SENP1表达水平与脑胶质瘤病情进展的关联及作用机制的初探

发布时间：2018-05-01 05:32

本文选题：脑胶质瘤 + SENP1　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：检测SUMO特异性蛋白酶1(SENP1)在正常组织及不同级别星型胶质瘤组织中的表达情况，分析其表达水平与病情进展的关联，采用RNA干扰技术在胶质瘤细胞株U87MG中敲低SENP1的表达，研究其对U87MG细胞增殖和凋亡的影响及可能的分子调控机制。方法：采用RT-qPCR和Western blotting方法检测SENP1在正常对照（3例）及星形细胞胶质瘤（28例）标本中mRNA水平和蛋白水平的表达差异。设计并合成特异性针对SENP1基因的siRNA，按照2.5×105个/孔、体积2ml接种U87MG细胞于6孔板中，12h后转染si-NC(对照组)、si-1、 si-2(敲低SENP1组)，待细胞融合度达90%以上后，使用Lipofectamine2000(Invitrogen,USA)转染细胞，继续培养48h后，采用RT-qPCR和Western blotting方法检测SENP1的mRNA和蛋白表达情况。同时在胶质瘤细胞株U87MG沉默SENP1后应用AnnexinⅤ-FITC/碘化丙啶(PI)双染色法在流式细胞仪上通过检测细胞表面的磷脂酰丝氨酸(PS)评估凋亡细胞的数量。最后，，在U87MG细胞中敲低SENP1，通过蛋白印迹实验检测胞内IκBα、p38的蛋白表达情况和二者的磷酸化水平，以及通路下游靶因子p65和bcl-2蛋白水平的表达情况。结果：通过qPCR检测收集的31例脑组织标本中发现：与正常脑组织相比，人脑星型胶质瘤中SENP1的mRNA表达水平明显升高(P0.05)，其随着肿瘤恶性程度的增高而增高，在高级别星形细胞胶质瘤(Ⅲ,Ⅳ级,WHO)中增高的水平更为显著(P0.01)；通过蛋白印迹实验从蛋白水平检测SENP1在正常组织和不同级别之间蛋白水平的差异，结果显示其与mRNA水平类似。转染SENP1siRNA后，U87MG细胞SENP1mRNA表达受到抑制(P0.01)，蛋白表达水平降低。通过细胞凋亡分析发现，与si-NC(对照组)相比、转染si-1、si-2(敲低SENP1组)的U87MG细胞，其凋亡明显增加，凋亡率统计学分析与对照组相比显著增加（P0.05）。在U87MG细胞中敲低SENP1，通过蛋白印迹实验检测分析IκBα、p65和bcl-2在蛋白水平的表达情况，发现IκBα蛋白在两种细胞株中的表达水平无变化，与对照组相比，IκBα的磷酸化水平明显降低且其下游靶因子p65和抗凋亡蛋白bcl-2的表达亦随之降低，而在U87MG细胞中敲低SENP1后检测p38MAPK通路的激活情况，p38的磷酸化水平没有发生明显的变化。结论： 1SENP1在脑胶质瘤标本中的表达差异与肿瘤的发生发展密切相关，可以作为临床监测和评价预后的重要指标。 2SENP1可以通过调控NF-κB通路介导的抗凋亡途径，即通过调控IκBα蛋白的磷酸化水平，影响其下游基因p65、bcl-2的表达，进而调节U87MG细胞的增殖或凋亡，从而进一步揭示了SENP1在促进癌症发生发展过程中可能的新的分子生物学机制。
[Abstract]:Objective: to detect the expression of SUMO specific protease 1 (SENP1) in normal tissue and different grade Astroglioma tissues, and to analyze the relationship between the expression level and the progression of the disease. The expression of SENP1 in the glioma cell line U87MG was knocked out by RNA interference technique, and the effect of the expression on the proliferation and apoptosis of U87MG cells and the possible molecules were studied. Regulation mechanism.
Methods: RT-qPCR and Western blotting were used to detect the difference in the expression of mRNA and protein levels in normal control (3 cases) and astrocytoma (28 cases). The specific siRNA of the SENP1 gene was designed and synthesized. The U87MG cells were inoculated in the 6 hole plate according to the 2.5 x 105 / pores, and the volume 2ml was transfected to si-NC (control group) after 12h. ), SI-1, Si-2 (knocking low SENP1 group), after cell fusion was over 90%, Lipofectamine2000 (Invitrogen, USA) transfected cells and 48h were continued. RT-qPCR and Western blotting methods were used to detect SENP1 mRNA and protein expression. In flow cytometry, the number of apoptotic cells was evaluated by detecting phosphatidylserine (PS) on the cell surface. Finally, SENP1 was knocked down in U87MG cells. The protein expression of intracellular I kappa B alpha, p38 and the level of phosphorylation of the two persons were detected by Western blot, and the expression of the target factor p65 and bcl-2 protein level in the downstream pathway was expressed. Situation.
Results: in 31 specimens of brain tissue collected by qPCR, it was found that the level of mRNA expression of SENP1 in human Astroglioma was significantly higher than normal brain tissue (P0.05), which increased with the increase of tumor malignancy, and increased significantly in advanced astrocytoma (grade III, grade IV, WHO) (P0.01). The difference in protein level between normal tissue and different levels of SENP1 was detected from protein level by Western blot. The results showed that the protein level was similar to that of mRNA. After transfection of SENP1siRNA, the expression of SENP1mRNA in U87MG cells was inhibited (P0.01) and protein expression level decreased. The transfection of SI-1, compared with si-NC (control group), was found through apoptosis analysis. The apoptosis of U87MG cells in the group of Si-2 (group SENP1) increased significantly and the apoptosis rate was significantly increased (P0.05) compared with the control group. The expression of I kappa B a, p65 and Bcl-2 in the protein level was detected in U87MG cells, and the expression level of I kappa B alpha protein in the two cell lines was not changed. Compared with the control group, the phosphorylation level of I kappa B alpha was obviously decreased and the target factor p65 and the expression of anti apoptotic protein Bcl-2 were also decreased, and the activation of p38MAPK pathway was detected after the low SENP1 in U87MG cells, and there was no significant change in the phosphorylation level of p38.
Conclusion:
The difference of expression of 1SENP1 in glioma specimens is closely related to the occurrence and development of tumors. It can be used as an important indicator for clinical monitoring and prognosis.
2SENP1 can regulate the anti apoptosis pathway mediated by NF- kappa B pathway, that is, by regulating the phosphorylation level of I kappa B alpha protein, affecting the expression of the downstream gene p65, Bcl-2, and then regulating the proliferation or apoptosis of U87MG cells, thus further revealing the possible new molecular biological mechanism of SENP1 in the process of promoting the development of cancer.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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