结肠癌来源外泌体对自然杀伤细胞miR-18a和NKG2D表达的影响

doi:10.3877/cma.j.issn.1674-0793.2021.03.003

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。

关键词: 结肠肿瘤, 外泌体, 自然杀伤细胞, MicroRNA-18a, NKG2D

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.

Key words: Colon neoplasms, Exosomes, Natural killer cells, MicroRNA-18a, NKG2D

表1 qPCR引物序列

基因	引物序列
miR-18a	F:5’-AAGGTGCATCTAGTGCAGATAG-3’
miR-18a	R:5’-GTGCAGGGTCCGAGGT-3’
U6	F:5’-CGCTTCGGCAGCACATATAC-3’
U6	R:5’-AACGCTTCACGAATTTGCGT-3’

表1 qPCR引物序列

基因	引物序列
miR-18a	F:5’-AAGGTGCATCTAGTGCAGATAG-3’
miR-18a	R:5’-GTGCAGGGTCCGAGGT-3’
U6	F:5’-CGCTTCGGCAGCACATATAC-3’
U6	R:5’-AACGCTTCACGAATTTGCGT-3’

图1 结肠癌SW480细胞来源的外泌体鉴定　A为透射电子显微镜观察结果（×50 000）；B为Western blotting检测结果

图2 自然杀伤细胞的鉴定

表2 CDEs对NK细胞活力的影响（±s，%，n=3）

组别	孵育24 h	孵育48 h
NC组	100.00	100.00
20 mg/L CDEs组	99.86±8.45	99.75±7.63
40 mg/L CDEs组	99.71±7.22	99.60±5.57
80 mg/L CDEs组	99.56±6.10	99.47±6.16
F值	0.003	0.005
P值	1.000	0.999

表2 CDEs对NK细胞活力的影响（±s，%，n=3）

组别	孵育24 h	孵育48 h
NC组	100.00	100.00
20 mg/L CDEs组	99.86±8.45	99.75±7.63
40 mg/L CDEs组	99.71±7.22	99.60±5.57
80 mg/L CDEs组	99.56±6.10	99.47±6.16
F值	0.003	0.005
P值	1.000	0.999

图3 流式细胞术检测NC组和不同浓度CDEs组自然杀伤细胞CD107a的表达情况

表3 CDEs对NK细胞TNF-α、IFN-γ水平的影响（±s，ng/L，n=3）

组别	TNF-α	IFN-γ
NC组	53.29±3.84	47.74±1.15
20 mg/L CDEs组	37.98±2.31^a	32.56±2.26^a
40 mg/L CDEs组	29.33±3.62^a	24.04±2.05^a
80 mg/L CDEs组	25.15±5.64^a	18.47±1.78^a
F值	28.567	141.366
P值	<0.001	<0.001

表3 CDEs对NK细胞TNF-α、IFN-γ水平的影响（±s，ng/L，n=3）

组别	TNF-α	IFN-γ
NC组	53.29±3.84	47.74±1.15
20 mg/L CDEs组	37.98±2.31^a	32.56±2.26^a
40 mg/L CDEs组	29.33±3.62^a	24.04±2.05^a
80 mg/L CDEs组	25.15±5.64^a	18.47±1.78^a
F值	28.567	141.366
P值	<0.001	<0.001

图4 流式细胞术检测自然杀伤细胞表面NKG2D受体表达

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

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