Exosome-derived MiR-20a Inhibit Apoptosis Of TAMs By Targeting BCL2L11 In Nasopharyngeal Carcinoma

Objective: It's proved that there are large amount of miRNAs in nasopharyngeal carcinoma(NPC)derived exosomes,and some of them were significantly up-regulated.The present study plan to predict the potential targets of aforementioned miRNAs and relative pathways facilitated by bioinformatics databases and software.We also plan to investigate whether some miRNAs and corresponding target genes are involved in the apoptosis process of tumorassociated macrophages,which is important in growth,invasion and metastasis of tumor.Furthermore,if possible,a preliminary exploration will be conducted on its regulatory mechanism.Methods: Target mi RNAs and their target genes were identified by bioinformatics methods first.Targetscan,MicroCosm and mi RTarBase online databases were used to predict the target mRNA of miRNAs that were up-regulated in nasopharyngeal carcinoma.Predicted intersection of these three databases was getting first,then target mRNAs related in apoptosis pathway and their corresponding miRNAs were screened by UniProt online database.The miRanda online database / software was used to predict the mi RNA-mRNA interaction loci.The exosomes were isolated and identified by electron microscopy,which were located in the serum of nasopharyngeal carcinoma patients and in the medium of cultured nasopharyngeal carcinoma cells.The expression of candidate mi RNAs in exosomes was detected by realtime quantitative PCR.And the primary cultured macrophages isolated from peripheral blood of healthy donors were treated with the exosomes obtained according to the method described above.These cells were observed whether had ingested exosomes.And the changes of apoptosis index and apoptosis pathways related proteins expression were detected then after exosomes ingestion.Finally,mi R-20 a mimics and inhibitors were used to transfect macrophages in vitro.Apoptotic index and apoptotic pathway-associated protein were detected after the intervention.Results: The bioinformatics analysis showed that mi R-20 a was closely related to apoptosis,which was significantly up regulated in the nasopharyngeal carcinoma serum and in the nasopharyngeal carcinoma cell culture medium.The target genes related to apoptosis were BCL2L11 and CASP2(encoding Bim and caspase-2,respectively).Transmission electron microscopy confirmed that large quantity of exosomes was isolated from the serum of nasopharyngeal carcinoma patients and the culture medium of 5-8F cells.These exosomes could be engulfed by macrophages in vitro.The apoptotic indexes of macrophages treated with exosomes isolated from 5-8F cell culture medium and serum from nasopharyngeal carcinoma patients were significantly lower than that of macrophages treated with exosomes isolated from the blank control group,NP69 cell culture medium and healthy donor's serum.The protein expression of Caspase-2 and Bim protein was detected by Western blot.There was no significant difference between groups on Caspase-2 expression.Bim expression of macrophages treated with exosomes isolated from the 5-8F cell culture medium and nasopharyngeal carcinoma patients was significantly lower than other groups.The expression of miR-20 a in exosomes was detected by realtime fluorescence quantitative PCR.The results showed that miR-20 a in the exosomes extracted from the 5-8F cell culture medium was significantly higher than that in NP69 cell culture medium.Meanwhile,the levels of miR-20 a in the exosomes extracted from serum of patients with nasopharyngeal carcinoma were significantly higher than that of healthy donors.mi R-20 a mimics and inhibitors were used to transfect into macrophages revealed following results: 1)the apoptotic index of the miR-20 a mimic group was significantly lower than that of the control group,while the apoptosis index of the inhibitor group was not significantly different from that of the control group.2)Compared with the control group,the Bim expression in the miR-20 a mimic group was significantly reduced,while the Bim protein in the inhibitor group was not significantly different from the control group.3)Compared with the control group,the Cleaved Caspase-9 protein content of the miR-20 a mimic group was significantly reduced,while the cleaved Caspase-9 protein of the inhibitor group was not significantly different from the control group.4)Compared with the control group,the cleaved Caspase-3 protein content of the miR-20 a mimic group was significantly reduced,while the Cleaved Caspase-3 protein of the inhibitor group was not significantly different from the control group.Conclusion: This study demonstrates that nasopharyngeal carcinoma cells can overexpress miR-20 a,which was subsequently transferred into macrophages via exosomes,thereby inhibiting apoptosis of them.The underlying mechanism might be suppression of Bim-Caspase-9-Casepase-3 pathway activation by miR-20a.