【作者】 柳昊；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 研究背景和目的：骨桥蛋白(Osteopontin, OPN)是一种分泌型钙结合的磷酸化糖蛋白。在多种高转移性的恶性肿瘤细胞和患者的血清中均有OPN的过度表达。OPN诱导肿瘤细胞的浸润和转移与基质金属蛋白酶MMP-2和MMP-9的活化密切相关,OPN通过IKK (I kappa B kinase)酶依赖的信号通道诱导NF-κB (nuclear factor kappaB)介导的MMP-2和MMP-9的活化。本研究旨在探讨OPN在人前列腺癌PC-3细胞的增殖和侵袭过程中所发挥的作用,为IKK-1和IKK-2酶在NF-κB介导的信号通道中可能的功能提供实验依据。方法：构建四种含OPNshRNA的真核表达载体,分别转染人前列腺癌PC-3细胞,然后采用半定量RT-PCR方法筛选能显著抑制OPN基因表达的重组质粒。建立稳定转染OPNshRNA重组质粒的PC-3细胞株,并且应用不同浓度的IKK抑制剂Ⅶ分别抑制IKK-1和IKK-2两种酶的活性,然后采用Real-time PCR和Western blot分别检测OPN、MMP-2和MMP-9三种mRNA和蛋白的表达水平,ELISA法检测加入OPN蛋白培养的PC-3细胞的条件培养基中MMP-2、MMP-9的浓度。流式细胞术、MTT法和Transwell实验分别检测细胞生长周期变化、细胞增殖和和侵袭能力变化。应用成瘤实验观察OPN shRNA表达载体对PC-3细胞在裸鼠体内的生长潜能的影响。结果：RT-PCR方法成功筛选出能显著抑制前列腺癌PC-3细胞中OPN表达的OPN shRNA重组质粒PGPU6/GFP/Neo-OPN2,建立了稳定传代的细胞株PCs, PC/Vect和PC/OPN2。与PCs组相比,转染重组质粒的细胞PC/OPN2中OPN、MMP-2和MMP-9三种蛋白的表达量分别下降了55.22%、51.71%和28.35%(P<0.05),PC/OPN2细胞的增殖、迁移和侵袭能力受到明显抑制。此外,特异性地抑制IKK-2酶的活性能显著降低MMP-2和MMP-9的表达水平,而抑制IKK-1酶的活性对MMP-2和MMP-9的表达水平没有明显影响。结论：OPN shRNA表达载体介导的OPN基因沉默不仅能下调OPN的表达而且能抑制MMP-2和MMP-9的分泌,OPN shRNA对前列腺癌PC-3细胞的增殖、迁移和侵袭能力具有抑制作用,而且该过程与MMP-2和MMP-9两种酶的表达减少密切相关,此外,在NF-κB介导的OPN诱导MMP-2和MMP-9活化的过程中IKK-2酶的活性发挥了重要的作用。更多还原

【Abstract】 Objective:Substantial data have linked osteopontin (OPN), a secreted phosphoglycoprotein, with tumor progression and metastatic spread. OPN regulates cell migration and invasion in a variety of cancers and induces the activities of matrix metalloproteinase (MMP)-2 and MMP-9. It has been reported that OPN induce matrix metalloproteinase (MMP)-2 and MMP-9 activations through nuclear factor kappaB (NF-κB)-mediated signaling pathways. This study was to investigate the role of OPN in the proliferation and invasion of human prostate cancer PC-3 cells, and provide clues about the possible functions of IκB kinase (IKK) in OPN-induced MMP-2/9 activitions by nuclear factor kappaB (NF-KB)-mediated signaling pathways. Methods: Four sorts of OPN short-hairpin RNA (shRNA) recombinant plasmids were constructed and transfected into PC-3 cells. The most highly functional shRNA recombinant plasmid was selected by RT-PCR for further studies, then after the stablely transfected cell line were established, the different concentrations of IKK inhibitors were used to inhibit the activities of IKK-1 and IKK-2. The mRNA and protein expression levers of OPN, MMP-2 and MMP-9 were detected by real-time PCR and Western blot. MMP-2 and MMP-9 protein levels in the medium of the cells cultured by OPN were detected by ELISA. The different cell cycles, cell proliferation and invasion abilities of PC-3 cells were detected by flow cytometry, MTT and Transwell chamber assays, respectively. The tumor formation assay were used to observe the growth situation of PC-3 cells in vivo. Results:The most highly functional shRNA recombinant plasmid (PGPU6/GFP/Neo-OPN2) was selected by RT-PCR assay. It can obviously inhibit OPN expression in PC-3 cells and the stablely transfected cell line, PCs, PC/Vect and PC/OPN2 were established. Compared with untreated cells(PCs), the protein expression levers of OPN, MMP-2 and MMP-9 in PC-3 cells transfected with recombinant plasmids (PC/OPN2) were reduced by 55.22%,51.71% and 28.35%, respectively, thereby resulting in suppression of the proliferation, migration and invasion of PC/OPN2 cells. Moreover, the inhibition of IKK-2 inhibited the expressions of MMP-2 and MMP-9, while the inhibition of IKK-1 had no influence on the expressions of MMP-2 and MMP-9. Conclusions:A short hairpin RNA expression vector-mediated OPN gene silencing can not only inhibit OPN expression in PC-3 cells but also decrease the expression levels of MMP-2 and MMP-9. OPN shRNA recombinant plasmid can inhibit the malignant physiological behaviors of PC-3 cells and these processes are associated with the activities of MMP-2 and MMP-9. Moreover, IKK-2 may play a crucial role in the OPN-induced activations of MMP-2 and MMP-9 via NF-κB mediated signaling pathways.更多还原