The Investigation Of The Effect Of MiR-34a Regulating The B Cell Redifferentiation On CIA Process

Objective:Our previous studies have illustrated that miR-34a promotes the development of collagen induced arthritis?CIA?by reversing the balance of Treg/Th17 via targeting Foxp3.At the same time,we have also confirmed that the expression of miR-34a in serum of RA patients has been increased.Foxp1 is the key transcription factor for the B cell development and differentiaiton.It has been reported that miR-34a can interfere with the development of B lymphocytes via inhibiting the expression of Foxp1.In this study,we constructed miR-34a knockout mice by using CRISPR/Cas9 technique.To explore the effect of miR-34a on the redifferentiation and development of B cells in rheumatoid arthritis,A CIA models were performed in miR-34a knockout mice and transgenic mice and used to verified.Methods:1.According to the website:http://crispor.tefor.net/crispor.cgi,the sgRNA sequence of miR-34a specific target site was selected.The cas9 mRNA and sgRNA were synthesized by Sangon Biotech?Shanghai?Co,Ltd.They were microinjected into the cytoplasm of the fertilized eggs of DBA1/J mice and cultured in a specific cell culture medium.When the cells reached the two-cell stage embryo,they were transferred into ICR surrogacy female mice about 10 weeks old for breeding.2.The ratio of total T and B lymphocytes was detected in immune organs of 8-12 weeks miR-34a^-/-DBA1/J mice by flow cytometry?FCM?.3.To establish CIA mouse model,the mice of the wild type DBA1/J mice, miR-34a^-/-DBA1/J mice,the wild type C57BL/6 mice and miR-34a TG C57BL/6 mice about 6-8 weeks were used.The emulsions of bovine type II collagen?C?? and Freund's complete adjuvant?CFA?were injected at 3 cm from the root of the tail on the first day,and immunized again at 1-2 cm from the root of the tail on the day 21.From the day 0 of immunization,the mice's weight was verified every 3 days.After the 2^nd immunization on the day 21,the general state of mice were checked every 2 days and the clinical scores of four ankle joints were evaluated and recorded by taking photos.The ankle joints inflammation state was evaluated by pathological section with HE staining and the joints restore was recorded by molybdenum target photography until 42^nd day.4.Isolating the lymphocytes from lymph nodes and spleen of CIA model mice,and the surface markers and ratio of total B cells and subpopulation,such as GC B cells,MZ B cells and plasma cells were detected by FCM.Results:1.The miR-34a^-/-DBA1/J mice were successfully constructed by using CRISPR/cas9 technique.That was no significant change in CD4⁺,CD8⁺T cell population,but the proportion of total B cells increased significantly in immune organs of miR-34a^-/-DBA1/J mice by FCM.The results indicated that miR-34a may has no significantly effect on the redifferentiation and development of CD4⁺,CD8⁺Tcells,but it may affect the redifferentiation and development of B cells.2.The CIA models of miR-34a knockout mice and transgenic mice were set up.The onset of arthritis was about at the day 25 in CIA model mice groups.The degree of inflammation and swelling of the joints in the miR-34a TG C57BL/6 CIA model group were more serious than that in the wild type C57BL/6 CIA model group. The state of arthritis in DBA1/J CIA model group was more serious than that in miR-34a^-/-DBA1/J CIA model group.It was consistent with the scores of inflammation,imaging and pathological examination.3.The proportion of total B cells was decreased in miR-34a TG C57BL/6 mice,but the inflammatory reaction was more serious in miR-34a TG C57BL/6 CIA model mice.In miR-34a^-/-DBA1/J mice,the proportion of total B cells increased,the inflammatory degree of miR-34a^-/-DBA1/J CIA model mice was decreased and the period of inflammatory was much long than wild type and miR-34a TG C57BL/6 CIA model mice.4.The proportions of GC B and MZ B cells was decreased in miR-34a^-/-DBA1/J CIA mice,but the proportions of GC B and MZ B cells in miR-34a TG C57BL/6 CIA mice was increased.Conclusion:In the CIA model,miR-34a may promote the progression of CIA diseases by regulating the redifferentiation and development of B cells.It can provide new research ideas for the basic,clinical research and diagnosis and treatment of RA.