核转录因子RelB在前列腺癌中的作用及其机制的探讨

发布时间：2018-06-10 17:31

本文选题：前列腺癌 + RelB　；参考：《苏州大学》2014年硕士论文

【摘要】：目的本研究试图阐明NF-κB家族成员中的RelB在前列腺癌细胞中的作用以及探讨潜在的生物学机制。方法用Western blot法检测三种前列腺癌细胞株中NF-κB家族成员的蛋白表达情况。建立稳定转染RelB-siRNA的DU145细胞株。Western blot法检测RelB沉默后细胞的NF-κB家族其它成员的蛋白表达情况。用xCELLigence系统实时动态观察RelB沉默后细胞生长，，以及细胞迁移和细胞侵袭的变化。用流式细胞术检测RelB沉默后细胞的细胞周期、细胞增殖以及细胞凋亡的变化。用qRT-PCR技术检测基因的mRNA水平的表达。用细胞划痕实验观察RelB沉默后细胞迁移能力的变化。用明胶酶谱MMP活性检测方法检测RelB沉默后细胞的MMP2、MMP9活性变化。结果RelA、p50、RelB和p52蛋白在雄激素非依赖型前列腺癌细胞株DU145和PC-3中呈持续性高表达，RelB在DU145细胞中的表达最为显著。在DU145细胞株中分别稳定转染pSilencer3.1-siRelB质粒和pSilencer3.1质粒，成功获得实验组RelB低表达DU145-siRelB细胞和对照组DU145-control细胞。RelB的沉默对细胞中NF-κB家族其它成员无明显影响。DU145-siRelB细胞的生长速度明显慢于DU145-control细胞，并且迁移、侵袭能力均较DU145-control细胞显著减弱。细胞划痕实验也表明了DU145-siRelB细胞的迁移能力明显较DU145-control细胞弱。DU145-siRelB细胞与DU145-control细胞在细胞增殖和细胞周期上无明显差异。DU145-siRelB细胞的凋亡细胞百分比比DU145-control细胞明显增多。DU145-siRelB细胞中的bcl-2基因mRNA表达水平较DU145-control细胞明显下调，其余相关基因Bcl-xl、ICAP1、Bax、A20、Bim、Mcl-1mRNA表达水平无明显变化。DU145-siRelB细胞中的ITGB1基因的mRNA和蛋白质水平的表达均较DU145-control细胞显著下调。DU145-siRelB细胞中的MMP2、MMP9蛋白质水平的表达均较DU145-control细胞显著下调，同时明胶酶谱MMP活性检测方法检测显示DU145-siRelB细胞MMP2和MMP9的表达显著低于DU145-control细胞。结论在雄激素非依赖型前列腺癌细胞株中，RelB在DU145细胞中表达最为显著。DU145-siRelB细胞的生长明显慢于DU145-control细胞，并且迁移、侵袭能力均较DU145-control细胞显著减弱。RelB的缺失显著抑制了前列腺癌细胞株DU145细胞的生长，与细胞凋亡增多相关，bcl-2基因的下调可能是潜在的原因之一。RelB缺失显著抑制了肿瘤细胞的迁移和侵袭能力，ITGB1基因表达的下调以及MMP2和MMP9活力的减弱是潜在的原因之一。
[Abstract]:Objective to elucidate the role of RelB from NF- 魏 B family in prostate cancer cells and to explore the potential biological mechanisms. Methods Western blot assay was used to detect the protein expression of NF- 魏 B family members in three prostate cancer cell lines. A stable DU145 cell line transfected with RelB-siRNA was established. Western blot assay was used to detect the protein expression of other members of NF- 魏 B family after the silencing of RelB. XCELLigence system was used to observe the cell growth, cell migration and cell invasion after RelB silencing. Cell cycle, cell proliferation and apoptosis were detected by flow cytometry. The mRNA level of the gene was detected by qRT-PCR. The cell migration ability was observed by cell scratch assay after RelB silencing. The changes of MMP2MMP9 activity were detected by gelatinase assay. Results the expression of RelAp50A RelB and p52 protein in androgen independent prostate cancer cell lines DU145 and PC-3 was significantly higher than that in DU145 cells. After stable transfection of pSilencer3.1-siRelB and pSilencer3.1 plasmids in DU145 cell line, the silencing of DU145-siRelB cells in experimental group and DU145-siRelB cells in control group had no significant effect on the growth rate of DU145-siRelB cells compared with that of DU145-control cells, and the growth rate of DU145-siRelB cells was significantly slower than that of DU145-control cells. Moreover, migration and invasion ability of DU 145-control cells were significantly decreased compared with those of DU145-control cells. Cell scratch assay also showed that the migration ability of DU145-siRelB cells was significantly weaker than that of DU145-control cells. There was no significant difference in cell proliferation and cell cycle between DU145-siRelB cells and DU145-siRelB cells. The percentage of apoptotic cells in DU145-siRelB cells was significantly higher than that in DU145-control cells. The expression of bcl-2 mRNA in DU145-control cells was significantly lower than that in DU145-control cells. The mRNA and protein levels of ITGB1 gene in DU145-siRelB cells were significantly down-regulated than those in DU145-RelB cells, and the expression of MMP2mMP9 in DU145-RelB cells was significantly lower than that in DU145-control cells, and the expression of MMP2mMP9 in DU145-RelB cells was significantly lower than that in DU145-control cells. The expression of MMP2 and MMP9 in DU145-siRelB cells was significantly lower than that in DU145-control cells. Conclusion the expression of MMP2 and MMP9 in androgen independent prostate cancer cells is the most significant in DU145 cells. Significantly slower than DU145-control cells, The migration and invasion ability of DU145-control cells were significantly lower than those of DU145-control cells. The loss of RelB significantly inhibited the growth of prostate cancer cell line DU145. The down-regulation of bcl-2 gene associated with increased apoptosis may be one of the potential reasons. The deletion of RelB significantly inhibits the migration and invasion of tumor cells. The down-regulation of ITGB1 gene expression and the decrease of MMP2 and MMP9 activity are one of the underlying reasons.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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