Lung Cancer-derived Exosomal MicroRNAs Target RNF-216 And EHMT-1 And Inhibit Activation Of Natural Killer Cells

Background Natural killer(NK)cells play important roles in anti-tumor immune responses.However,NK cells are often inhibited in the tumor microenvironment.One of mechanisms for tumor immune escape is that tumor-derived exosomes and their components interact with NK cells and negatively regulate activation of NK cells in the tumor microenvironment.In our previous study,2 novel exosomal miRNAs(tentatively named as miRNA-A and miRNA-B)were identified in the serum of lung cancer patients.In the current study,we focus on their inhibitory effects on NK cell activation via in vitro experiments and observation of human lung cancer tissues.Objective Suppressive effects of miRNA-A and miRNA-B on NK cell activation and mechanisms of action will be studied.Methods 1)Increased expression of miRNA-A and miRNA-B in lung cancer cells and in the exosomes were confirmed by qPCR;2)After transfection of miRNA-A/B mimics into NK92 mi,a human NK cell line,and activation by combined stimulation with PMA,ionomycin and IL-18,real-time quantitative PCR,ELISA,immunofluorescence and flow cytometry were applied to detect changes in the signature molecules of NK activation including cytokines TNF-α,IFN-γ,IL-2 and the surface receptor NKG2 D for determine if miRNA-A/B had inhibitory effects on NK cell activation and for determining the optimal doses and time-points of their action.3)The ratios of IFN-γ-positive NK cells(activated NK cells)in the lung cancer tissues from patients with high or low plasma concentration of miRNA-A/B were analyzed by immunofluorescence staining to judge the relationship of miRNA-A/B concentrations with NK cell activation;4)After prediction of target-genes,qPCR analysis of the predicted genes and bioinformatics analysis,candidate targeted genes(RNF-216 and EHMT-1)shared by both miRNA-A and miRNA-B were determined;5)Impacts of knock-down of RNF-216 or EHMT-1 by si RNAs on NK cell activation were observed to verify whether the targeted genes were associated with inhibitory roles of miRNA-A/B in NK cell activation.Results1)Comparison of expression of miRNA-A and miRNA-B by qPCR between lung cancer tissues and nearby normal tissues,isolated exosomes from lung cancer cell lines or from normal bronchial epithelial cell lines,indicated increased expression of miRNA-A and miRNA-B in lung cancer cells and existence in the exsomes;2)The exosomes isolated from the culture medium of H1975,a lung cancer cell line,showed a suppressive effect on NK cell activation;3)The optimal doses of miRNA-A mimics and miRNA-B mimics were 100 pmol and50pmol,respectively,and the optimal observation time was 24 h after NK92 mi was stimulated.Under these conditions,miRNA-A mimics and miRNA-B mimics had the best inhibitory effects.4)Under the optimal conditions,transfection of miRNA-A mimics or miRNA-B suppressed the expression of the signature molecules of NK cell activation(TNF-α,IFN-γ,IL-2 and NKG2D),as detected by qPCR.ELISA indicated decreased concentrations of TNF-α,IFN-γ and IL-2 in the culture medium.Flow cytometry showed the proportion of IFN-γ-positive cells declined in the miRNA-A mimics or miRNA-B transfected NK cells,and similar results were also observed by immunofluorescent staining.These results demonstrated that miRNA-A and miRNA-B were able to suppress NK activation in vitro.5)The ratio of IFN-γ-positive cells(activated NK cells)in CD56-positive cells(total NK cells)in the lung cancer tissues of patients with high expression of miRNA-A/B was 12.88 %,significantly lower than that of patients with low expression(56.18 %),suggesting that exosomal miRNA-A/B are negative regulators of NK cell activation in the tumor microenvironment.6)qPCR detection of predicted target genes and bioinformatics analysis indicated that RNF-216 and EHMT-1 were candidate target genes shared by both miRNA-A and miRNA-B7)Similar to transfection of miRNA-A or miRNA-B,knock-down of RNF-216 or EHMT-1 by si RNAs resulted in a decrease in the expression of TNF-α,IFN-γ,IL-2 and NKG2 D,as manifested by qPCR and ELISA analyses.Also,flow cytometry analysis and immunofluorescent staining showed that the ratio of IFN-γ-positive cells in CD56-positive cells declined in the si RNA-treated NK92 mi cells.Taken together,RNF-216 and EHMT-1 are potential target genes,and are down-regulated by miRNA-A and miRNA-B,leading to suppression of NK cell activation.Conclusion1)lung cancer-derived exosomal miRNA-A/B suppress NK cell activation in both in vitro experiments and observation of human lung cancer specimens;2)It is plausible that miRNA-A/B target RNF-216 and EHMT-1 and down-regulate their expression,thus,resulting in inhibition of NK cell activation;3)Theses results indicate that lung cancer-derived exosomes and their containing miRNAs act on NK cells in the tumor microenvironment and suppress NK cell activation and cytotoxic ability.This is probably one of mechanisms for tumor immune escape,and will provide theoretical basis for immunotherapy.