Effect and mechanism of circular RNA SEC24A on proliferation and apoptosis of synovial fibroblasts in osteoarthritis

doi:10.12307/2025.908

Abstract

Abstract: BACKGROUND: Synovitis is involved in all stages of osteoarthritis and is a key factor contributing to the development of osteoarthritis. Studies have shown that circular RNA (circRNA) plays an important role in the proliferation, apoptosis and extracellular matrix degradation of synovial cells and chondrocytes.
OBJECTIVE: To observe the effects of circRNA SEC24A on the interleukin-1β-induced proliferation, apoptosis, and expression of inflammatory factors in human synovial fibroblasts.
METHODS: Human synovial fibroblasts were divided into four groups, including control group, interleukin-1β group, empty vector group, and sh-circSEC24A group. Except for the control group, the other three groups were induced with 10 ng/mL interleukin-1β for 24 hours to establish inflammatory cell models; the empty vector group and sh-circSEC24A group were infected with empty vector virus and lentiviral vector knocking down circSEC24A. CCK-8 assay was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. ELISA was used to detect the levels of matrix metalloproteinase-9, matrix metalloproteinase-13, interleukin-6, and tumor necrosis factor-α in cell supernatant. Western blot assay was used to detect the relative expression levels of Bax, Bcl-2, matrix metalloproteinase-9, matrix metalloproteinase-13, casepase3, cleaved-casepase3, casepase8, and cleaved-casepase8 proteins in cells.
RESULTS AND CONCLUSION: (1) qRT-PCR results showed that compared with the normal group, the expression of circSEC24A in human synovial fibroblasts induced by interleukin 1β was significantly up-regulated. (2) The absorbance value of cells in the sh-circSEC24A group detected by CCK-8 assay was significantly higher than that of interleukin 1β group and empty vector group (P < 0.05). The apoptosis rate of sh-circSEC24A group detected by flow cytometry was significantly lower than that of interleukin 1β group and empty vector group (P < 0.05). (3) The levels of tumor necrosis factor α and interleukin 6 in the supernatant of human synovial fibroblasts in the sh-circSEC24A group detected by ELISA were significantly lower than those in the interleukin 1β group and the empty vector group (P < 0.01, P < 0.001). (4) Western blot assay results showed that compared with the interleukin 1β group and the empty vector group, the expression of the pro-apoptotic factor Bax protein in the sh-circSEC24A group significantly decreased, and the expression of the anti-apoptotic factor Bcl-2 protein increased significantly (P < 0.05); apoptosis and related activating factors cleaved-casepase3 and cleaved-casepase8 protein expressions were both reduced (P < 0.05). (5) ELISA and western blot assay results showed that compared with the interleukin 1β group and the empty vector group, the sh-circSEC24A group had lower levels of matrix metalloproteinase 9 and matrix metalloproteinase 13 protein (P < 0.05). These findings indicated that the expression of circSEC24A was abnormally increased in human synovial fibroblasts induced by interleukin 1β. Knocking down circSEC24A expression could promote the proliferation of human synovial fibroblasts and inhibit apoptosis, inflammatory factor release, and extracellular matrix degradation, suggesting that circSEC24A may be an important intervention target for early osteoarthritis.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: circular RNA, circRNA, circSEC24A, inflammation, synovial fibroblast, apoptosis, proliferation, extracellular matrix, engineered tissue construction

CLC Number:

Zhou Lijun, Zhang Keyuan, Xu Feihu, Wang Xi, Yu Li, Dong Shiming, Xu Junyu, Guo Yufeng, Ma Hairong, Ding Hong. Effect and mechanism of circular RNA SEC24A on proliferation and apoptosis of synovial fibroblasts in osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(24): 5086-5092.

Figures/Tables 5

2.1 白细胞介素1β 诱导炎症滑膜成纤维细胞模型向人滑膜成纤维细胞中加入0，5，10，20 ng/mL的白细胞介素1β溶液，培养6，12，24，48 h后，CCK-8检测结果显示10 ng/mL白细胞介素1β诱导下细胞活性最低(P < 0.001)，见图1A。ELISA结果显示10 ng/mL白细胞介素1β诱导24 h组肿瘤坏死因子α和白细胞介素6水平最高(P < 0.001)，见图1B，C。以下实验选择白细胞介素1β诱导的最佳质量浓度为10 ng/mL，诱导最佳时长为24 h。 2.2 circSEC24A在白细胞介素1β诱导的人滑膜成纤维细胞中高表达 qRT-PCR结果进一步验证了炎症模型建立成功，10 ng/mL白细胞介素1β诱导人滑膜成纤维细胞24 h组肿瘤坏死因子α和白细胞介素6的mRNA表达量最高(P < 0.001)，见图2A，B。经10 ng/mL白细胞介素1β诱导后的滑膜成纤维细胞相较于对照组，circSEC24A显著高表达(P < 0.01)，见图2C，说明circSEC24A可能参与骨关节炎的发生发展。 2.3 敲低circSEC24A对白细胞介素1β诱导的人滑膜成纤维细胞增殖能力的影响 2.3.1 circSEC24A稳转株筛选细胞转染48 h后，荧光显微镜下观察有绿色荧光表达，见图3A。qRT-PCR 检测结果显示，与sh-circSEC24A#1组和sh-circSEC24#2组相比，sh-circSEC24#3组circSEC24A的mRNA表达显著下调(P < 0.001)，见图3B，其中sh-circSEC24A#3病毒序列的敲低效果最好，因此sh-circSEC24A#3作为稳定敲低circSEC24A的滑成纤维细胞株(sh-circSEC24A组)。 2.3.2 敲低circSEC24A对白细胞介素1β诱导的人滑膜成纤维细胞增殖的影响 CCK-8实验显示，sh-circSEC24A组的吸光度值在24，48，72 h均高于白细胞介素1β组，具有时间依赖性(P < 0.01)，敲低circSEC24A可以增强白细胞介素1β诱导的人滑膜成纤维细胞的增殖能力，见图4。 2.4 敲低circSEC24A对白细胞介素1β诱导的人滑膜成纤维细胞炎症因子表达的影响 ELISA结果显示：与对照组相比，白细胞介素1β组人滑膜成纤维细胞上清液中肿瘤坏死因子α和白细胞介素6水平显著升高(P < 0.001，P < 0.000 1)；与白细胞介素1β组和空载体组相比，sh-circSEC24A组人滑膜成纤维细胞上清液中肿瘤坏死因子α和白细胞介素6水平显著降低(P < 0.01，P < 0.001)，见图5A，B。同时，qRT-PCR结果显示：与对照组相比，白细胞介素1β组人滑膜成纤维细胞中肿瘤坏死因子α和白细胞介素6 mRNA表达显著升高(P < 0.001)；与白细胞介素1β组和空载体组相比，sh-circSEC24A组人滑膜成纤维细胞中肿瘤坏死因子α和白细胞介素6 mRNA表达显著降低(P < 0.05，P < 0.01)，见图5C，D。 2.5 circSEC24A敲低对白细胞介素1β诱导的人滑膜成纤维细胞凋亡的影响流式细胞术检测结果显示，与对照组相比，白细胞介素1β组细胞总凋亡率呈现显著上升趋势(P < 0.01)；与白细胞介素1β组相比，sh-circSEC24A组细胞总凋亡率呈现显著下降趋势(P < 0.01)。Western blot检测结果显示，与对照组相比，白细胞介素1β组人滑膜成纤维细胞中Bax、cleaved-caspase3、cleaved-caspase8蛋白表达显著升高(P < 0.001)，Bcl-2蛋白表达显著降低(P < 0.01)；与白细胞介素1β组和空载体组相比，sh- circSEC24A组人滑膜成纤维细胞中Bax、cleaved-caspase3、cleaved-caspase8蛋白表达显著降低(P < 0.001，P < 0.01)，Bcl-2蛋白表达显著升高(P < 0.01)，见图6。因此，敲低circSEC24A可以降低白细胞介素1β诱导的人滑膜成纤维细胞的凋亡能力。 2.6 敲低circSEC24A对白细胞介素1β诱导的人滑膜成纤维细胞中基质金属蛋白酶表达的影响 ELISA检测结果显示：与对照组相比，白细胞介素1β组人滑膜成纤维细胞上清液中基质金属蛋白酶9和基质金属蛋白酶13水平显著升高(P < 0.001)；与白细胞介素1β组和空载体组相比，sh-circSEC24A组人滑膜成纤维细胞上清液中基质金属蛋白酶9和基质金属蛋白酶13水平显著降低(P < 0.01)，见图7A。同时，Western blot检测结果显示：与对照组相比，白细胞介素1β组人滑膜成纤维细胞中基质金属蛋白酶9和基质金属蛋白酶13 mRNA表达显著升高(P < 0.001)；与白细胞介素1β组和空载体组相比，sh-circSEC24A组人滑膜成纤维细胞中基质金属蛋白酶9和基质金属蛋白酶13 mRNA表达显著降低(P < 0.01)，见图7B。因此，敲低circSEC24A可以减缓白细胞介素1β诱导的人滑膜成纤维细胞的细胞外基质分解。"

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