Jab1基因靶向RNA干扰对喉癌生长抑制的实验研究

发布时间：2018-06-24 02:05

  本文选题：喉癌 + Jab1　； 参考：《郑州大学》2010年硕士论文

【摘要】： 喉鳞状细胞癌(Laryngeal Squamous Cell Carcinoma, LSCC)和其它肿瘤一样,是一种多基因性、多步骤、多阶段的发生过程,发病机制复杂,治疗困难。目前,喉癌治疗以手术切除或放疗为主,尽管取得了较大进展,但手术致残率高、并发症多,常规放疗的毒副作用大,疗效差,特别是晚期肿瘤病人,转移率高,病人生存率低。因此,急需寻找有效、简便的治疗方法。随着喉癌相关基因及分子生物学研究的发展,喉癌基因治疗的相关研究也在不断深入。 对肿瘤相关基因在转录、转录后和翻译等水平特异性的阻断能够诱导肿瘤细胞向成熟方向转化或凋亡。RNA干扰((RNA interference, RNAi)现象广泛存在于各种生物体中,能够在哺乳动物细胞内产生特异性的基因沉默,在后基因组研究中得到广泛应用,是分子靶向抗癌治疗的分子学基础。最近,RNA干扰作为一种抗癌治疗的方法已经进入实验阶段,预计将发展成为一种基因治疗的药物。 Jab1 (C-Jun activation domain- binding protein 1)位于COP9(COP9signalosome, CSN)信号复合体的第五亚单位,早期研究证明Jabl是c-Jun激活区的辅助激活因子,后来研究发现Jab1也是激活蛋白-1(activator protein 1, AP-1)的辅助因子,最近研究证实Jab1和多种蛋白的出核转运降解有关,例如p27kip1(kinase inhibit protein 1, P27)、P53、Smad4。Jab1作为一个潜在的致癌基因在多种肿瘤疾病的发展中起着调节器的作用。 P27是细胞周期素依赖的激酶抑制因子,通常在细胞核内发生作用,Jab1可以与P27发生作用并改变其细胞定位使其出核降解,从而导致肿瘤细胞无限增殖,Jab1与P27在各种肿瘤细胞的研究中表明存在负相关性。 在我们前期的实验中发现Jab1在人类喉癌细胞中高表达,与P27的表达存在负相关性,并利用RNAi技术沉默喉癌细胞中活跃的癌基因Jab1的表达,通过RT-PCR、流式细胞学、MTT的方法检测到其能达到阻止或减缓肿瘤细胞生长的目的。在本研究中,我们通过应用RNA干扰技术来研究Jab1的低表达对喉癌细胞的细胞株(Hep-2细胞)在体外增殖的影响,使用一系列的干扰方式,例如,短发夹施工干扰RNA (shRNA)的质粒和非特异性质粒抑制Jab1的表达，，通过WST-8检测细胞增殖的能力,通过Western Blotting检测基因的表达。为了观察shRNA质粒在体内引起Jab1的下调对肿瘤生长的影响,我们将Hep-2肿瘤细胞在裸鼠皮下注射建立人喉癌异种移植瘤模型。模型建立后经与Jab1shRNA质粒和对照组治疗,肿瘤的生长受到了抑制。这些结果为人类喉癌治疗提供了新的实验数据。 目的：利用RNA干扰技术,构建Jab1基因靶向shRNA质粒,观察目的基因的沉默效应及对Hep-2肿瘤细胞的影响。 方法：设计合成以Jab1基因为靶向目标的shRNA,将其定向克隆到真核表达载体pGenesill.1中形成重组质粒；利用脂质体介导的方法将质粒转染至人喉癌细胞株(Hep-2),通过WST-8、Western Blotting的方法对细胞凋亡及蛋白水平的表达变化进行检测,分析shRNA干扰质粒对喉癌细胞的影响。 结果：成功构建了Jab1基因靶向的pJab1以及阴性对照pKB真核重组质粒。WST-8结果示pJab1重组质粒能显著抑制Hep-2细胞的增殖活性,与对照组相比,显著差异(p0.001)。Western Blotting结果示Hep-2-pJab1细胞Jab1蛋白表达量明显降低,P27蛋白表达量明显升高,与对照组相比,差异显著(p0.001)。 结论：构建靶向Jab1癌基因的shRNA干扰质粒能够下调Jab1基因的表达,上调p27基因的表达,抑制人喉癌细胞株Hep-2细胞在体外的增殖。 目的：探讨Jab1重组质粒对人喉癌Hep-2细胞裸鼠移植瘤生长的影响。 方法：建立了喉癌Hep-2细胞裸鼠皮下移植瘤模型,通过瘤体内注射pJab1, pKB, PBS,观察瘤体增长情况,通过免疫组化方法、RT-PCR、Western Blotting等观察瘤体内Jab1、P27的mRNA水平及蛋白水平的变化。 结果：成功建立喉癌Hep-2细胞裸鼠皮下移植瘤模型；pJab1质粒组的肿瘤体积为(267.60±88.19)mm3,瘤重为(0.49±0.03)g,显著低于错意序列对照组和空白对照组；免疫组化结果显示实验组瘤内的Jabl蛋白的表达水平降低,显著低于对照组,P27蛋白的表达水平增高,显著高于对照组；RT-PCR结果显示实验组瘤内Jab1的mRNA水平显著降低,P27的mRNA无明显变化；Western Blotting结果显示实验组瘤内Jab1蛋白表达量明显减少,P27蛋白表达量明显增高。 结论：建立了喉癌Hep-2细胞裸鼠皮下移植瘤模型；pJab1干扰质粒明显降低裸鼠肿瘤组织内Jab1基因的表达并抑制瘤体的生长；pJab1质粒能有效抑制Jab1基因的表达,有望成为临床治疗喉癌的新途径。
[Abstract]:Laryngeal Squamous Cell Carcinoma (LSCC), like other tumors, is a multi gene, multistep, multi-stage process, complicated and difficult to treat. At present, surgical resection or radiotherapy is the main treatment for larynx cancer. Although great progress has been made, there are high morbidity, complications and conventional radiotherapy. Therefore, it is urgent to find effective and simple treatment methods. With the development of the related genes and molecular biology of larynx cancer, the related research on the gene therapy of larynx cancer is also in deep.
Specific blocking of tumor related genes at transcriptional, post transcriptional and translation levels can induce tumor cells to transform or apoptotic.RNA interference (RNA interference, RNAi) in various organisms, which can produce specific gene silencing in mammalian cells, and can be widely used in post genome research. Ubiquitous application is the sub molecular basis for molecular targeting anticancer therapy. Recently, RNA interference, as a method of anticancer therapy, has entered the experimental stage and is expected to develop into a drug for gene therapy.
Jab1 (C-Jun activation domain- binding protein 1) is located in the fifth subunit of the COP9 (COP9signalosome, CSN) signal complex. Early studies showed that Jabl was an auxiliary activating factor in the c-Jun activation region. Nuclear transport and degradation, such as p27kip1 (kinase inhibit protein 1, P27), P53, Smad4.Jab1, as a potential oncogene, play the role of regulators in the development of a variety of tumor diseases.
P27 is a cyclin dependent kinase inhibitor, which usually acts in the nucleus. Jab1 can interact with P27 and change its cell location to cause nuclear degradation, which leads to the proliferation of tumor cells. Jab1 and P27 have negative correlation in the study of various tumor cells.
In our previous experiments, we found that Jab1 was highly expressed in human larynx cancer cells and had a negative correlation with the expression of P27. The expression of active oncogene Jab1 in laryngeal cancer cells was silenced by RNAi technique. The purpose of detecting the growth of tumor cells could be detected by RT-PCR, flow cytology and MTT. In this study, We used RNA interference to study the effect of low expression of Jab1 on the proliferation of cell line (Hep-2 cells) in laryngeal cancer cells in vitro. We use a series of interference methods, for example, the plasmid and non specific plasmids that interfere with RNA (shRNA) and non specific plasmids to suppress Jab1, and the ability to detect cell proliferation through WST-8 and through Western Blo. The expression of the gene was detected by tting. In order to observe the effect of the downregulation of the shRNA plasmid on the tumor growth in the body, we injected the Hep-2 tumor cells into the nude mice to establish a xenograft tumor model of human larynx. After the establishment of the model, the growth of the tumor was inhibited by the treatment of the Jab1shRNA plasmid and the control group. These results were the human larynx. Cancer treatment provides new experimental data.
Objective: to construct Jab1 gene targeting shRNA plasmid using RNA interference technology, observe the silencing effect of target gene and its effect on Hep-2 tumor cells.
Methods: the shRNA was designed and synthesized with the target of Jab1 gene. The recombinant plasmid was cloned into the eukaryotic expression vector pGenesill.1, and the plasmid was transfected into the human larynx cell line (Hep-2) by liposome mediated method. The expression of apoptosis and protein level was detected by WST-8 and Western Blotting. The effects of shRNA interference plasmids on laryngeal cancer cells were analyzed.
Results: the Jab1 gene targeted pJab1 and the negative control pKB eukaryotic recombinant plasmid.WST-8 showed that the pJab1 recombinant plasmid could significantly inhibit the proliferation activity of Hep-2 cells. Compared with the control group, the significant difference (p0.001).Western Blotting results showed that the Jab1 protein expression of Hep-2-pJab1 cells decreased obviously, and the expression of P27 protein was obvious. The difference was significant compared with the control group (p0.001).
Conclusion: the construction of shRNA interfering plasmid for targeting Jab1 oncogene can downregulate the expression of Jab1 gene, up regulate the expression of p27 gene and inhibit the proliferation of Hep-2 cells in human larynx cell line in vitro.
Objective: To investigate the effect of recombinant plasmid Jab1 on the growth of human laryngeal carcinoma Hep-2 cells transplanted in nude mice.
Methods: a subcutaneous tumor model of Hep-2 cells in larynx cancer cells was established. The tumor growth was observed by injection of pJab1, pKB and PBS in the tumor. The changes of Jab1 in the tumor, mRNA level and protein level in the tumor were observed by immunohistochemical method, RT-PCR, Western Blotting and so on.
Results: the model of subcutaneous transplantation of Hep-2 cells in laryngeal carcinoma was successfully established. The tumor volume of the pJab1 plasmid group was (267.60 + 88.19) mm3, and the tumor weight was (0.49 + 0.03) g, significantly lower than the wrong sequence control group and the blank control group. The immunohistochemical results showed that the expression level of Jabl protein in the experimental group was lower, significantly lower than the control group, P27 egg. The level of white expression was higher, significantly higher than that in the control group. The RT-PCR results showed that the mRNA level of Jab1 in the experimental group was significantly decreased and the mRNA of P27 was not significantly changed. The Western Blotting results showed that the expression of Jab1 protein in the experimental group was significantly reduced and the expression of P27 protein was significantly increased.
Conclusion: the subcutaneous transplanted tumor model of Hep-2 cells in larynx cancer cells was established. The pJab1 interference plasmid obviously reduced the expression of Jab1 gene in the tumor tissues of nude mice and inhibited the growth of the tumor. The pJab1 plasmid could effectively inhibit the expression of Jab1 gene, and it is expected to be a new way to treat the cancer of the larynx.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.65

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