OTUD7B对乳腺癌细胞增殖、迁移、凋亡及血管内皮生长因子表达的影响

发布时间：2018-04-16 05:31

本文选题：LV3-si-hOTUD7B + pEGFP-hOTUD7B　；参考：《河北大学》2017年硕士论文

【摘要】：目的:乳腺癌对世界女性造成的身心伤害早已不容乐观。乳腺癌的发生、发展是由多基因、多通路、多反应相互作用的结果,其发病率和病死率呈现逐年上升的趋势。肿瘤的增殖速率、发生转移及转移部位不同直接影响着乳腺癌患者的生存期。近年来乳腺癌患者的发病年龄趋向于年轻化,发生侵袭、转移的机率大大增加,导致许多患者就诊时已经失去了手术时机,直接影响着患者总生存期。所以抑制肿瘤的生长、阻断肿瘤的转移,对提高患者的生存率、改善生活质量发挥着至关重要的作用。然而肿瘤血管生成是引发恶性肿瘤发生、发展、转移的根本源头,所以抑制乳腺癌细胞的血管生成对治疗乳腺癌具有极其重大的意义。其中核因子-Kappa B(NF-κB)可调节血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,而去泛素化酶对于NF-κB的调节至关重要。有研究发现NF-κB不仅能够在胃癌、甲状腺癌、乳腺癌中促进VEGF的表达,还能够促进淋巴结的转移。去泛素化酶OTUD7B能够阻止NF-κB激活,而NF-κB能够调节癌细胞迁移、侵袭、血管内皮生长因子的表达等。所以明确去泛素化酶OTUD7B与乳腺癌细胞增殖、迁移、凋亡及VEGF表达之间的关系,可能为治疗乳腺癌提供新的方法。方法:构建慢病毒小干扰RNA沉默载体(LV3-si-h OTUD7B)及对照载体(LV3-NC),构建带有绿色荧光蛋白标签的人OTUD7B表达质粒(pEGFP-h OTUD7B)及对照质粒(pEGFP-CI)。用包含LV3-si-h OTUD7B、LV3-NC、pEGFP-h OTUD7B、pEGFP-CI慢病毒液分别感染MCF-7细胞。MTS法检测OTUD7B对MCF-7细胞增殖的影响;细胞划痕实验评价OTUD7B对MCF-7细胞迁移能力的影响;流式细胞术PI染色法检测OTUD7B对MCF-7细胞周期的影响;蛋白质印迹法(Western blot)检测OTUD7B对MCF-7细胞细胞质NF-κBP65和细胞核NF-κBP65的表达水平的调节;RT-PCR检测OTUD7B对VEGF m RNA的相对表达水平的影响;ELISA检测OTUD7B对MCF-7乳腺癌细胞分泌VEGF的调节。应用SPSS19.0统计软件进行处理,所有数据以均数±标准差(?)表示,均数比较采用t检验或单因素方差分析(One-way ANOVA),以P0.05为差异有统计学意义。结果:通过MTS法检测细胞增殖显示,转染pEGFP-h OTUD7B后,MCF-7乳腺癌细胞增殖受到抑制,转染LV3-si-h OTUD7B后,MCF-7乳腺癌细胞增殖得到促进。细胞划痕实验表明,转染pEGFP-h OTUD7B后,MCF-7乳腺癌细胞迁移受到抑制,转染LV3-si-h OTUD7B后,MCF-7乳腺癌细胞迁移能力升高。流式细胞术PI染色检测细胞周期结果提示pEGFP-h OTUD7B可调节MCF-7细胞周期阻滞在G1期。Western blot结果分析显示,与阴性对照组及正常对照组相比,pEGFP-h OTUD7B能够下调MCF-7乳腺癌细胞核蛋白NF-κBP65的表达水平,而细胞质内NF-κBP65却无明显差异。RT-PCR结果提示,在m RNA水平,pEGFP-h OTUD7B抑制了VEGF的表达。ELISA法检测MCF-7细胞条件培养基中的VEGF含量结果显示,与阴性对照组及正常对照组相比,pEGFP-h OTUD7B组条件培养基中VEGF含量低。结论:OTUD7B能够抑制MCF-7乳腺癌细胞增殖、迁移能力,可调节MCF-7乳腺癌细胞阻滞于G1期,同时还可抑制MCF-7乳腺癌细胞对VEGF的表达和分泌。
[Abstract]:Objective: breast cancer in the world women's physical and psychological harm caused already is not optimistic. Breast cancer development by multiple genes, multiple channels, multiple reaction interaction, the incidence rate and mortality rate is rising year after year. The proliferation rate of tumor, metastasis and metastasis of breast cancer directly affects different parts the survival of the patients. In recent years, the age of onset of breast cancer patients tend to be younger, invasion, metastasis rate increased significantly, resulting in many patients have lost the opportunity of operation, directly affect the overall survival of patients. So the inhibition of tumor growth, tumor metastasis, to improve the survival rate of the patients. Play a crucial role in improving the quality of life. However, tumor angiogenesis is caused by malignant tumor occurrence, development and metastasis of the fundamental source, so the inhibition of breast cancer cell to treat angiogenesis Has the extremely significant significance of treatment of breast cancer. The nuclear factor -Kappa B (NF- K B) can regulate vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) and the expression of deubiquitinase for regulating vital NF- kappa B. Studies have found that NF- K B can not only in gastric cancer, thyroid cancer, expression the promotion of VEGF in breast cancer, can also promote lymph node metastasis. Deubiquitinase OTUD7B prevented NF- kappa B activation, and NF- K B can regulate cancer cell migration, invasion, vascular endothelial growth factor expression. So clearly deubiquitinase OTUD7B and breast cancer cell proliferation, migration, the relationship between the expression of VEGF and apoptosis, may provide a new method for the treatment of breast cancer. Methods: to construct lentiviral vector silencing by small interfering RNA (LV3-si-h OTUD7B) and control vector (LV3-NC), constructed a green fluorescent protein tag OTUD7B expression plasmid (pE GFP-h OTUD7B) and the control plasmid (pEGFP-CI) containing LV3-si-h OTUD7B. LV3-NC, pEGFP-h, OTUD7B, pEGFP-CI lentivirus infected MCF-7 cells was detected by.MTS OTUD7B on the proliferation of MCF-7 cells; evaluation of cell scratch test OTUD7B on MCF-7 cells migration effects; flow cytometry with PI staining was detected on OTUD7B MCF-7 cell cycle; Western blotting (Western blot) regulating the level of OTUD7B expression was detected in MCF-7 cell cytoplasm and nucleus of NF- NF- kappa BP65 kappa BP65; the relative expression level of RT-PCR effects on VEGF M detection of OTUD7B RNA; ELISA OTUD7B VEGF on the secretion regulation of detection of human breast cancer cell line MCF-7 by statistical software SPSS19.0. For processing, all data to mean + standard deviation (?) said, were compared by t test or ANOVA (One-way ANOVA), with P0.05 as the difference was statistically significant. Results: Cell proliferation was detected by MTS method showed that the transfection of pEGFP-h OTUD7B, the proliferation of MCF-7 breast cancer cells was inhibited by transfection of LV3-si-h, OTUD7B, MCF-7 breast cancer cell proliferation was promoted. The scratch test indicates that the cells transfected with pEGFP-h, OTUD7B, MCF-7 in breast cancer cell migration was inhibited after transfection of LV3-si-h, OTUD7B, MCF-7 in breast cancer cell migration.. PI staining flow cytometry to detect the cell cycle results suggest that pEGFP-h OTUD7B may regulate MCF-7 cell cycle arrest in G1 phase,.Western blot results showed that compared with the negative control group and normal control group, the expression level of pEGFP-h OTUD7B could downregulate MCF-7 breast cancer cell nuclear protein NF- kappa BP65, and cytosolic NF- kappa BP65 but no significant difference of.RT-PCR results suggest that m in RNA level, pEGFP-h OTUD7B inhibited the expression of VEGF.ELISA detected by MCF-7 cell conditioned culture medium VEGF content. The results show that, with the negative control group and normal control group compared to pEGFP-h group OTUD7B conditioned medium with low content of VEGF. Conclusion: OTUD7B can inhibit MCF-7 proliferation, migration of breast cancer cells, can regulate breast cancer MCF-7 cell arrest in G1 phase, and can also inhibit the secretion of MCF-7 breast cancer cells on the expression of VEGF.

【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

【参考文献】