RACK1在胶质瘤中的表达及其功能的实验研究

发布时间：2018-06-26 14:04

本文选题：胶质瘤 + RACK1　；参考：《中南大学》2014年博士论文

【摘要】：背景：脑胶质瘤是中枢神经系统中最常见的恶性肿瘤。手术彻底切除困难,且其生长迅速,术后易复发,复发间隔时间短,预后差。高级别胶质瘤平均生存期仍然在一年左右。胶质瘤生物遗传学及分子生物学的研究进展,揭示了胶质瘤的发生发展同样是一个复杂的生物学过程,涉及到许多相关基因的异常表达。因此积极地探索胶质瘤的发生发展的分子生物学机制对指导临床治疗具有重要的意义。已有研究证实了RACK1基因在细胞的生长、分化、恶变、转移、侵袭等方面发挥了关键的调节作用,但该基因如何调控并作用于神经上皮组织,与胶质瘤发生,发展的关系究竟如何,以及与胶质瘤生物学行为的相关性等问题,目前国内外尚未见系统的研究和报道。我们推测RACK1基因可能参与了胶质瘤的发生发展过程并发挥了重要的调节作用,RACK1基因可能具有成为胶质瘤靶向治疗新靶点的应用前景,可能给临床基因诊断及预后评价提供参考。目的：探讨RACK1基因在人脑胶质瘤组织中的表达情况及与病理分级关系。通过瞬时和稳定RNA干扰技术在人胶质瘤细胞株中下调RACK1的表达,探讨RACK1对胶质瘤细胞生长、增殖、迁移、凋亡等多种生物学特性的影响,并初步分析其发挥作用的分子生物学机制,结合临床病理初步评价其临床应用前景。方法：对收集的45例各级别临床胶质瘤及10例正常脑组织标本以及U87-MG, CHG-5细胞株进行Real-Time PCR和Western-Blot分别检测RACK1mRNA和蛋白的表达水平,证实了RACK1在人脑胶质瘤中的异常高表达,然后用siRNA瞬时干扰U87-MG, CHG-5细胞株中RACK1的基因,观察RACK1基因干扰前后胶质瘤细胞株生物学特性(增殖,侵袭,凋亡等)的改变,再通过慢病毒为载体稳定转染U87-MG细胞株后,RACK1表达下调后进行裸鼠成瘤试验,于体内进一步证实RACK1对胶质瘤增殖的调控作用,最后初步探讨其调控胶质瘤的发生发展的机制,并结合临床病理资料,来评价RACK1用于人脑胶质瘤中基因诊断及治疗的潜在价值。结果：结果显示低级别胶质RACK1表达较正常脑组织增高有统计学差异(P0.05),高级别胶质瘤和正常脑组织统计学差异更为显著(P0.01),低级别与高级别胶质瘤之间亦有统计学差异(P0.05)RACK1的表达增高和胶质瘤病理级别正相关。U87-MG和CHG-5细胞株中RACK1的表达也较正常明显升高。RACK1经RNAi后,细胞的抗凋亡及迁移侵袭能力形成明显受到抑制(P0.01),并降低了细胞株的增殖率(P0.05),裸鼠成瘤试验中,经慢病毒介导的shRNA干扰后,U87-MG细胞株成瘤与对照组同时期内瘤体相比明显减小,成瘤速度也明显减慢,结果有统计学差异(P0.01)。RACK1经siRNA干扰后,无论U87-MG还是CHG-5其Bcl-2的表达均明显下降,Bax的表达显著升高(P0.01), Bcl-2/Bax比值也明显下降,siRNA下调了RACK1的表达后检测Src/Akt磷酸化,两者在两组细胞株中均显著的表达下调(P0.01和P0.05)。同时证实RACK1可能与胶质瘤的大小,血运,累及部位有一定的相关性(P0.05) 结论： 1. RACK1在胶质瘤中高表达； 2. RACK1在体外及体内试验中均能正向调控胶质瘤的增殖,侵袭及抗凋亡；3. RACK1可能通过Bcl-2/Bax, Src/Akt相关的信号传导通路发挥作用。
[Abstract]:Background: glioma is the most common malignant tumor in the central nervous system. It is difficult to be excised thoroughly, and its growth is rapid. The recurrence of glioma is easy after operation. The recurrence interval is short and the prognosis is poor. The average survival time of the high grade glioma is still about one year. The progress of the biological genetics and subbiology of glioma has revealed the hair of glioma. It is also a complex biological process that involves the abnormal expression of many related genes. Therefore, it is of great significance to actively explore the molecular mechanism of the development of glioma to guide clinical treatment. The research has proved that the RACK1 gene plays a role in cell growth, differentiation, malignant transformation, metastasis, invasion and so on. It is a key regulatory role, but how the gene regulates and regulates the relationship between the neuroepithelial tissue, the relationship with the occurrence of glioma, the relationship between the development of glioma, and the correlation with the biological behavior of glioma. There is no systematic research and report at home and abroad. We speculate that the RACK1 gene may be involved in the development of glioma. The RACK1 gene may have the prospect of becoming a new target for targeting glioma, and may provide a reference for clinical gene diagnosis and prognosis evaluation.
To investigate the expression of RACK1 gene in human glioma tissue and its relationship with pathological classification. Through transient and stable RNA interference technique, the expression of RACK1 in human glioma cell lines is downregulated, and the effects of RACK1 on the growth, proliferation, migration and apoptosis of glioma cells are discussed, and the molecules that play a role are preliminarily analyzed. Biological mechanism, combined with clinical pathology, preliminary evaluation of its clinical application prospects.
The expression levels of RACK1mRNA and protein were detected in 45 cases of clinical gliomas and 10 normal brain tissue specimens and U87-MG, CHG-5 cell lines, Real-Time PCR and Western-Blot respectively, which confirmed the abnormal high expression of RACK1 in human glioma, and then interfered with U87-MG, RACK1 gene in CHG-5 cell lines by siRNA transient. The changes of the biological characteristics (proliferation, invasion, apoptosis, etc.) of glioma cell lines before and after the interference of RACK1 gene were observed, and then the U87-MG cell line was transfected steadily through the lentivirus as the carrier, and the expression of RACK1 was downregulated after the downregulation of the tumor. The regulation of RACK1 on the proliferation of glioma was further confirmed in the body. Finally, the regulation of glioma was preliminarily discussed. To evaluate the potential value of RACK1 in gene diagnosis and treatment of human gliomas by combining the mechanism of occurrence and development with clinicopathological data.
Result:
The results showed that the expression of low grade glial RACK1 was significantly higher than that of normal brain tissue (P0.05). The statistical difference between high grade glioma and normal brain tissue was more significant (P0.01), and there was a statistically significant difference between low grade and high grade glioma (P0.05), the higher expression of RACK1 and the positive correlation between.U87-MG and CHG-5 cells in the pathological grade of glioma. The expression of RACK1 in the plant was also significantly higher than that of the normal.RACK1. After RNAi, the anti apoptosis and migration and invasion ability of the cells were obviously inhibited (P0.01), and the proliferation rate of cell lines was reduced (P0.05). In the nude mouse tumor test, after the slow virus mediated shRNA interference, the adult tumor of U87-MG cell line was significantly reduced compared with the control group in the same period. After siRNA interference, the expression of Bcl-2 in U87-MG or CHG-5 decreased significantly, the expression of Bax increased significantly (P0.01) and Bcl-2/Bax ratio decreased significantly (P0.01) and siRNA decreased after siRNA interference. SiRNA decreased the RACK1 expression and detected Src/Akt phosphorylation. Both were significant in the two groups of cell lines. The expression of P0.01 (P0.05 and RACK1) was down regulated. It was also confirmed that RACK1 may be related to the size, blood flow and location of glioma (P0.05).
Conclusion:
1. RACK1 was highly expressed in glioma.
2. RACK1 in vitro and in vivo can positively regulate the proliferation and invasion of glioma.
Anti apoptosis; 3. RACK1 may play a role through Bcl-2/Bax and Src/Akt related signal transduction pathways.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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