Yonsei Med J.  2019 Jun;60(6):500-508. 10.3349/ymj.2019.60.6.500.

MiR-218-5p Suppresses the Killing Effect of Natural Killer Cell to Lung Adenocarcinoma by Targeting SHMT1

• Yang Q
• Li J
• Hu Y
• Tang X
• Yu L
• Dong L
• Chen D

• KMID: 2446954
• DOI: http://doi.org/10.3349/ymj.2019.60.6.500

Abstract

PURPOSE
Lung adenocarcinoma (LA) is one of the major types of lung cancer. MicroRNAs (miRNAs) play an essential role in regulating responses of natural killer (NK) cells to cancer malignancy. However, the mechanism of miR-218-5p involved in the killing effect of NK cells to LA cells remains poorly understood.
MATERIALS AND METHODS
The expression of miR-218-5p was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Serine hydroxymethyl transferase 1 (SHMT1) level was detected by qRT-PCR or western blots. Cytokines production of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were detected by ELISA. The killing effect of NK cells to LA cells was investigated using lactate dehydrogenase cytotoxicity assay kit. The interaction of miR-218-5p and SHMT1 was probed by luciferase activity assay. Xenograft model was established to investigate the killing effect of NK cells in vivo.
RESULTS
miR-218-5p was enhanced and SHMT1 was inhibited in NK cells of LA patients, whereas stimulation of interleukin-2 (IL-2) reversed their abundances. Addition of miR-218-5p reduced IL-2-induced cytokines expression and cytotoxicity in NK-92 against LA cells. Moreover, SHMT1 was negatively regulated by miR-218-5p and attenuated miR-218-5p-mediated effect on cytotoxicity, IFN-γ and TNF-α secretion in IL-2-activated NK cells. In addition, miR-218-5p exhaustion inhibited tumor growth by promoting killing effect of NK cells.
CONCLUSION
miR-218-5p suppresses the killing effect of NK cells to LA cells by targeting SHMT1, providing a potential target for LA treatment by ameliorating NK cells function.

Keyword

Lung adenocarcinoma; natural killer cells; miR-218-5p; SHMT1

MeSH Terms

Adenocarcinoma*
Blotting, Western
Cytokines
Enzyme-Linked Immunosorbent Assay
Heterografts
Homicide*
Humans
Interleukin-2
Killer Cells, Natural*
L-Lactate Dehydrogenase
Luciferases
Lung Neoplasms
Lung*
MicroRNAs
Necrosis
Real-Time Polymerase Chain Reaction
Serine
Transferases
Cytokines
Interleukin-2
L-Lactate Dehydrogenase
Luciferases
MicroRNAs
Serine
Transferases

Figure

• 

  Fig. 1 MiR-218-5p and SHMT1 expressions were ectopic in NK cells of LA patients (n=20). (A) Expression of miR-218-5p was detected by qRT-PCR in NK cells of control or LA patients. (B) SHMT1 mRNA level was examined in NK cells in control and LA groups. (C and D) Abundance of SHMT1 protein was investigated by western blots. *p<0.05, LA vs. control group. LA, lung adenocarcinoma; SHMT1, serine hydroxymethyl transferase 1; qRT-PCR, quantitative real-time polymerase chain reaction; NK, natural killer.

• 

  Fig. 2 IL-2 induced activation of NK cells and dysregulation of miR-218-5p and SHMT1 expression. NK-92 cells were treated by IL-2 (20 ng/mL) for 24 h. (A and B) TNF-α and IFN-γ production levels were determined by ELISA in NK-92 cells with or without IL-2 stimulation. (C) Effect of IL-2 on miR-218-5p expression was investigated in NK-92 cells. (D) SHMT1 protein expression was detected in NK-92 cells after or before IL-2 treatment. *p<0.05, IL-2-treated group vs. control group. IL-2, interleukin-2; NK, natural killer; SHMT1, serine hydroxymethyl transferase 1; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ.

• 

  Fig. 3 Enrichment of miR-218-5p blocked the killing effect of NK cells to LA cells. (A) Altered abundance of miR-218-5p was detected in IL-2-avtivated NK-92 cells with miR-218-5p or NC transfection. (B and C) IFN-γ and TNF-α secretion were examined in NK-92 cells after IL-2 and miR-218-5p treatment. (D) Killing effect of NK-92 to LA cells was evaluated by LDH cytotoxicity assay kit, and the effect of miR-218-5p on killing effect was detected in NK-92 cells transfected with miR-218-5p mimics. *p<0.05, IL-2-treated group vs. control group, and IL-2+miR-218-5p group vs. IL-2+NC group. NK, natural killer; LA, lung adenocarcinoma; IL-2, interleukin-2; NC, negative control; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; LDH, lactate dehydrogenase.

• 

  Fig. 4 SHMT1 was a target of miR-218-5p. (A) Bioinformatics assay indicated putative binding sites of miR-218-5p and 3′-UTR of SHMT1 by TargetScan. (B and C) Wt or mut of SHMT1-transfected 293T cells were used to investigate luciferase activity by co-transfection of miR-218-5p or anti-miR-218-5p, respectively. (D and E) Effect of miR-218-5p on SHMT1 expression was detected in NK-92 cells at mRNA and protein level. (F and G) Abundances of SHMT1 mRNA and protein were altered in NK-92 cells after anti-miR-218-5p transfection. *p<0.05, miR-218-5p group vs. NC group, and anti-miR-218-5p group vs. anti-NC group. SHMT1, serine hydroxymethyl transferase 1; NK, natural killer.

• 

  Fig. 5 Introduction of SHMT1 reversed miR-218-5p-mediated inhibitory role in the killing effect of NK-92 to LA cells. (A) Expression of miR-218-5p was examined in NK-92 cells after treatment. (B) Ectopic SHMT1 protein level was detected in miR-218-5p or SHMT1-transfected NK-92 cells after IL-2 insult. (C and D) Effect of SHMT1 on IFN-γ and TNF-α expression in IL-2-activated NK-92 cells was investigated. (E) Cytotoxicity of NK-92 with SHMT1 transfection to A549 cells was evaluated. *p<0.05, IL-2-treated group vs. control group, IL-2+miR-218-5p group vs. IL-2+NC group, and IL-2+miR-218-5p+SHMT1 group vs. IL-2+miR-218-5p+pcDNA group. SHMT1, serine hydroxymethyl transferase 1; NK, natural killer; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; IL-2, interleukin-2.

• 

  Fig. 6 Abrogation of miR-218-5p enhanced the killing effect of NK to LA cells in vivo. IL-2-treated LNK cells were transfected with anti-miR-218-5p and introduced into LA tumor in nude mice (n=8 per group). (A) Tumor volume was examined every three days. (B and C) Expression of SHMT1 protein was detected in the tumor tissues. *p<0.05, anti-miR-218-5p group vs. anti-NC group. NK, natural killer; LA, lung adenocarcinoma; IL-2, interleukin-2; LNK, mouse NK cell line; SHMT1, serine hydroxymethyl transferase 1.

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