hCaMKⅡNα对结肠肿瘤分泌免疫抑制因子的抑制作用

The inhibitory effect of hCaMKⅡNα on the production of immunosuppressive factors in colon cancer cells in vitro

目的:探讨钙离子/钙调素依赖性蛋白激酶Ⅱ抑制蛋白α(human calcium/calmodulin-dependent protein kinaseⅡinhibitory alpha,hCaMKⅡNα)对结肠肿瘤细胞分泌免疫抑制因子的影响及其作用机制。方法:将hCaMKⅡNα基因表达载体(pKⅡNα)或siRNA(si-KⅡNα)转染至结肠肿瘤细胞(LoVo细胞、SW620细胞和HT29细胞),形成过表达或干扰表达细胞。RT-PCR检测结肠癌LoVo细胞过表达hCaMKⅡα后白细胞介素-8(interleukin-8,IL-8)、白细胞介素-10(interleukin-10,IL-10)和血管内皮生长因子(vascular endothelial cell growth factor,VEGF)mRNA表达水平,ELISA法检测转染pKⅡNα或si-KⅡNα对SW620和LoVo细胞中VEGF、PGE2、IL-8和IL-10分泌的影响。为观测细胞外调节蛋白激酶1/2(extracellular regulated protein kinases,ERK1/2)在hCaMKⅡNα介导的细胞因子调控中的作用,利用U0126抑制HT-29细胞中ERK1/2活性后,检测hCaMKⅡNα表达下调对VEGF和IL-8分泌的影响。结果:hCaMKⅡ可显著抑制结肠癌LoVo细胞中VEGF、IL-8和IL-10mRNA水平的表达;可抑制SW620和LoVo细胞中IL-8和VEGF蛋白的分泌,但对PGE2和IL-10的分泌没有影响。相应地,利用RNA干扰技术下调hCaMKⅡNα表达可显著上调HT29细胞中IL-8和VEGF的分泌;并且发现MEK1/2活性的抑制可完全阻断hCaMKⅡNα对IL-8的影响,但只能部分阻断对VEGF的影响。结论:hCaMKⅡNα通过抑制ERK活性下调结肠肿瘤细胞VEGF和IL-8的分泌,在结肠肿瘤免疫逃逸中起到负相调控作用。

Objective： To investigate the effect of（ human calcium/calmodulin-dependent protein kinase Ⅱ inhibitory alpha（ hCaMKⅡN-α） on the production of immunosuppressive factors in colon cancer cells and the mechanisms underlying the effect in vitro. Methods： Overexpression and silencing of the hCaMKⅡN-α gene in human colon adenocarcinoma（ LoVo,SW620 and HT29） cells were achieved by transfection with a hCaMKⅡN-α-expressing plasmid（ pKⅡN-α） and an siRNA（ si-KⅡN-α） vector,respectively. Messenger RNA levels of interleukin-8（ IL-8）,interleukin-10（ IL-10） and vascular endothelial cell growth factor（ VEGF） in LoVo cells transfected with pKⅡN- were analyzed by RT-PCR. Protein levels of IL-8,IL-10 and VEGF in SW620 and LoVo cells transfected with pKⅡN- and in HT29 cells transfected with si-KⅡN-were determined by ELISA. The differences in IL-8 and VEGF protein levels in HT29 cells transfected with pKIN-α in the presence or absence of U0126（ 10 M）,a selective ERK1 /2 inhibitor,were analyzed to elucidate the role of ERK1 /2 in hCaMKⅡN-α-mediated IL-8 and VEGF production. Results： Overexpression of hCaMKⅡN- significantly decreased the mRNA abundance and protein levels of VEGF and IL-8（ P 〈 0. 05） but not PGE2（ P 〉 0. 01）. Silencing of hCaMKⅡN-by siRNA significantly increased the secretion of VEGF and IL-8 in HT29 cells,but had no effect on the secretion of PGE2 and IL-10. U0126 treatment resulted in a complete reversion of increased IL-8 secretion but only a partial reversion of increased VEGF secretion in HT29 cells overexpressing hCaMKⅡ-α. Conclusion： Our observations suggest that hCaMKⅡ- may inhibit the secretion of VEGF and IL-8 and thus down-regulate the immune response in rectal tumor cells through an ERK signaling pathway-dependent mechanism.