Study On The Mechanism Of CircRNA And MiRNA Related To NK92-MI Cell Activated By Neuropeptide SP

Objective: NK cells,which account for about 10¹5% of circulating lymphocytes,not only immunologically monitor tumors and pathogen-infected cells,but also secrete cytokines to exert immunomodulatory effects.They are the most important components of the immune system.Eukaryotic cells express a large number of micro RNA and circ RNA and play an important regulatory role at the RN A level.Unlike linear RNA,circ RNAs are closed loop structures that do not contain a 3’ or 5’ end,and are spliced in two special ways,including reverse splicing and ligation intron ligation,and are not affected by RNA exonuclease.Therefore,the expression is not easy to degrade.Diversification of regulatory functions of circ RNA: Indirect regulation of target gene expression via mi RNAs based on sponge action.Direct regulation of RNA,this effect is achieved by the base complement between the two nucleotides.It can bind to inhibit certain functional proteins.Synthetic the protein,this effect is the same as common exon-coding genes.Substance P has been widely studied as a neuropeptide,but the most important function is a universal biological factor between the neurological,endocrine,and immune systems.Some previous studies in our laboratory have shown that SP can induce the expression of NK92-MI cell activating receptor NKRs in a certain concentration range,and significantly enhance the killing and proliferation activity.At present,the involvement of SP in the detailed activation of NK92-MI cells at the RNA level,including circ RNA and mi RNA molecular level studies,has not been reported at home and abroad.Based on the previous work of this experiment,the Illumina Hi Seq TM sequencing platform was used to detect and screen the expression of circ RNA before and after NK92-MI cells were treated with SP.Bioinformatics software was used to predict the target mi RN A that differentially expressed circ RNA and then subjected to enrichment analysis.The mi RN As related to NK92-MI cell activation were screened for detection,and their relationship with the expression of activating receptor m RN A（NKG2D,NKp46）and perforin m RNA expression in killer media was analyzed.Finally,it was determined that circ RNA and mi RN A affect NK92-MI in SP.The specific aspects of cell function provide the basis for further research.Methods: Illumina Hi Seq TM platform was used to detect and screen the differential expression of circ RNAs before and after NK92-MI cells were treated with SP.After being verified by q RT-PCR,bioinformatics information was enriched and analyzed;q RT-PCR method was used to detect the expression of mi RNA27 a and mi RNA30 e,and bioinformatics analysis was performed.The target m RNA was detected by q RT-PCR to detect the expression of NK92-MI cell activating receptors NKG2 D and NKp46 m RNA.The expression of NK92-MI cell degranulation marker CD107 a was detected by flow cytometry and q RT-PCR was used to detect NK cell killing media perforin.Results:（1）After NK92-MI cell were treated with SP,two molecules,circ RNA₀0027978 and circ RNA₀0015274,which were down-regulated,were selected and verified by q RT-PCR.After NK92-MI was treated with SP,a total of 54 differentially expressed genes were detected.A total of 28 differentially expressed transcripts were detected,all down-regulated.（2）Bioinformatics prediction circ RNA₀0027978 regulated a total of 1245 mi RN As,circ RNA₀0015274 regulated mi RN A a total of 880.According to predictions and literature queries,we selected mi RN A27 a and mi RN A30 e for further study.（3）SP stimulation of NK92-MI resulted in decreased expression of mi RN A27 a and mi RN A30 e.Bioinformatics predicts 959 target genes for mi RN A27 a and 1182 target genes for mi RN A30 e.（4）SP stimulation of NK92-MI cell resulted in a significant increase in the expression of activating receptors including NKG2 D and NKp46 m RNA.（5）Resting NK92-MI cell showed little or no expression of CD107 a.After stimulation with SP,the expression of CD107 a and killer media m RN A was significantly increased.Conclusion: The expression of circ RNA₀0027978 and circ RNA₀0015274 in NK92-MI down-regulated after treatment by SP.The related expression of mi RN A27 a and mi RN A30 e also decreased,while the activating receptors including NKG2 D,NKp46 m RNA was enhanced,and the expression of perforin m RNA in killer media was enhanced and the expression of cell degranulation marker CD107 a increased significantly.Therefore,we believe that circ RNA₀0027978,circ RNA₀0015274,mi RNA27 a,and mi RN A30 e are involved in SP activation of NK92-MI at the RN A level,and enhance their regulatory mechanisms for killing function.