结肠癌来源外泌体对自然杀伤细胞miR-18a和NKG2D表达的影响

Effect of colon cancer derived exosomes on the expression of miR-18a and NKG2D in natural killer cells

目的基于miR-18a/NKG2D探究结肠癌来源外泌体(CDEs)对自然杀伤(NK)细胞杀伤作用的影响。方法取人结肠癌SW480细胞培养后收集CDEs并进行鉴定;从健康志愿者外周血中分离NK细胞并采用IL-2刺激其增殖,流式细胞仪进行鉴定;将CDEs与NK细胞共培养后采用CCK-8法检测NK细胞活力;流式细胞术分析CD107a表达,酶联免疫吸附法(ELISA)检测肿瘤坏死因子α(TNF-α)和干扰素γ(IFN-γ)的表达,评估NK细胞对SW480细胞的杀伤功能;流式细胞术测定NK细胞表面NKG2D受体表达水平;实时荧光定量PCR(qPCR)检测NK细胞中miR-18a的表达。结果电子透射显微镜下CDEs呈圆盘状、双层膜囊泡状结构,Western blotting可检测到外泌体阳性标志蛋白CD63、CD81的表达;IL-2刺激后CD3-、CD56+的NK细胞比例显著增加(P<0.05);CDEs并未明显影响NK细胞生长活力(P=0.999),与阴性对照组相比,经CDEs干预的NK细胞CD107a表达、TNF-α、IFN-γ、NKG2D受体表达水平明显降低(P<0.05),miR-18a表达明显增加(P<0.05),且呈一定的浓度依赖性。结论CDEs可能通过上调miR-18a的表达、下调NKG2D表达,抑制NK细胞的杀伤作用来介导结肠癌细胞的免疫逃逸。

Objective To explore the effect of colon cancer derived exosomes (CDEs) on the killing effect of natural killer (NK) cells based on miR-18a/NKG2D.Methods Human colon cancer SW480 cells were cultured,and CDEs were collected and identified.NK cells were isolated from peripheral blood of healthy volunteers and stimulated by IL-2,then the proliferation was identified by flow cytometry.After CDEs were co-cultured with NK cells,the NK cell viability was detected by CCK-8 method.The expression of CD107a was analyzed by flow cytometry,the expression of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected by enzyme-linked immunosorbent assay (ELISA),the cytotoxicity of NK cells to SW480 cells was evaluated.The expression level of NKG2D receptor on NK cells was measured by flow cytometry and miR-18a in NK cells by quantitative PCR (qPCR).Results CDEs were disk-shaped and double membranous vesicular structure under electron transmission microscope.Western blotting could detect the expression of positive marker protein CD63 and CD81 in exosome.After IL-2 stimulation,the proportion of CD3-,CD56+ NK cells increased significantly (P<0.05);CDEs did not significantly affect the growth activity of NK cells (P=0.999).Compared with the negative control group,the expression levels of CD107a,TNF-α,IFN-γ and NKG2D receptor in NK cells intervened by CDEs were significantly decreased (P<0.05),the expression of miR-18a was significantly increased (P<0.05),in a concentration dependent manner.Conclusion CDEs may mediate the immune escape of colon cancer cells byup-regulating the expression of miR-18a,down-regulating the expression of NKG2D and inhibiting the killing effect of NK cells.