RNF135通过ERK调控细胞周期促进胶质母细胞瘤细胞增殖、迁移的体内和体外研究

发布时间：2018-05-13 15:34

本文选题：恶性胶质瘤 + RNF135基因　；参考：《大连医科大学》2015年博士论文

【摘要】：目的：探讨RNF135基因与胶质母细胞瘤发生、发展及预后的联系,并通过体内及体外实验研究RNF135基因对胶质母细胞瘤细胞U87的增殖、迁移及细胞周期的影响以及RNF135基因对U87细胞作用的机制,为胶质母细胞瘤提供新的基因治疗靶点。方法：首先对28例胶质瘤组织和12例正常的脑组织采用实时定量PCR与Western Blot实验检测组织中RNF135基因的表达差异。采用免疫组化实验比较123例胶质瘤和12例正常脑组织中RNF135蛋白的表达情况,(标本来源2004年7月到2009年6月,在大连医科大学附属第一医院手术的胶质瘤患者,正常脑组织为外伤、脑出血手术患者术区周边非功能区脑组织,所有标本获得都经过大连医科大学附属第一医院医学伦理委员会批准),用统计学分析RNF135基因表达与患者年龄、性别、胶质瘤病理组织类型、预后及生存时间是否有相关性。然后通过构建shRNF135稳转的U87胶质母细胞瘤细胞系,研究RNF135基因对胶质母细胞瘤细胞U87增殖、迁移及细胞周期的影响及机制。通过使用慢病毒包装shRNA转染U87细胞对RNF135基因进行沉默,在体外建立稳定低表达RNF135基因的U87细胞系,同时通过慢病毒包装LVTHMGFP转染U87细胞作为对照组,慢病毒转完后通过流式细胞术筛选转染成功的细胞继续培养,培养的细胞用实时定量PCR与Western Blot实验验证RNF135基因的表达以确定稳定低表达RNF135基因的U87细胞系模型建立成功。实时定量PCR与Western Blot实验验证成功后首先检测干扰RNF135基因表达对U87细胞增殖的影响,细胞活性用MTT实验检测,通过划痕实验与Boyden小室迁移实验验证干扰RNF135表达后U87细胞迁移能力的变化情况。利用Western Blot实验验证RNF135基因与细胞通路p38及ERK之间的关系,利用细胞周期实验解释RNF135低表达对细胞周期的影响,通过Western Blot实验检测细胞周期相关蛋白的表达情况。最后通过荷瘤裸鼠模型在体内验证干扰RNF135基因表达对U87细胞在裸鼠体内成瘤的影响。结果：临床病例的统计分析表明RNF135基因与胶质瘤患者预后及生存时间均有明显的相关性。在所有调查的123例胶质瘤患者当中,RNF135基因明显高表达。在小于50岁的63名胶质瘤患者中RNF135基因高表达的概率为69.8%略微低于50岁以上的60名患者中RNF135基因高表达的概率(70%)。在73名女性胶质瘤患者中RNF135基因高表达的概率为74%,而在50名男性胶质瘤患者中RNF135基因高表达的概率为63%,女性患者的概率略微高于男性患者的概率。在9名少突胶质瘤患者中RNF135基因高表达的概率为77.8%,在60名间变性星型细胞瘤患者中RNF135基因高表达的概率为60%而在40名胶质母细胞瘤的患者中RNF135基因高表达的概率也为60%。按世界卫生组织对胶质瘤恶性程度的分类来看,恶性程度越高的胶质瘤中RNF135基因高表达的概率越高。Ⅲ和Ⅳ级的恶性胶质瘤患者的RNF135基因高表达的概率78%明显高于Ⅰ和Ⅱ级的恶性胶质瘤患者中RNF135基因高表达的概率34.8%。甚至Ⅰ和Ⅱ级的恶性胶质瘤患者中RNF135基因低表达的概率为65.2%,远高于RNF135基因高表达的概率22%。经SPSS统计分析发现,胶质瘤中RNF135的表达情况与患者性别、年龄、病理类型没有相关性,P值分别为0.985、0.236和0.766。WHO胶质瘤病理分级中,Ⅱ和Ⅳ级病理中的RNF135表达较I和II级的高,经统计学分析,p0.05。综合以上实验结果可知,胶质瘤的恶性程度与RNF135的异常表达情况相关。高恶性程度的胶质瘤更容易发生RNF135基因的高表达。而Ⅲ和Ⅳ级的恶性胶质瘤,尤其是Ⅳ级恶性胶质瘤(多形性胶质母细胞瘤)往往预后很差而且患者生存时间很短。因此RNF135基因与胶质瘤的发生与预后均有密切的联系。同样的,在体外实验中我们发现RNF135基因在胶质瘤组织中的表达明显高于正常脑组织。我们用Western blot方法,分析在28个胶质瘤样本中RNF135蛋白表达量,明显高于正常的脑组织样本中RNF135蛋白表达量。以上结果说明胶质瘤细胞尤其是Ⅲ和Ⅳ级的恶性胶质瘤细胞往往发生RNF135基因程高表达。这种高表达不仅表现在RNF135基因的mRNA水平,而且蛋白水平也表现出一致的结果。在用慢病毒转染shRNA干扰RNF135基因在U87细胞中的表达后,实时定量PCR显示RNF135基因的mRNA表达水平及Western Blot实验显示RNF135基因的蛋白较空载对照组的明显降低,p0.05统计学有意义。以上的实时定量PCR结果与Western Blot实验表明稳定低表达RNF135基因的U87细胞系建立成功。RNF135基因被干扰后U87细胞的增殖速度明显低于对照组,这说明高表达的RNF135基因有助于U87细胞的增殖。而划痕实验与Boyden小室迁移实验同时显示RNF135基因被干扰后U87细胞的迁移速度明显低于对照组,这说明高表达的RNF135基因有助于U87细胞的迁移。Western Blot实验显示稳定低表达RNF135基因的U87细胞中的Erkl/2及P-Erkl/2的表达量显著降低而p38及P-p38的表达量没有明显变化,这说明干扰RNF135基因后对U87细胞产生的抑制功能很可能是通过抑制ERK的磷酸化来实现的。细胞周期实验表明干扰RNF135基因会引起U87细胞阻滞在G0/G1期,这说明干扰RNF135基因是通过阻滞细胞周期从而抑制细胞增殖。而对细胞周期相关蛋白的Western Blot实验的结果显示,干扰RNF135基因后细胞细胞周期蛋白依赖性激酶CDK4的表达降低,而细胞周期抑制蛋白p27和p21的表达增多从而导致细胞周期阻滞在G0/GI期。荷瘤裸鼠实验中稳定低表达RNF135基因的U87细胞所成的瘤块明显小于对照组。说明干扰RNF135基因在裸鼠体内抑制U87细胞的增殖。结论：RNF135基因与胶质瘤的发生与预后均有密切的联系,干扰RNF135基因可以使U87细胞阻滞在G0/G1期从而抑制细胞增殖及细胞的迁移能力。干扰RNF135基因后对U87细胞产生的抑制作用很可能是通过抑制ERK的磷酸化实现的,干扰RNF135基因通过抑制细胞周期蛋白依赖性激酶CDK4并上调细胞周期抑制蛋白p27和p21的表达从而导致细胞周期阻滞在G0/G1期,以上的研究表明RNF135基因有望作为治疗胶质母细胞瘤的新靶点。
[Abstract]:Objective: To investigate the relationship between the RNF135 gene and the occurrence, development and prognosis of glioblastoma, and to study the effect of RNF135 gene on the proliferation, migration and cell cycle of glioblastoma cell U87 and the mechanism of RNF135 gene on U87 cells in vivo and in vitro, and provide new target for gene therapy for glioblastoma. Methods: the expression of RNF135 gene in the tissues of 28 glioma and 12 normal brain tissues was measured by real-time quantitative PCR and Western Blot. The expression of RNF135 protein in 123 gliomas and 12 normal brain tissues was compared by immunohistochemistry. (the source from July 2004 to June 2009, in Dalian medicine. The patients with glioma surgery in the first hospital affiliated to the University, the normal brain tissue for trauma, the peripheral nonfunctional brain tissue around the operation area of the patients with cerebral hemorrhage, all the specimens were approved by the medical ethics committee of the First Affiliated Hospital of Dalian Medical University. The RNF135 gene expression was statistically analyzed with the patient's age, sex, glioma pathology group. The effect and mechanism of the RNF135 gene on the proliferation, migration and cell cycle of glioblastoma cells by the construction of a shRNF135 stable U87 glioblastoma cell line. The RNF135 gene was silenced by transfecting U87 cells with the lentivirus package shRNA, and the construction of the RNF135 gene was built in vitro. The U87 cell line with low expression of RNF135 gene was established, and U87 cells were transfected through the lentivirus package LVTHMGFP as the control group. After the lentivirus was completed, the transfected cells were screened by flow cytometry to continue to be cultured. The cultured cells used real-time quantitative PCR and Western Blot to verify the expression of RNF135 gene to determine the stable low expression of RNF. The U87 cell line model of the 135 gene was successfully established. The effect of interference RNF135 gene expression on the proliferation of U87 cells was first detected by real-time quantitative PCR and Western Blot experiments. The cell activity was tested by MTT test, and the migration ability of U87 cells after RNF135 expression was verified by scratch test and Boyden chamber migration experiment. Western Blot experiment was used to verify the relationship between RNF135 gene and cell pathway p38 and ERK, and the effect of RNF135 low expression on cell cycle was explained by cell cycle experiment. The expression of cell cycle related proteins was detected by Western Blot experiment. Finally, the interference of RNF135 gene expression to U87 was verified by the model of tumor bearing nude mice in vivo. Results: the statistical analysis of clinical cases showed that the RNF135 gene was significantly correlated with the prognosis and survival time of the patients with glioma. In all 123 patients with glioma, the RNF135 gene was highly expressed. The probability of high expression of RNF135 gene in 63 glioma patients less than 50 years old The probability of high expression of RNF135 gene (70%) in 60 patients younger than 50 years old (70%). The probability of high expression of RNF135 gene in 73 female glioma patients was 74%, while the probability of high expression of RNF135 gene in 50 male glioma patients was 63%, and the probability of female patients was slightly higher than that of male patients. In 9 oligodendrocytes, the probability of the high expression of RNF135 gene was slightly higher than that of the male patients. The probability of high expression of RNF135 gene in tumor patients was 77.8%. The probability of high expression of RNF135 gene was 60% in 60 anaplastic astrocytoma patients and the probability of high expression of RNF135 gene in 40 patients with glioblastoma was also based on the classification of the malignant degree of the glioma by WHO, the higher the malignancy of glioma The higher probability of high expression of RNF135 gene in the tumor was higher. The probability of high expression of RNF135 gene in patients with grade III and IV malignant gliomas was significantly higher than the probability of high expression of RNF135 gene in patients with grade I and II malignant gliomas. The probability of low expression of RNF135 gene in patients with grade I and grade II malignant gliomas was 65.2%, far higher than RNF1. The probability of high expression of 35 gene 22%. showed that there was no correlation between the expression of RNF135 in glioma and the sex, age and pathological type of the patients. The P value was 0.985,0.236 and 0.766.WHO glioma pathological classification, and the RNF135 expression in grade II and IV was higher than that of I and II. By statistical analysis, p0.05. integrated experiments. The results show that the malignant degree of glioma is related to the abnormal expression of RNF135. The high malignant glioma is more likely to have high expression of RNF135 gene. And grade III and IV malignant glioma, especially grade IV malignant glioma (pleomorphic glioblastoma) often have poor prognosis and the patient's survival time is very short. So RNF135 base There is a close relationship with the occurrence and prognosis of glioma. Similarly, we found that the expression of RNF135 gene in glioma tissue is obviously higher than that of normal brain tissue in vitro. We use Western blot method to analyze the expression of RNF135 protein in 28 glioma samples, which is significantly higher than that of the normal brain tissue samples of RNF135 protein. The above results indicate that glioma cells, especially grade III and grade IV malignant glioma cells often have high expression of RNF135 gene process. This high expression is not only shown in the mRNA level of the RNF135 gene but also in the protein level. After the expression of the RNF135 gene in the U87 cells by the lentivirus transfected to the RNF135 gene, Real-time quantitative PCR showed the mRNA expression level of RNF135 gene and Western Blot experiment showed that the protein of RNF135 gene was significantly lower than that of the empty control group, and P0.05 statistics was significant. The above real-time quantitative PCR results and Western Blot experiments showed that the U87 fine cell line of stable low expression RNF135 gene was established. The proliferation rate of the cells was significantly lower than that of the control group, which indicated that the high expression of RNF135 gene was helpful to the proliferation of U87 cells. The scratch test and the Boyden cell migration experiment showed that the migration speed of U87 cells was significantly lower than that of the control group after the RNF135 gene was disturbed, which indicated that the high expression of the RNF135 gene was helpful to the migration of.Western Bl in U87 cells. The OT experiment showed that the expression of Erkl/2 and P-Erkl/2 in U87 cells with stable low expression of RNF135 gene decreased significantly and the expression of p38 and P-p38 did not change significantly. This indicated that the inhibition of U87 cells after the interference of RNF135 gene was likely to be realized by inhibiting the phosphorylation of ERK. Cell cycle experiments showed that interference RNF135 base U87 cells are blocked at G0/G1 stage, which indicates that the interference of RNF135 gene is by blocking cell cycle to inhibit cell proliferation. The result of Western Blot experiment on cell cycle related proteins shows that the expression of cell cycle protein dependent kinase CDK4 in cell cell is reduced after the interference of RNF135 gene, and cell cycle inhibitor protein p27 and P The increased expression of 21 resulted in cell cycle arrest in G0/GI phase. In nude mice, the tumor blocks of U87 cells with low expression of RNF135 gene in nude mice were significantly smaller than those of the control group. It indicated that the interference of RNF135 gene in nude mice to inhibit the proliferation of U87 cells in nude mice. Conclusion: there is a close relationship between the occurrence and prognosis of the RNF135 gene and glioma, and the interference is closely related to the prognosis. RNF135 gene can inhibit U87 cells in G0/G1 phase and inhibit cell proliferation and cell migration. The inhibition effect on U87 cells after interference with RNF135 gene may be achieved by inhibiting the phosphorylation of ERK, interfering with the RNF135 gene by inhibiting the cyclin dependent kinase CDK4 and up regulation of the cell cycle inhibitor protein. The expression of p27 and p21 leads to cell cycle arrest in G0/G1 phase. The above studies suggest that RNF135 gene is expected to be a new target for the treatment of glioblastoma.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.4

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