Mechanism Of Regulation On CD8~+ T Cells Mediated By Methionine Enkephalin Via Binding Opioid Receptor

Objective:The goal of this study was to investigate how MENK could regulate the functions of CD8⁺ T cells and to explore the relationship between this regulation and opioid receptor expression.In vitro experiments were conducted to investigate the effect of MENK on the function of CD8⁺ T cells by regulating opioid receptor subunits.In vivo experiment were conducted to investigate how MENK affect the transcriptome of the spleen CD8⁺ T cells by using C57BL/6 mice S180 subcutaneous tumor-burdened model,then the different genomic expressions of lncRNA,mRNA and signaling pathway enrichment were analyzed by bioinformatics,and differences in gene expression were verified by real-time PCR.Methods:The CD8⁺ T cells were sorted out and purified from splenocytes of C57BL/6mice by immune-magnetic negative selection.The purity of CD8⁺ T cells was up to 95%and the subsequent experiments were carried out with the purified cells.To study the effect of MENK on CD8⁺ T cells,the expressions of opioid receptors were detected at both RNA and protein levels.In order to block the mu opioid receptor,the stimulated CD8⁺ T cells were incubated with CTAP at the concentration of 4.5μM,450 nM,45 nM and 4.5 nM for 48 h.Then the expressions of MOR and DOR were measured by RT-qPCR and western blot.For blocking delta opioid receptor,the cells were treated with NTI at concentration of 10μM,1μM,100 nM and 10 nM.In order to antagonize both of the mu and delta opioid receptors,NTX was used to treat cells for 48 h at the concentration of 13μM,1.3μM,130 nM and 13 nM.To assess the phenotypes and functions of CD8⁺ T cells,both intracellular and extracellular biomarkers were measured by flow cytometry.To examine the effect of MENK on cell proliferation,the purified CD8⁺ T cells stained by CFSE were stimulated with anti-CD3 and anti-CD28,and co-cultured with different MENK combinations for 3 days,and then the treated cells were examined for proliferation by flow cytometry.2.C57BL/6 tumor-bearing mice model was used to study the effects of MENK in vivo.Immunomagnetic bead sorting was used to purify CD8⁺ T cells in spleen by negative enrichment.High-throughput RNA transcriptome sequencing was used to analyze the effects of MENK on lncRNA and mRNA of CD8⁺ T cells in spleen of tumor-bearing mice.Cuffdiff software was used to analyze the differential expression of the spliced transcript,and a negative binomial distribution-based model was used.The screening condition was P-adjust<0.05.GOseq R package was used to analyze the GO（gene ontology）enrichment of mRNA and lncRNA target genes for differentially expressed genes.The screening condition was that the corrected P value was less than 0.05,showing statistical difference,and GO was significantly enriched.KOBAS software was used to test the statistical enrichment of differential expression mRNA genes or lncRNA target genes in KEGG pathways.The differently expressed genes enriched in KEGG were confirmed by real-time PCR,and the main target genes were as follows:CCnd3,Cdc7,Anapc4,Smc3,Atm in cell cycle pathway（P=0.005）;Pten,Atr,Casp8,Ccnd3,Atm in P53 pathway（P=0.007）;Rasgrp2,Pak2,Bad,Ets-1,Rap1b,Rala,Tbk1,Rasa3 in Ras pathway（P=0.028）;Kidins220,Rap1b,Matk,Prkcd,Camk2b,Bad,Rps6ka3 in Neurotrophin signaling pathway（P=0.005）;classic opioid receptors MOR,DOR.The major differently expressed lncRNA detected were:GM27008,Snhg6,Tug1,Pvt1.Results:1.MENK could promote the expression of opioid receptors on the surface of CD8⁺ T cells in vitro.At the RNA level,MENK could promote the expression of MOR and DOR at the RNA level,and the optimal concentration was 10^-10 M,the relative expression of MOR was 2.03±0.21,and the relative expression of DOR was 1.89±0.45,with statistically significant difference（P<0.05）.The CTAP treatment could significantly down-regulated the RNA and protein expression levels of MOR,and the relative protein expression levels of each experimental concentration respectively were 1.13±0.02,0.62±0.01,0.85±0.01,0.62±0.05,and 0.63±0.01,with statistically significant differences（P<0.05）.At the RNA level,NTI could significantly inhibited the expression of DOR,while up-regulated the expression of MOR（P<0.01）.At the concentration of 1μM,the relative expression of DOR was significantly down-regulated,and the corresponding relative expression was 0.75±0.04,while the relative expression of MOR was up-regulated,and the corresponding relative expression was 2.88±0.37,with statistically significant difference（P<0.01）.The protein expression of the DOR was significantly down-regulated at the concentration of 1μM,the relative expression level of the protein band relative toβ-actin was 0.56±0.02;the relative expression level of the MOR protein band was 1.09±0.05.NTX could significantly inhibited the expression of both MOR and DOR at the RNA level.At the concentration of 1.3μM,the expression level of MOR relative toβ-actin was 0.28±0.01,and the expression level of DOR relative toβ-actin was 0.35±0.03 at the RNA level（P<0.01）.The expression levels of MOR and DOR were respectively 0.44±0.02 and 0.51±0.03 at the protein level（P<0.01）.The expression of CD28,PD-1,CTLA-4,FasL and intracellular GrzB of CD8⁺ T cells in the MENK treatment group were significantly increased（P<0.01）,and the expression levels of the above indicators were significantly decreased after the selective blocking of MOR（CTAP+MENK）or DOR（NTI+MENK）,while the expression levels of CD28,PD-1,CTLA-4,FasL and GrzB were further decreased after the combined blocking of MOR and DOR（NTX+MENK）.The proliferation of CD8⁺ T cells was significantly enhanced in MENK group（P<0.01）;blocking MOR（CTAP+MENK）or DOR（NTI+MENK）alone could not block the proliferation effect of MENK on CD8⁺ T cells,while the proliferation effect of MENK on CD8⁺ T cells disappeared after both MOR and DOR were blocked.2.Based on transcriptome splicing results,a total of 845 genes with noncoding potential were obtained after strict screening.Subsequently the cluster analysis of GO and KEGG enrichment analysis were performed on differentially expressed transcripts.After MENK treatment,there were 8 up-regulated genes and 5down-regulated genes in differentially expressed lncRNA;there were 3 up-regulated genes and 7 down-regulated genes in differentially expressed TUCP;for differentially expressed mRNA,there were 301 up-regulated genes and 240 down-regulated genes.The GO enrichment analysis results of differential lncRNA target genes were mainly as follows:GO:0005251（delayed rectifier potassium channel activity）,GO:0008076（voltage-gated potassium channel complex）,GO:0034705（potassium channel complex）,GO:0005249（voltage-gated potassium channel activity）,GO:0034702（ion channel complex）,GO:0043269（regulation of ion transport）;the GO enrichment analysis results of differential mRNA target genes were mainly as follows:GO:0005634（nucleus）,GO:0044260（Cellular macromolecule metabolic process）,GO:0044428（nuclear part）,GO:0005622（intracellular）,GO:0031981（nuclear lumen）,GO:0044237（Cellular metabolic process）.The KEGG enrichment analysis results of differential lncRNA target genes were mainly as follows:Thyroid cancer（Mmu05216）,Cholinergic synapse（Mmu04725）,Bladder cancer（Mmu05219）,FoxO signaling pathway（Mmu04068）,Endometrial cancer（Mmu05213）,Acute myeloid leukemia（Mmu05221）;the KEGG enrichment analysis results of differential mRNA target genes were mainly as follows:Ribosome（Mmu03010）,Viral carcinogenesis（Mmu05203）,Amphetamine addiction（Mmu05031）,P53 signaling pathway（Mmu04115）,Neurotrophin signaling pathway（Mmu04722）,Alzheimer’s disease（Mmu05010）.The differentially expressed genes were confirmed by real-time PCR,and the differentially expressed mRNAs were bcl-2,Bax,Rap1b,Pak2,Rasa3,Bad,Pten,Rasgrp2,MOR,DOR,from which the up-regulated mRNAs were bcl-2,Pak2,MOR,DOR,while the down-regulated mRNAs were Bax,Rap1b,Rasa3,Bad,Pten,Rasgrp2;the differentially expressed lncRNAs were GM27008and Pvt1,and both of them were verified up-regulated,with statistically significant difference（P<0.05）.Conclusion:1.MENK could enhanced the proliferation and functions of CD8⁺ T cells through up regulating mu and delta opioid receptors.This effect of MENK could be cancelled while one or both of the receptors would be blocked.MENK might play a vital role in immune functions via precise regulation to subunits of opioid receptors.2.MENK could significantly inhibit the mouse S180 sarcoma;MENK treatment could increase the proportion of CD8⁺ T cells in spleen and lymph nodes in tumor-bearing mice;MENK treatment could significantly affect the expressions of transcriptome mRNA and lncRNA of CD8⁺ T cells in the spleen of tumor-bearing mice.