Figure 4. miR-27b regulates ASC-mediated suppression of CFSE-labeled allogeneic CD4⁺ T cell proliferation.

(A) ASCs from Lewis rats were transfected with α-miR-27b or α-miRNC (25 µg/ml) for 24 h. Cells were harvested and the relative expression levels of CXCL12 mRNA were measured. Western blot analysis was also performed for CXCL12 and actin (as internal control). (B) A total of 2.5×10⁻⁴ purified splenic T cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE). The cells were then cultured with Con A (1 µg/ml) (as 100% proliferation) and Con A + with different ratios of ASCs (1∶10, 1∶5, 1∶1, ASCs:T cells) with α-miR-27b or α-miRNC transfection. The cells were then stained with the anti-CD4 antibody, and proliferation was analyzed by FACS. A decrease in CFSE staining is an indication of proliferation. We quantified the percentage of CD4⁺ cells that have low levels of CFSE labeling. (C) Knocking out CXCL12 by siRNA transfection significantly impaired the suppressive actions of ASCs on T cell proliferation. Data are expressed as the mean ± SD of three independent experiments. *p<0.05; **p<0.01 compared with untreated ASCs.