Effects of programmed cell death protein 4 targeting by microRNA-21-5p on the proliferation and apoptosis of peripheral blood B lymphocytes in patients with systemic lupus erythematosus / 中华风湿病学杂志

ABSTRACT

Objective:To investigate the regulatory effect of miRNA-21-5p on peripheral blood B lymphocyte proliferation and apoptosis in systemic lupus erythematosus (SLE) patients by targeting program-med cell death protein 4 (PDCD4) .Methods:Thirty patients with SLE diagnosed clinically in our hospital were enrolled. Peripheral blood lymphocyte (PBL) was extracted by gradient centrifugation, and B cells were separated by magnetic beads. The proportion of B lymphocyte in peripheral blood was detected by flow cytometry. The cells were divided into five groups by electrotransfection blank control group, miRNA-21-5p negative control (NC) group, miRNA-21-5p group and miRNA-21-5p inhibitor group, PDCD4 negative control group and PDCD4 siRNA group. Cells were collected 48 hours after transfection. The expression levels of miRNA-21 and PDCD4 were detected by real-time polymerase chain reaction (RT-PCR). Western blotting assay was used to detect the expression of PDCD4 in cells of each group. The targeting relationship between miRNA-21-5p and PDCD4 was verified by double luciferase target experiment. Flow cytometry was used to detect the apoptosis of cells in each group, and CCK-8 method was used to detect the proliferation of cells in each group. Western blotting and RT-PCR were used to detect the expression levels of Fas, FasL, CD40 and CD40L, respectively. Independent sample t test was used to compare the data between the two groups; single factor analysis of variance was used to analyze the results of multiple samples; chi square test was used to compare the positive rate of anti dsDNA antibody. Results:The levels of serum complement [C3 (0.85±0.11) g/L and C4 (0.54±0.09) g/L] in patients with SLE were lower ( t=7.524, P<0.05; t=38.471, P<0.05) than [C3 (1.16±0.17) g/L and C4 (1.57±0.09) g/L] in healthy controls. The levels of anti-dsDNA antibodies (47%), IgG(15.46±0.75) g/L, and IgA (2.68±0.20) g/L were increased than the levels of anti-dsDNA antibodies (17%), IgG (11.95±0.80) g/L, and IgA (2.16±0.11) g/L in healthy controls ( χ2=4.427, P<0.05; t=15.218, P<0.05; t=10.125, P<0.05). The proportion of B lymphocyte [(6.78±0.29)%] and the expression levels of miRNA21-5p (7.52±0.59) in peripheral blood of SLE patients was significantly higher than the proportion of B lymphocyte [(2.03±0.24)%] and the expression levels of miRNA21-5p (3.60±0.62) in healthy controls ( t=59.064, P<0.05; t=19.317, P<0.05), while the expression levels of PDCD4 gene (1.54±0.35) in peripheral blood of SLE patients was significantly lower than that (4.42±0.42) in healthy controls ( t=19.126, P<0.05). Compared with the blank control group and the miRNA-21-5p NC group, cell proliferation in the miRNA-21-5p Inhibitor group was inhibited, and the proportion of apoptotic cells increased ( F=5.244, P<0.05; F=37.903, P<0.05). Compared with the blank control group and PDCD4 NC group, cell proliferation in PDCD4 siRNA group was significantly enhanced, and apoptotic rate decreased ( F=5.956, P<0.05; F=25.431, P<0.05). The results of double luciferase reporter gene assay showed that PDCD4 is the target gene of miRNA-21-5p. Conclusion:miRNA-21-5p may promote the proliferation of peripheral blood B lymphocyte in SLE patients by inhibiting the expression of PDCD4, leading to abnormal lymphocyte apoptosis. miRNA-21-5p can be used as a new target gene for the treatment of systemic lupus erythematosus.