Objective To investigate the role of long non-coding RNA (LncRNA) G-quadruplex forming sequence (GSEC) in Brucellar spondylitis (BS) and its regulatory mechanism on macrophage polarization in BS.
Methods A total of 36 newly diagnosed BS patients admitted from May 2021 to May 2024 and 30 healthy individuals undergoing physical examination during the same period were enrolled as study subjects. Venous blood samples were collected from all participants for subsequent use. Cultured human monocytic cells (THP1) were divided into the following groups: control group cells cultured in medium supplemented with 5% serum from healthy people and 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48-hour differentiation, BS group (cells cultured in medium with 5% serum from BS patients and 50 ng/mL PMA for 48-hour differentiation), sh-NC group (transfected with control shRNA vector sh-NC), sh-GSEC group transfected with lentiviral vector carrying shRNA targeting LncRNA GSEC(sh-GSEC), sh-GSEC+LV-NC group co-transfected with sh-GSEC lentiviral vector and a control overexpression vector (LV-NC), and sh-GSEC+LV-CTCF group co-transfected with sh-GSEC lentiviral vector and a lentiviral overexpression vector for CCCTC-binding factor (LV-CTCF). The expression levels of LncRNA GSEC and CTCF mRNA in differentiated macrophages from each group were detected by real-time quantitative polymerase chain reaction (RT-qPCR). CTCF protein expression was analyzed by western blot. The binding between LncRNA GSEC and CTCF was determined by RNA immunoprecipitation (RIP). Serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), IL-4, IL-10, and transforming growth factor-β (TGF-β) in participants, as well as their levels in cell culture supernatants from each group, were measured by enzyme-linked immunosorbent assay (ELISA). The proportions of M1-type and M2-type macrophages in each group were analyzed by flow cytometry.
Results The content of M1-type macrophages in the peripheral blood of BS patients was upregulated compared with that of healthy people, while the content of M2-type macrophages was decreased compared with that of healthy people, the differences were statistically significant (P < 0.001). ELISA showed that compared with healthy people, the levels of pro-inflammatory factors (IL-1β, IL-6 and TNF-α) in the serum of BS patients were upregulated, while the levels of anti-inflammatory factors (IL-4, IL-10 and TGF-β) were decreased, the differences were statistically significant (P < 0.001). The RT-qPCR results showed that the expression level of LncRNA GSEC in the peripheral blood of BS patients was (4.05±0.26), which was higher than (1.02±0.11) of the healthy people, and the difference was statistically significant (P < 0.001). The RT-qPCR results showed that the expression level of LncRNA GSEC in the sh-GSEC group was (0.32±0.07)%, which was lower than (1.02±0.09)% in the sh-NC group, and the difference was statistically significant (P < 0.001). Flow cytometry analysis showed that the content of M1-type macrophages in the sh-GSEC group was lower than that in the sh-NC group, and the content of M2-type macrophages was higher, the differences were statistically significant (P < 0.01). The ELISA test results showed that the levels of pro-inflammatory factors (IL-1β, IL-6 and TNF-α) in the supernatant of macrophages in the sh-GSEC group were lower than those in the sh-NC group, while the levels of anti-inflammatory factors (IL-4, IL-10 and TGF-β) were higher, the differences were statistically significant (P < 0.01). Western blot analysis showed that the expression level of CTCF protein in the peripheral blood of BS patients was upregulated compared with that of healthy people, and the difference was statistically significant (P < 0.001). RIP assay results confirmed that LncRNA GSEC was significantly more enriched in complexes captured by the CTCF antibody compared to those captured by the IgG antibody (P < 0.001). The RT-qPCR results showed that the expression level of CTCF mRNA in macrophages of the sh-GSEC+LV-CTC group was (3.53±0.47), which was higher than that of the sh-GSEC+LV-NC group (1.01±0.05) and the sh-GSEC group (1.02±0.04), the differences were all statistically significant (P < 0.01). The Western blot results showed that the level of CTCF protein in macrophages of the sh-GSEC+LV-CTCF group was (3.12±0.27), which was higher than that of the sh-GSEC+LV-NC group (1.03±0.06) and the sh-GSEC group (1.01±0.04), the differences were all statistically significant (P < 0.01). The results of flow cytometry showed that the content of M1-type macrophages in the sh-GSEC group was lower than that in the sh-NC group, and the content of M2-type macrophages was higher, the differences were statistically significant (P < 0.01); The content of M1-type macrophages in the sh-GSEC+LV-CTCF group was higher than that in the sh-GSEC+LV-NC group, while the content of M2-type macrophages was lower, and the difference was statistically significant (P < 0.01).
Conclusion LncRNA GSEC promotes the occurrence of BS by regulating CTCF expression and driving macrophage polarization towards the M1 phenotype, providing new insights for understanding the pathology of BS and for developing novel diagnostic and therapeutic biomarkers.
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