亲环素A在非小细胞肺癌转移中的作用及其相关机制的初步探讨

发布时间：2018-05-19 01:35

本文选题：肺肿瘤 + 转移　；参考：《北京市结核病胸部肿瘤研究所》2017年博士论文

【摘要】：背景与目的:肺癌因其高发病率和死亡率,在中国乃至世界范围内依然是一个重大的健康问题。肺癌普遍发病隐匿,早期症状不明显,因此在发病初期易被忽视。加之肺癌的早期诊断及治疗进展缓慢,5年生存率仅为18%。大部分肺癌患者在初次就诊时,已经发生了转移,这也是目前肺癌疗效不佳的一个主要原因。作为肿瘤的六大生物学特性之一,“转移”一直以来都是导致肿瘤复发以及患者死亡的主要原因。因此,研究肿瘤发生转移的机制,阐明参与肿瘤转移的信号通路,对控制肿瘤的复发和转移有着重要的意义。亲环素A作为最早被发现的亲环素家族成员,在肺癌、肝癌、胰腺癌、乳腺癌等多种肿瘤组织中均呈高表达状态。研究发现,亲环素A在这些肿瘤的发生和发展过程中均发挥着重要的作用。其中,肿瘤的转移更是与亲环素A的表达水平有着密切的关系。然而,目前针对亲环素A在肺癌的转移过程中的作用及其相关机制的研究尚比较少。本课题组前期研究发现,通过RNA干扰和使用抑制剂的方法,抑制非小细胞肺癌细胞中亲环素A的表达水平后,细胞的迁移和侵袭能力均受到了抑制,这提示我们,亲环素A可能能够促进非小细胞肺癌细胞的迁移和侵袭。随后我们发现,在抑制了非小细胞肺癌细胞中亲环素A的表达水平之后,MAPK信号通路中的一个重要分子——P38的磷酸化水平也受到了明显抑制,这提示我们亲环素A对非小细胞肺癌细胞的迁移和侵袭能力的调控,可能是通过P38 MAPK信号通路来实现的。因此,本课题将分别通过体外和体内实验来进一步研究亲环素A在非小细胞肺癌转移过程中的作用,并对其作用机制进行初步探讨。方法:首先,在“亲环素A在非小细胞肺癌转移过程中的作用”的研究中,我们通过慢病毒感染的方法,在两种人非小细胞肺癌细胞H1299和A549中构建亲环素A低表达细胞株,并在此基础上构建亲环素A过表达的细胞株,及其对照细胞;然后通过划痕实验和Transwell实验的方法,观察亲环素A的表达水平对两种人非小细胞肺癌细胞H1299和A549的迁移和侵袭能力的影响;同时通过Western-blot的方法来观察亲环素A的表达水平的变化对EMT相关蛋白表达水平的影响。最后通过动物实验的方法,构建非小细胞肺癌小鼠转移模型,来进一步探讨亲环素A的表达水平对非小细胞肺癌转移的影响。接着,在“亲环素A在非小细胞肺癌转移过程中的作用机制的探讨”的研究中,我们使用P38抑制剂SB203580,分别对亲环素A低表达和高表达的非小细胞肺癌细胞进行处理,一方面通过细胞迁移和侵袭实验,观察两种抑制剂在亲环素A表达水平不同的情况下,对非小细胞肺癌细胞迁移和侵袭能力的影响;另一方面通过Western-blot的方法来检测两种抑制剂在亲环素A表达水平不同的非小细胞肺癌细胞中,对EMT相关蛋白表达水平的影响。结果:1.在A549和H1299两种非小细胞肺癌细胞中,通过慢病毒感染,成功构建了亲环素A低表达(CypA-KD)和过表达(CypA-KD-OE)细胞株,及其各自的对照细胞;2.划痕实验结果显示,与对照组H1299 NC细胞相比,H1299 Cyp A-KD组细胞的迁移能力明显降低(P0.01),而H1299 Cyp A-KD-OE组细胞的迁移能力也明显高于对照组细胞H1299 CypA-KD-Vector(P0.01);Transwell迁移实验结果同样表明,与对照组细胞H1299/A549 NC相比,H1299 CypA-KD组(P0.001)和A549 CypA-KD(P0.05)组细胞的迁移能力也显著降低,同时H1299CypA-KD-OE组细胞(P0.001)和A549 CypA-KD-OE组细胞(P0.05)的迁移能力较对照组细胞H1299/A549 CypA-KD-Vector相比明显增高;然而,Transwell侵袭实验的结果则显示,无论是在A549还是H1299细胞中,亲环素A的表达水平对两者的侵袭能力都没有明显的影响;3.Wester-blot的结果显示,亲环素A的表达水平降低后,β-catenin,Vimentin和Snail蛋白的表达水平在H1299和A549细胞中都受到了明显的抑制,而随后在亲环素A过表达之后,三者的表达水平较对照组又明显升高。4.非小细胞肺癌小鼠转移模型的结果显示,14周后,A549组和A549CypA-KD-OE组的各自有5/5和5/5只小鼠肺部出现了转移病灶,而A549CypA-KD组肺部出现转移病灶的小鼠数量为0/5只;此外,在A549和A549CypA-KD-OE组中,各有一只小鼠出现了骨转移。5.在H1299和A549细胞中,亲环素A表达水平降低后,P38的磷酸化水平都出现了明显的降低,而当亲环素A的表达水平再次上调时,P38的磷酸化水平也随之上升,差异均具有统计学意义(P0.05);6.使用P38抑制剂SB203580处理细胞后,H1299 CypA-KD-OE细胞的迁移能力较对照组(DMSO)明显降低(划痕实验P0.01,Transwell迁移实验P0.01),而H1299 CypA-KD细胞的迁移能力与对照组(DMSO)相比,没有显著的统计学差异;7.经P38抑制剂SB203580处理后,H1299 CypA-KD-OE组细胞中,β-catenin,Vimentin和Snail蛋白的表达水平于对照组(DMSO)相比均受到明显的抑制,并且差异具有统计学意义(P0.05);而在H1299 Cyp A-KD组的细胞中,与对照组相比,三者的表达水平并未明显受到SB203580的影响。结论:1.体外实验的结果表明,亲环素A能够明显促进非小细胞肺癌细胞的迁移能力,同时能够促进细胞发生EMT;2.动物实验的结果表明,亲环素A能够促进非小细胞肺癌在体内发生转移;3.P38是亲环素A介导的非小细胞肺癌转移过程中的重要因素,因此亲环素A在非小细胞肺癌转移过程中的作用能够通过P38 MAPK信号通路来实现的。
[Abstract]:Background and objective: lung cancer is still a major health problem in China and in the world because of its high incidence and mortality. Lung cancer is generally insidious and early symptoms are not obvious, so it is easily ignored in the early stage of the disease. In addition, the early diagnosis and treatment of lung cancer are slow, and the 5 year survival rate is only 18%. most of the lung cancer patients. As one of the six major biological characteristics of cancer, "metastasis" has been the main cause of tumor recurrence and the death of the patients. Therefore, the mechanism of tumor metastasis and the signal pathways involved in tumor metastasis have been studied. As a member of the most early discovered cyclophilin family, cyclophilin A is highly expressed in various tumor tissues, such as lung cancer, liver cancer, pancreatic cancer, and breast cancer. It is found that cyclophilin A plays an important role in the development and development of these tumors. The metastasis is closely related to the level of the expression of cyclophilin A. However, there are few studies on the role of cyclophilin A in the metastasis of lung cancer and its related mechanisms. In our previous study, we found that the expression of cyclophilin A in non-small cell lung cancer cells was suppressed by RNA interference and the use of inhibitors. After leveling, cell migration and invasion were inhibited, which suggests that cyclophilin A may be able to promote the migration and invasion of non small cell lung cancer cells. Subsequently, we found that after inhibiting the expression of cyclophilin A in non-small cell lung cancer cells, a key molecule in the MAPK signaling pathway, phosphorylated water of the P38 It is also significantly inhibited, which suggests that the regulation of the migration and invasion of non small cell lung cancer cells by cyclophilin A may be achieved through the P38 MAPK signaling pathway. Therefore, this topic will further study the role of cyclophilin A in the metastasis of non-small cell lung cancer through in vitro and in vivo experiments. Methods: first, in the study of the role of cyclophilin A in the metastasis of non-small cell lung cancer, we constructed a cyclophilin A low expression cell line in two human non-small cell lung cancer cells, H1299 and A549 by the method of lentivirus infection, and on this basis, we construct a cyclophilin A overexpressed cell. The effects of the expression level of cyclophilin A on the migration and invasion of two human non-small cell lung cancer cells H1299 and A549 were observed by scratch test and Transwell test, and the expression level of the expression level of cyclophilin A on the expression level of EMT related proteins was observed by Western-blot method. Finally, the effect of the expression level of cyclophilin A on the metastasis of non-small cell lung cancer was further explored by the method of animal experiment to further explore the effect of the expression level of cyclophilin on non-small cell lung cancer. Then, in the study of the mechanism of the role of cyclophilin A in the metastasis of non-small cell lung cancer, we use the P38 inhibitor SB203580. Do not treat the low expression and high expression of non small cell lung cancer cells with cyclophilin A. On the one hand, the effects of two inhibitors on the migration and invasion ability of non small cell lung cancer cells in different levels of cyclophilin A expression are observed by cell migration and invasion experiments. On the other hand, two kinds of cells are detected by Western-blot method. The effect of inhibitors on the expression level of EMT related proteins in non small cell lung cancer cells with different levels of cyclophilin A. Results: 1. in two non small cell lung cancer cells of A549 and H1299, the low expression of cyclophilin A (CypA-KD) and overexpression (CypA-KD-OE) cell lines and their respective control cells were successfully constructed, and their respective control cells were constructed, 2. The results of the scratch test showed that compared with the control group H1299 NC cells, the migration ability of the cells in the H1299 Cyp A-KD group was significantly lower (P0.01), while the migration ability of the H1299 Cyp A-KD-OE group was significantly higher than the H1299 CypA-KD-Vector (P0.01) in the control group, and the results of the migration experiment also showed that the cells were compared with the control group. The cell migration ability of 99 CypA-KD group (P0.001) and A549 CypA-KD (P0.05) group also decreased significantly, while the migration ability of H1299CypA-KD-OE group (P0.001) and A549 CypA-KD-OE group cell (P0.05) was significantly higher than that of the control group cell H1299/A549 CypA-KD-Vector. In H1299 cells, the expression level of cyclophilin A has no significant influence on the invasiveness of the two. 3.Wester-blot results showed that the expression level of cyclophilin A decreased, and the expression level of beta -catenin, Vimentin and Snail protein was significantly inhibited in H1299 and A549 cells, and then after the expression of cyclophilin A, three The expression level of the.4. non small cell lung cancer mice was significantly higher than that in the control group. After 14 weeks, the A549 and A549CypA-KD-OE groups had 5/5 and 5/5 mice with metastatic lesions, while the number of mice with metastatic lesions in the A549CypA-KD group was 0/5 only; in addition, in the A549 and A549CypA-KD-OE groups, Each mouse had bone metastasis.5. in H1299 and A549 cells. After the level of cyclophilin A expression decreased, the phosphorylation level of P38 decreased significantly. When the expression level of cyclophilin A was up, the phosphorylation level of P38 increased, the difference was statistically significant (P0.05); 6. the P38 inhibitor SB203580 was used. After the cells, the migration ability of H1299 CypA-KD-OE cells was significantly lower than that of the control group (DMSO) (scratch test P0.01, Transwell migration test P0.01), while the migration ability of H1299 CypA-KD cells was no significant difference compared with the control group (DMSO); 7. The expression level of ntin and Snail protein was significantly inhibited compared with the control group (DMSO), and the difference was statistically significant (P0.05); in the cells of the H1299 Cyp A-KD group, the expression level of the three was not significantly affected by SB203580. Conclusion: the results of 1. in vitro showed that the cyclophilin A could be significantly promoted. The migration of non small cell lung cancer cells can also promote the cell formation of EMT. 2. animal experiments showed that procyclin A could promote the metastasis of non-small cell lung cancer in the body; 3.P38 was an important factor in the metastasis of non small cell lung cancer mediated by cyclophilin A. Therefore, cyclophilin A is in the process of metastasis of non-small cell lung cancer. Its role can be achieved through the P38 MAPK signaling pathway.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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