Translation of circ-PTPN22 in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and its significance
- VernacularTitle:circ-PTPN22在系统性红斑狼疮患者外周血单个核细胞中的翻译及意义
- Author:
Zhiting ZHONG
1
;
Qingqing MIAO
;
Zhuyan JIANG
;
Mengjie ZHANG
;
Jun TANG
Author Information
- From: Chinese Journal of Dermatology 2020;53(7):525-532
- CountryChina
- Language:Chinese
- Abstract: Objective:To assess the translation activity of circ-PTPN22, and to investigate its relationship with systemic lupus erythematosus (SLE) .Methods:From May 10, 2019 to September 30, 2019, whole blood samples were collected from 6 female patients with SLE and 9 healthy female controls in the Department of Rheumatology and Immunology, Integrated Traditional Chinese and Western Medicine, and Physical Examination Center of Southwest Hospital, respectively. Peripheral blood mononuclear cells (PBMCs) were isolated from these blood samples, and PBMCs from 3 cases of SLE and 3 healthy controls were sorted into T, B and NK cells by using magnetic beads. The circ-PTPN22 internal ribosomal entry site (IRES) sequence and protein sequence translated from it were predicted in the circRNADb database, rabbit anti-circ-PTPN22pro polyclonal antibodies were prepared against the specific amino acid sequence at the circ-PTPN22 splice site, and Western blot analysis was performed to determine the protein expression of circ-PTPN22pro in PBMCs and T, B and NK cell subsets of the healthy controls and patients with SLE. Cultured Jurkat cells were divided into 4 groups to be transfected with recombinant lentiviral vectors carrying circ-PTPN22-FLAG, circ-PTPN22-NC-FLAG, circ-PTPN22-shRNA-FLAG, circ-PTPN22-shRNA-NC-FLAG respectively, with the normally cultured cells as the cell control group. Then, Western blot analysis was performed to determine the protein expression of circ-PTPN22pro in Jurkat cells, flow cytometry to evaluate the effect of circ-PTPN22 on cell activation and apoptosis. Statistical analysis was carried out by using two-way repeated measures analysis of variance and two-independent-sample t test. Results:Based on the circRNADb database, circ-PTPN22 was predicted to have a translation ability, and Western blot analysis showed that the relative molecular mass of circ-PTPN22pro was 20 000. Forty-eight hours after transfection, circ-PTPN22pro expression was significantly higher in the circ-PTPN22-FLAG group than in the circ-PTPN22-NC-FLAG group and cell control group. At 24, 48 and 72 hours after transfection, the interleukin 2 (IL-2) expression was significantly lower in the circ-PTPN22 group (22.20% ± 8.92%, 31.10% ± 5.88%, 53.20% ± 10.25%, respectively) than in the circ-PTPN22-NC Group (30.90% ± 11.00%, 51.23% ± 10.70%, 69.67% ± 9.00%, respectively; F = 284.881, P = 0.003) , but significantly higher in the circ-PTPN22-shRNA group (35.57% ± 8.79%, 78.10% ± 10.08%, 88.63% ± 3.89%, respectively) than in the circ-PTPN22-shRNA-NC group (26.73% ± 4.92%, 41.03% ± 10.45%, 41.33% ± 4.96%, respectively; F = 293.818, P = 0.003) . After 48, 72 and 96 hours after transfection, the apoptosis rate was significantly higher in the circ-PTPN22 group than in the circ-PTPN22-NC group ( F = 81.287, P = 0.012) , as well as in the circ-PTPN22-shRNA group than in the circ-PTPN22-shRNA-NC group ( F = 111.813, P = 0.009) . The SLE group showed decreased (almost no) circ-PTPN22pro expression in PBMCs compared with the healthy control group. The circ-PTPN22pro expression in T and B cells was significantly lower in the SLE group than in the healthy control group ( t = 3.047, 4.806, both P <0.05) , and there was no significant difference in the circ-PTPN22pro expression in NK cells between the two groups ( t = 0.582, P > 0.05) . Conclusions:Circ-PTPN22 can be translated into circ-PTPN22pro protein, and can inhibit the activation of Jurkat cells. The circ-PTPN22pro expression is lower in PBMCs of the SLE patients than in those of the healthy controls, suggesting that circ-PTPN22 may be related to the occurrence and development of SLE.
