Expression And Function Of Chicken CircMYO1B/miR-155-5p/CTLA-4 In Different Immune Responses Affected By Stress-induced Immunosuppression

Cytotoxic T-lymphocyte associated antigen 4（CTLA-4）negatively regulates T cell activation and plays an important role in immune and inflammatory responses.However,function and mechanism of chicken CTLA-4 in immune regulation have not been reported.In this study,Hy-Line Brown chicken was selected as the model animal,and the CTLA-4 gene was cloned by RT-PCR and analyzed by bioinformatic methods.At the same time,three classic avian vaccines（Inactivated avian influenza vaccine,Newcastle disease attenuated vaccine and infectious bursal attenuated vaccine）were used to prepare the immune chicken models,and dexamethasone was used to the preparation of stress-induced immunosuppressed chicken model.Firstly,the expression levels and changes of circulating miR-155-5p,the targeting miRNA of CTLA-4 m RNA,were analyzed at 10 time points in the three kinds of immune chicken models and stress-induced immunosuppressed chicken models,and the key change time points of serum circulating miR-155-5p were screened out.Then,the expression characteristics of CTLA-4 gene,miR-155-5p and the "sponge" circMYO1 B of miR-155-5p in seven tissues（heart,liver,spleen,bursa of Fabricius,thymus,glandular stomach and cecal tonsils）at the above critical time points were compared and analyzed.The results were as follows:Firstly,the full-length coding sequence of CTLA-4 gene of Hy-Line Brown chicken was cloned and analyzed by sequence alignment and bioinformatic methods.The results showed that the full-length coding sequence of CTLA-4 was 618 bp,encoding 205 amino acids,and it was highly conserved among different species.Meanwhile,the partial sequence of circMYO1B（126bp）was identified,and the analysis of tissue expression profile showed that circMYO1 B was expressed in various tissues of Hy-Line Brown chicken.Secondly,in the different vaccine immunization models and stress-induced immunosuppressed models,the changes of serum circulating miR-155-5p were the same overall,and they all had significant changes with different degrees on 2dpi,5dpi and 21 dpi.It was speculated that these three time points were the key time points when the serum circulating miR-155-5p changed significantly under the different treatment conditions.Subsequently,the tissue expression characteristics of circMYO1 B,miR-155-5p and CTLA-4under different treatment conditions at these three time points were identified.The results showed that circMYO1 B,miR-155-5p and CTLA-4 gene actively participated in the regulation of immune function.In different vaccine immune response models,the spleen and glandular stomach（2dpi）,liver,bursa of Fabricius,and cecal tonsils（5dpi）,and heart and liver（21dpi）were mainly affected by circMYO1 B,miR-155-5p and CTLA-4 in different immune groups.In the stress-induced immunosuppressed model,the heart,spleen,bursa of Fabricius and glandular stomach（2dpi）,liver,spleen,bursa of Fabricius,thymus,glandular stomach and cecal tonsils（5dpi）as well as liver,bursa of Fabricius,and glandular stomach and cecal tonsil（21dpi）were the key tissues that circMYO1 B,miR-155-5p and CTLA-4significantly responed to the immunosuppression.In stress-induced immunosuppression affecting the immune responses,the heart,liver,bursa of Fabricius and cecal tonsils（2dpi）,liver,spleen,bursa of Fabricius,glandular stomach and cecal tonsils（5dpi）,as well as the heart,liver,and cecal tonsils and cecal tonsil（21dpi）were the key tissues for circMYO1 B,miR-155-5p and CTLA-4 responsing to stress-induced immunosuppression affecting the immune responses induced by different vaccines.Finally,the expression regulation relationships of circMYO1B/miR-155-5p/CTLA-4regulatory network between different treatment groups in vivo were comprehensively analyzed,and it was found that there was a significant game regulation relationship among circMYO1 B,miR-155-5p and CTLA-4 in multiple tissues at different time points in different treatment groups.For example,in the glandular stomach（2dpi）,heart and thymus（5dpi and21dpi）,as well as the spleen and cecal tonsil（21dpi）,the ce RNA regulatory network might participate in the immune response to different vaccines in the above key organs;heart,bursa of Fabricius,thymus and cecal tonsils were the key tissues in which the ce RNA regulatory network significantly responded to the stress-induced immunosuppression at these three time points;the bursa of Fabricius（2dpi）,cecal tonsils（2dpi and 5dpi）and liver（21dpi）were the possible key tissues in which circMYO1B/miR-155-5p/CTLA-4 regulatory network was actively involved in stress-induced immunosuppression affecting the immune responses to different vaccines.In this study,the CTLA-4 gene,circMYO1 B and miR-155-5p sequences were cloned respectively,and the expression characteristics,biological functions and endogenous regulatory relationships in vivo of the three factors were discussed under the conditions of stress-indced immunosuppression affecting different vaccine immune responses.And the key time points and tissues of CTLA-4 gene,circMYO1 B and miR-155-5p with significantly differential expression were obtained,which could provide theoretical references for in-depth study of the function and mechanism of the circMYO1B/miR-155-5p/CTLA-4 regulatory network in the regulation of immune function.