Experimental Study Of Mmu_circ₀000451 Promoting Mouse Th17 Cell Differentiation

Objective:To screen potential circular RNAs?circRNAs?with differential expressions in induced Th17 cells in vitro;to clarify the regulatory effect of one circRNA on Th17 cell differentiation and to analyze its potential regulatory mechanism.Methods:?1?Na?ve CD4⁺T cells isolated from the spleen of wild-type mice were separated by using an immunomagnetic separation method,and cell purity was detected by flow cytometry?FCM?.Th17 cells were induced in 48-well plates that were pre-coated with anti-CD3 mAb?2?g/mL?and anti-CD28 mAb?2?g/mL?overnight at cell density of 5×10⁵/well.The induction system included TGF-??2ng/mL?,IL-6?50ng/mL?,IL-23?50ng/mL?,anti-IFN-?mAb?5?g/mL?,and anti-IL-4 mAb?5?g/mL?.After cell culture at 37?with 5%CO₂ for 72h and subsequent continuous culture with cell stimulation cocktail?1?L/well?for 5h.The induction ratio of Th17 cells was detected by FCM and the expression level of IL-17A in the supernatant of Th17 cells was tested by enzyme linked immunosorbent assay?ELISA?.The above cells were involved in high-throughput sequencing?Hangzhou 1GENE Technology Co.,Ltd?for circRNA detection to analyze circRNA expression in the induced Th0 and Th17 cells,and to screen potential circRNA with differential expressions.Quantitative real-time PCR?qRT-PCR?was then used to analyze the expression of the screened mmu_circ₀000451 in the induced Th17 cells and to verify the basic information of mmu_circ₀000451.?2?For a clarification of the role of mmu_circ₀000451 in Th17 cell differentiation,mmu_circ₀000451overexpression adenovirus vector was added in the culture system of inducing Th17 cells,and qRT-PCR was used to verify the efficiency of overexpression.The proportion of Th17 cells was detected by FCM and ELISA was used to measure the expression level of IL-17A.Moreover,the specific small interfering RNA?siRNA?of mmu_circ₀000451?mmu_circ₀000451 siRNA?was designed and added in the culture system of inducing Th17 cells,followed by the detection of interference efficiency using qRT-PCR.Afterwards,the proportion of Th17 cells was detected by FCM and the level of IL-17A secreted by Th17 cells was detected by ELISA.?3?In order to determine the possible mechanism of mmu_circ₀000451,fluorescence in situ hybridization?FISH?and isolation of nuclear and cytoplasmic RNA were carried out to detect the location of mmu_circ₀000451 in Th17 cells.?4?In order to determine the regulatory effect of mmu_circ₀000451 on the parental gene CDYL,qRT-PCR and Western blot were first used to detect the expression of CDYL in Th17 cells.Adding adenovirus vector overexpressing mmu_circ₀000451 to the induction system of Th17 cells,and verifying the overexpression efficiency and CDYL mRNA level by qRT-PCR;adding mmu_circ₀000451 siRNA to the induction system of Th17 cells,and verifying the knockdown efficiency and CDYL mRNA level by qRT-PCR;the CDYL protein level was detected by Western blot and Co-immunoprecipitation?CO-IP?was used to detect the binding capacity of CDYL and EZH2.?5?Add the EZH2 inhibitor GSK343 to the induction system of Th17 cells to detect the inhibitory effect by WB and the proportion of Th17 cells by FCM.?6?In order to explore the effect of mmu_circ₀000451 on disease progression and related indicators of CIA mice in vivo,we randomly divided DBA mice into three groups?simmu_circ₀000451 group,siNC group,and DBA group?,and used DBA mice to construct the CIA model.Mice were immunized on days 0 and 21,respectively.On the 19th day of the model construction process,5×106(simmu_circ₀000451 CD4+T cells,control CD4+T cells and PBS were injected into the corresponding group of DBA mice through the tail vein.The disease progression of the three groups of mice was observed separately?Clinical score,CIA incidence?.Flow cytometry was used to detect the proportion of Th17 cells in the spleen and lymph nodes.Results:?1?According to the results of FCM,the purity of Na?ve CD4⁺T cells from mouse spleen was 92.8%,and the proportion of induced Th17 cells was?12.66±0.23?%.High-throughput sequencing analysis revealed that there were 400 circRNAs expressed in Th0 cells and 310 in Th17 cells,of which 16 circRNAs were co-expressed in both Th0 and Th17 cells.Besides,the differential expression of mmu_circ₀000451 derived from chromodomain Y like protein?CDYL?is the most obvious among all differentially expressed circRNA.qRT-PCR detection showed that mmu_circ₀000451 was highly expressed in Th17 cells compared with Th0 cells?P<0.05?.Compared with linear CDYL,mmu_circ₀000451 was not easily degraded by ribonuclease R?RNase R??P>0.05?.Meanwhile,Sanger sequencing showed that the splicing site of mmu_circ₀000451 after head-to-tail splicing were consistent with those in the database?circbase,http://www.circbase.org/?.?2?The expression of mmu_circ₀000451 was significantly increased after mmu_circ₀000451 overexpression in Th17 cells?P<0.005?,with the proportion of Th17 cells detected to be 18.16±0.64%and 12.47±0.55%in the overexpression group and the control group,respectively.There are significant differences between the two groups?P<0.005?.The IL-17A concentration in the culture supernatant of the overexpression group was 2585±91.62pg/mL,and the concentration of the control group was 1751±70.34pg/mL.The overexpression group was significantly higher than the control group?P<0.005?.The expression of mmu_circ₀000451 was down-regulated after specific interference during Th17 cell induction?P<0.0001?.The proportion of Th17 cells in the interference group was 8.140±0.61%,and the proportion of Th17 cells in the control group was 13.25±0.25%.The interference group was significantly lower than the control group?P<0.05?;the concentration of IL-17A in the culture supernatant of the interference group was 1473±121.10pg/mL,the concentration of the control group was 2151±97.39pg/mL,the interference group was significantly lower than the control group?P<0.05?.?3?FISH and the isolation of nuclear and cytoplasmic RNA revealed that mmu_circ₀000451 was mainly located in the cytoplasm of Th17 cells.?4?Compared with Th0 cells,the expression of CDYL was increased in Th17 cells.When mmu_circ₀000451 overexpressed adenovirus was added during the induction process,CDYL mRNA was increased significantly?P<0.05?.During the induction process,specific interference mmu_circ₀000451 was added,CDYL mRNA and protein levels were decreased significantly?P<0.05?;EZH2 and CDYL The combination is also significantly weakened.?5?Adding the EZH2 inhibitor GSK343 to the Th17 cell induction system can significantly reduce the differentiation ratio of Th17 cells.The CIA incidence and average arthritis index of the CD4+T cell group with adoptive transfer simmu_circ₀000451 was significantly lower than that of the CD4+T cell group with adoptive transfer siNC and DBA group.There was no significant change in the proportion of Th17 cells in the spleen of each group?P>0.05?;the proportion of Th17 cells in the middle lymph nodes of the CD4+T cell group with adoptive transfer simmu_circ₀000451 was slightly lower than that of the CD4+T cell group with adoptive transfer siNC?P<0.05?.Conclusions:?1?The high expression of mmu_circ₀000451 in Th17 cells induced in vitro can promote the induced differentiation of Th17 cells.?2?mmu_circ₀000451 is mainly distributed in the cytoplasm of Th17 cells and promotes the expression of CDYL.?3?mmu_circ₀000451 induces the differentiation of naive CD4⁺T cells into Th17cells by strengthening the combination of CDYL and EZH2.?4?CD4⁺T cells with down-regulated expression of mmu_circ₀000451 can weaken the pro-inflammatory effect.