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

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

Abstract

Abstract:

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

Yonghui Zhong, Fuchuan Xie, Yisheng Wei. Effect of colon cancer derived exosomes on the expression of miR-18a and NKG2D in natural killer cells[J]. Chinese Archives of General Surgery(Electronic Edition), 2021, 15(03): 172-177.

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References 19

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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’

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’

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

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

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

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