摘要
目的研究钙离子/钙调蛋白依赖性蛋白激酶Ⅱγ(CaMKⅡγ)体内外促进结直肠癌细胞增殖的作用并探讨其机制。方法半定量RT-PCR法测定5种结直肠癌细胞系及20对结直肠癌组织及其配对癌旁组织中CaMKⅡγmRNA表达水平。用慢病毒载体pLenti6.3-MCS-IRES2-eGFP制备慢病毒颗粒Lenti-CaMKⅡγ,转染SW620细胞,建立稳定表达CaMKⅡγ结直肠癌细胞系SW620-CaMKⅡγ。测定SW620-CaMKⅡγ细胞的生长曲线及克隆形成能力。Western blotting检测SW620-CaMKⅡγ细胞IKKα、IKKβ、IKKγ、p-IKKα/β、P-IκB和IκB表达水平。免疫荧光检测SW620-CaMKⅡγ细胞NF-κB p65核浆及核内的表达情况。检测裸鼠移植瘤的瘤体积。结果 CaMKⅡγ在5种结直肠癌细胞系中的mRNA水平均高,在20对组织中有18对癌组织mRNA水平比癌旁组织高。慢病毒转染的CaMKⅡγ过表达细胞系增殖能力增强(P<0.05)。CaMKⅡγ能够激活细胞内的NF-κB信号通路,促进NF-κ3 p65进核。CaMKⅡγ过表达细胞的裸鼠移植瘤瘤体积大于阴性对照(P<0.05)。结论 CaMKⅡγ体内外均能促进结直肠癌细胞的增殖,并激活NF-κB通路。
Objective To investigate the effects of the y isoform of Ca2＋/calmodulin-dependent protein kinase II （CaMKIIy） on colorectal cancer （CRC） cell growth in vitro and in vivo and explore the mechanisms. Methods The mRNA levels of CaMKIIy in 5 CRC cell lines, tumor tissues and matched adjacent tissues from 20 CRC patients were examined by semi-quantitative RT-PCR. The lentiviral vector pLenti6.3-MCS-IRES2-eGFP was used to genrate the lentivirus particle Lenti-CaMKIIy for transfecting SW620 cells. The proliferation ability of the transfected SW620-CaMKIIy cells was assessed by growth curve and colony formation assay. The expression of IKKa, IKKβ, IKKy, p-IKKα/β, p-IKB and IkB of the transfected cells were determined by Western blotting, and the expression and localization of nuclear factor-kB （NF-lcB） p65 were detected by immunofluorescence, in nude mouse models bearing the transfected SW620-CaMKIIy cell xenograft, the tumor volume was measured twice a week. Results CaMKIIy mRNA showed high expressions in the 5 colorectal cancer cell lines. Eighteen of the 20 tumor tissues showed higher expressions of CaMKIIy than the adjacent non-tumor tissues. The proliferation of transfected SW620-CaMKIIy cells was enhanced significantly. CaMKIIy activated NF-KB signaling pathway and led to NF-lcB p65 nuclear translocation. In the tumor-bearing mouse model, the volume of the tumors generated by the transfected SW620-CaMKIIy cells was 1.46- and 1.68-fold higher than that of the tumors with the control cells at the 8th and 12th day respectively. Conclusion CaMKIIy can effectively promote the growth of colorectal cancer cells in vitro and in vivo by activating NF-kB signaling pathway.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2013年第5期649-653,共5页
Journal of Southern Medical University
基金
国家自然科学基金(30672381,30873095,81070420,81270601)
浙江省卫生高层次创新人才基金~~
关键词
钙离子/钙调蛋白依赖性蛋白激酶Ⅱ_γ
结直肠癌
核转当因子κB
细胞增殖
Ca2＋/calmodulin-dependent protein kinase II y
colorectal cancer
SW620 cells
nuclear transcription factor-KB
cellproliferation
