of apoptosis, the distribution of GRIM-19 was moved into the nucleus, and the GRIM-19 in the cytoplasm decreased obviously. In the clear apoptosis process, the GRIM-19 moved to the nucleus. Then, the effect of GRIM-19 on the apoptosis related signal transduction pathway is discussed. In order to ensure the smooth progress of the follow-up experiment, this study first successfully constructed a eukaryotic expression plasmid that can express GRIM-19, P GRIM-19, through the detection of RT-PCR and Western blotting, and proved that P GRIM-19 can significantly increase the table of GRIM-19 in tumor cells. Then, this study detected the activation of GRIM-19 expression on the related signaling pathway. The study found that the up-regulation of GRIM-19 inhibited the STAT3 signaling pathway and the expression of VEGF, Bcl-2, cyclin D1. In the process of apoptosis, the subcellular localization of GRIM-19 occurred from mitochondria to nucleus, and could cause STAT3 correlation. The inhibitory effect of signal transduction pathway. These results provide the possibility of GRIM-19 as a target for cancer treatment, and it has been reported that GRIM-19 can inhibit the growth of a variety of tumors, but there is no study on the role of GRIM-19 in oral squamous cell carcinoma. Therefore, this study attempts to reveal the effect of.M on the proliferation, apoptosis, metastasis and invasion of oral squamous cell carcinoma. The results of TT and clone formation showed that the up-regulation of GRIM-19 could inhibit the proliferation of oral squamous cell carcinoma cells. The results of acridine orange staining and Caspase activity showed that up regulation of GRIM-19 could promote the proliferation of oral squamous cell carcinoma cells. Cell migration experiments showed that the up regulation of GRIM-19 could inhibit the migration of oral squamous cell carcinoma; the Transwell invasion experiment showed that the up regulation of GRIM-19 was significant. The up-regulation of GRIM-19 can inhibit the invasion of oral squamous cell carcinoma cells. Further invasion mechanism shows that up regulation of GRIM-19 can reduce the expression of MMP-2 and MMP-9 in oral squamous cell carcinoma cells and inhibit cell invasion. On the basis of the above in vitro experiments, this study detected the effect of GRIM-19 on the growth of oral squamous cell carcinoma in mice. The tumor cells were inoculated subcutaneously in BALB nude mice. The weight and volume of the tumor were significantly decreased after the GRIM-19 up regulation, and the proliferation of splenocytes in mice was also significantly inhibited. It was further proved that the inhibition of GRIM-19 on oral squamous cell carcinoma and the detection of the cell apoptosis after taking out the tumor tissue. The TUNEL method showed that the GRIM-19 up regulation was up. The tumor cell apoptosis in the nude mouse model is obviously enhanced. Therefore, GRIM-19 has the potential to be the target of oral squamous cell cancer treatment. It has been proved that GRIM-19 has the potential to be the target of oral squamous cell cancer treatment, but it is very difficult to develop GRIM-19 activator in a short time at the current level of drug development. Therefore, the first search for GR Combined treatment of IM-19 with existing antitumor drugs may be a more feasible strategy for the current clinical application of GRIM-19. However, cisplatin has a good therapeutic effect in the existing oral squamous cell carcinoma treatment drugs, but its toxicity and drug resistance have greatly limited its further clinical application. Therefore, this study attempts to explore the effect of up regulation of GRIM-19 on the effect of cisplatin in the treatment of oral squamous cell carcinoma. First, the appropriate concentration of cisplatin is 10 u M, and the up-regulation of GRIM-19 can enhance the inhibition of cisplatin to the proliferation and cell migration of oral squamous cell carcinoma, and enhance the cisplatin. At the same time, the experimental results also showed that the weight and volume of GRIM-19 and cisplatin combined with cisplatin were significantly reduced in weight and volume compared with cisplatin alone, and it could further inhibit the proliferation of murine splenocytes. Therefore, the up-regulation of GRIM-19 can enhance the treatment of cisplatin to oral squamous cell carcinoma. To sum up, the subcellular location of GRIM-19 changes in the process of apoptosis, partly from mitochondria to the nucleus and can inhibit the activation of STAT3 related signal transduction pathway in the nucleus. At the same time, it is proved that GRIM-19 has the potential to be a target for the treatment of oral squamous cell carcinoma. Up regulation of cisplatin can enhance cisplatin to oral squamous cell carcinoma This study revealed the changes in the subcellular localization of GRIM-19 and its effect on the related signal transduction pathway, and further elaborated the mechanism of GRIM-19 in cell apoptosis, and the research on the therapeutic effect of GRIM-19 in oral squamous cell carcinoma expanded the potential clinical scope of GRIM-19 for the development of GRIM-19 related drugs. Preliminary data of preclinical trials were provided.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.8

【相似文献】