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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 calcium ion / calmodulin-dependent protein kinase Ⅱ inhibitory factor α (hCaMKⅡNα) on the secretion of immunosuppressive factors in human colon cancer cells and its mechanism. METHODS: hCaMKⅡNα gene expression vector (pKⅡNα) or siRNA (si-KⅡNα) were transfected into colon tumor cells (LoVo cells, SW620 cells and HT29 cells) to form overexpression or interference expression cells. The expression of interleukin-8 (IL-8), interleukin-10 (IL-10) and vascular endothelial cell (VEGF) in colon cancer LoVo cells after overexpression of hCaMKⅡα were detected by RT- The expression of VEGF, PGE2, IL-8 and IL-10 in SW620 and LoVo cells were detected by ELISA. To investigate the role of extracellular regulated protein kinase (ERK1 / 2) in the regulation of hCaMKⅡNα-mediated cytokines and the inhibition of ERK1 / 2 activity in HT-29 cells by U0126, the expression of hCaMKⅡNα was down-regulated Effect on VEGF and IL-8 Secretion. Results: hCaMKⅡinhibited the expression of VEGF, IL-8 and IL-10 mRNA in colon cancer LoVo cells, inhibited the secretion of IL-8 and VEGF protein in SW620 and LoVo cells, but not in secretion of PGE2 and IL-10 influences. Correspondingly, down-regulation of hCaMKⅡNα expression by RNA interference significantly up-regulated the secretion of IL-8 and VEGF in HT29 cells; and found that the inhibition of MEK1 / 2 activity completely blocked the effect of hCaMKⅡNα on IL-8, but only partially blocked Effect on VEGF. CONCLUSION: hCaMKⅡNα can downregulate the secretion of VEGF and IL-8 in colon tumor cells by inhibiting ERK activity, which plays a negative regulatory role in the immune escape of colon tumors.