BEX2基因在结直肠癌增殖和转移中的作用及其机制研究

发布时间：2018-06-04 15:50

本文选题：BEX2 + 结直肠癌　；参考：《浙江大学》2016年博士论文

【摘要】：研究目的Bex2(Brain expressed X-linked protein 2)蛋白属于IDPs(Instrinsically Disordered Proteins),包含128个氨基酸,在细胞的转录调节、周期、增殖等信号通路中发挥着重要作用。近年来,有研究报道BEX2与肿瘤发生有密切关系,且在不同类型的肿瘤中发挥不同的功能。目前BEX2在结直肠癌组织中的功能如何尚未见报道。深入探讨BEX2在结直肠癌中的功能和机制不仅可进一步探究结直肠癌发生发展中遗传学特征,也可为治疗结直肠癌提供新的靶点。研究内容和方法本课题从公共数据库GEO (Gene Expression Omnibus)采集GSE14333数据库中结直肠癌组织的BEX2的表达水平,分析其与结直肠癌临床分期的相关性,并利用定量PCR检测34例新鲜结直肠癌组织中的BEX2表达,验证BEX2与临床分期的关系。利用细胞增殖实验、克隆形成实验、细胞迁徙迁移实验、动物致瘤能力实验和动物体内肝转移模型验证BEX2对结直肠癌细胞的影响。同时,结合生物信息学分析、蛋白免疫印迹实验、基因芯片技术等探索BEX2的可能下游调控机制。研究结果我们第一次分析了BEX2表达水平与结直肠癌分期的关系,首次发现BEX2的表达与结直肠癌的TNM分期呈正相关。体内外实验证实BEX2敲除后能明显抑制结直肠癌细胞增殖,克隆形成能力和致瘤能力。BEX2敲除后MAPK通路中的JNK和c-Jun的磷酸化表达减弱,而Erk及p-38未发生改变。而在非沉默BEX2细胞中加入JNK选择性抑制剂SP600125后生长得到显著抑制,且生长受抑制程度和BEX2敲除组类似；同时,我们发现BEX2敲除后能明显抑制结直肠癌细胞发生迁移,盲肠原位肝转移模型和半脾肝转移模型结果均提示BEX2敲除后能显著延长BEX2敲除组肝转移模型老鼠的生存时间,且该过程是非EMT依赖的,高通量芯片比较BEX2敲除前后的基因差异,提示与MMP家族基因改变相关。研究结论与创新BEX2在不同类型的肿瘤中发挥不同的功能,而BEX2在结直肠癌中的研究尚未报道。深入探讨BEX2在结直肠癌中的功能和机制研究有助于进一步明确结直肠癌发生发展的分子机制,从为基因治疗提供了新的靶点。1.本文利用系统生物学分析提取了GEO数据库中数据,并用大量结直肠癌新鲜组织样本中验证了BEX2表达水平和结直肠癌临床病理特征,第一次发现BEX2的表达水平与结直肠癌TNM分期呈明显正相关。2.本文构建了BEX2基因敲除细胞系,第一次证明敲除BEX2后结直肠癌细胞增殖,克隆形成能力,动物致瘤能力明显降低,主要通过MAPK/JNK信号通路来抑制结直癌细胞增殖。3.本研究为进一步研究BEX2在结直肠癌肝转移中的作用提供基础。我们成功建立了大肠癌盲肠原位及半脾肝转移模型,发现BEX2敲除后抑制了结肠癌肝转移发生,且该过程是EMT非依赖的,基因芯片数据提示可能与MMP系列基因改变相关。
[Abstract]:Objective Bex2(Brain expressed X-linked protein 2) is a member of IDPs(Instrinsically Disordered proteins, which contains 128 amino acids and plays an important role in cell transcriptional regulation, cell cycle, proliferation and other signaling pathways. In recent years, it has been reported that BEX2 is closely related to tumorigenesis and plays different functions in different types of tumors. At present, the function of BEX2 in colorectal cancer tissues has not been reported. Further study on the function and mechanism of BEX2 in colorectal cancer can not only further explore the genetic characteristics of colorectal cancer, but also provide a new target for the treatment of colorectal cancer. Methods the expression level of BEX2 in colorectal cancer tissue was collected from GEO gene Expression Omnibus database, and the correlation between BEX2 expression and clinical stage of colorectal cancer was analyzed. Quantitative PCR was used to detect the expression of BEX2 in 34 cases of fresh colorectal cancer, and to verify the relationship between BEX2 and clinical stage. The effects of BEX2 on colorectal cancer cells were tested by cell proliferation assay, clone formation test, migration and migration test, tumorigenic ability test and liver metastasis model in vivo. At the same time, the possible downstream regulation mechanism of BEX2 was explored by bioinformatics analysis, Western blot assay and gene chip technology. Results for the first time, we analyzed the relationship between the expression of BEX2 and the staging of colorectal cancer. For the first time, we found that the expression of BEX2 was positively correlated with the TNM stage of colorectal cancer. In vitro and in vivo experiments showed that BEX2 knockout could significantly inhibit the proliferation of colorectal cancer cells. The expression of JNK and c-Jun phosphorylation in MAPK pathway was decreased after knockout of BEX2 and clone formation ability. However, Erk and p-38 did not change. SP600125, a selective inhibitor of JNK, was significantly inhibited in non-silencing BEX2 cells, and the inhibition was similar to that in BEX2 knockout group. At the same time, we found that BEX2 knockout could significantly inhibit the migration of colorectal cancer cells. The results of cecum in situ liver metastasis model and hemisplenic liver metastasis model showed that BEX2 knockout could significantly prolong the survival time of BEX2 knockout mice, and the process was independent of EMT. The gene differences before and after BEX2 knockout were compared by high throughput microarray. The results suggest that it is related to the gene change of MMP family. Conclusion BEX2 plays different roles in different types of tumors, while BEX2 in colorectal cancer has not been reported. Further study on the function and mechanism of BEX2 in colorectal cancer is helpful to clarify the molecular mechanism of colorectal cancer and provide a new target for gene therapy. The data from GEO database were extracted by systematic biological analysis. The expression level of BEX2 and clinicopathological features of colorectal cancer were verified by a large number of fresh tissue samples. It was found for the first time that the expression of BEX2 was positively correlated with the TNM stage of colorectal cancer. BEX2 gene knockout cell line was constructed in this paper. It was first demonstrated that the proliferation, clone formation and tumorigenic ability of colorectal cancer cells after BEX2 knockout were significantly decreased, mainly through the MAPK/JNK signaling pathway to inhibit the proliferation of colorectal cancer cells. 3. This study provides a basis for further study of the role of BEX2 in liver metastasis of colorectal cancer. We successfully established an in situ and hemipplegic liver metastasis model of colorectal cancer. It was found that BEX2 knockout inhibited the occurrence of liver metastasis of colon cancer and the process was EMT independent. The gene chip data suggested that this process might be related to the MMP gene series.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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