环状RNA hsa_circ_0006156在系统性红斑狼疮患者外周血中的表达及其对Raji细胞增殖、凋亡的影响

马庆; 李黎; 李文宇; 侯伟; 林有坤

doi:10.16190/j.cnki.45-1211/r.2024.04.013

环状RNA hsa_circ_0006156在系统性红斑狼疮患者外周血中的表达及其对Raji细胞增殖、凋亡的影响

The significance of circular RNA hsa_circ_0006156 expression in peripheral blood of SLE patients and its effect on proliferation and apoptosis of Raji cells

摘要

摘要: 目的：研究hsa_circ_0006156对Raji细胞增殖、凋亡的影响及其在系统性红斑狼疮（SLE）发生、发展中的作用。方法：收集广西医科大学第一附属医院皮肤性病科2021 年9 月至2023 年9 月收治的55 例SLE 患者（SLE 组），并以40 例健康对照者作为对照组（HC 组），分离出外周血单个核细胞（PBMC）及CD20⁺B 淋巴细胞，使用实时荧光定量聚合酶链反应（RT-qPCR）法检测hsa_circ_0006156表达水平；采用受试者工作特征曲线（ROC）分析hsa_circ_0006156的诊断效能。以Raji细胞为研究对象，使用慢病毒构建hsa_circ_0006156 过表达模型，将细胞分为hsa_circ_0006156 过表达组（OE 组）、转染空病毒载体的阴性对照组（NC 组）以及空白对照组（control组）。利用CCK8法检测0 h、6 h、12 h、18 h、24 h、30 h、36 h细胞450 nm 吸光度，计算OE组和NC 组增殖率。Western blotting 实验检测各组Bcl-2、cleaved caspase-3 凋亡蛋白表达水平。结果：与HC 组比较，SLE 组PBMC 和CD20⁺B 淋巴细胞中hsa_circ_0006156 表达量均明显降低（P< 0.001）。ROC 曲线分析显示，hsa_circ_0006156 区分SLE 和HC 的ROC 曲线下面积（AUC）为0.787 3（95%CI：0.697 5～0.877；P< 0.001），敏感度为97.5%，特异度为50.9%，对SLE诊断Cut-off 值为< 0.652。hsa_circ_0006156 表达量在无活动或轻度活动与中重度活动患者之间存在显著差异（P＝0.014 8）。CCK8 实验结果显示，在36 h 时，OE 组Raji 细胞增殖较NC 组受到明显抑制（P< 0.001）。Western blotting 检测结果表明，与NC 组和control 组比较，OE 组Bcl-2 蛋白表达水平降低，而cleaved caspase-3 蛋白表达水平升高（均P< 0.05）。结论：hsa_circ_0006156对SLE具有一定的诊断效能。hsa_circ_0006156可能是通过调节B细胞的增殖、凋亡影响SLE的发生、发展。

Abstract: Objective: To investigate the effect of hsa_circ_0006156 on the proliferation and apoptosis of Raji cells and its role in the occurrence and development of systemic lupus erythematosus (SLE). Methods: A total of 55 SLE patients (SLE group) admitted to the Department of Dermatology and Venereal Diseases, the First Affiliated Hospital of Guangxi Medical University from September 2021 to September 2023 were collected, and 40 healthy controls were taken as the control group (HC group). Peripheral blood mononuclear cells (PBMC) and CD20⁺B lymphocytes were isolated. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of hsa_circ_0006156, and receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of hsa_circ_0006156. Taking Raji cells as the research object, the hsa_circ_0006156 overexpression model was constructed by lentivirus. The cells were divided into hsa_circ_0006156 overexpression group (OE group), negative control group (NC group) transfected with empty viral vector, and blank control group (control group). CCK8 assay was used to detect the cell absorbance of 450 nm at 0 h, 6 h, 12 h, 18 h, 24 h, 30, 36 h, and the proliferation rate of OE group and NC group was calculated.Western blotting was used to detect the expression levels of Bcl-2 and cleaved caspase-3 apoptotic proteins in each group. Results: Compared with the HC group, the expression of hsa_circ_0006156 in PBMC and CD20⁺B lymphocytes in the SLE group was significantly decreased (P< 0.001). ROC curve analysis showed that the area under the ROC curve (AUC) of hsa_circ_0006156 distinguishing SLE and HC was 0.7873 (95% CI:0.6975-0.877; P< 0.001), sensitivity was 97.5%, specificity was 50.9%, and cut-off value for SLE diagnosis was less than 0.652. There was a significant difference in the expression of hsa_circ_0006156 between patients with no or mild activity and moderate to severe activity (P=0.0148). CCK8 assay showed that the proliferation of Raji cells in the OE group was significantly inhibited compared with the NC group at 36 h (P< 0.001). The results of western blotting showed that compared with the NC group and the control group, the expression level of Bcl-2 protein in the OE group was decreased, while the expression level of cleaved caspase-3 protein was increased (all P< 0.05). Conclusion: The hsa_circ_0006156 has certain diagnostic efficacy for SLE. It may affect the occurrence and development of SLE by regulating the proliferation and apoptosis of B cells.

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