Department of Orthopaedic Surgery, Chang Gung Memorial Hospital, No 5, Fu-Hsing Street, Linkou, Taoyuan, 333, Taiwan.
Department of Nursing, Chang Gung University of Science and Technology, Taoyuan, Taiwan.
J Orthop Surg Res. 2021 Jan 7;16(1):16. doi: 10.1186/s13018-020-02114-6.
MicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment.
NP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO/95% air and the hyperoxic cells were exposed to 100% O at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined.
Bioinformatics analysis indicated that the 3' untranslated region (UTR) of the Bax mRNA contained the "seed-matched-sequence" for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO.
Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.
微小 RNA（miRNA）在椎间盘（IVD）退变中起着至关重要的作用。在人退变髓核细胞中，miR-573 的表达水平下调，而 Bax 明显上调。在这项研究中，我们旨在探讨 miR-573 在高压氧（HBO）治疗后人退变髓核（NP）细胞中的作用。
从人退变椎间盘组织中分离 NP 细胞。对照组细胞在 5% CO/95%空气下维持，高氧组细胞在 2.5 个大气压的 100% O 下暴露。通过微阵列进行 miRNA 表达谱分析，并通过实时 PCR 进行验证，通过生物信息学和荧光素酶报告基因检测鉴定 miRNA 靶基因。测量 Bax 的 mRNA 和蛋白水平。使用 MTT 测定法检测 NPCs 的增殖。检测 Bax、cleaved caspase 9、cleaved caspase 3、pro-caspase 9 和 pro-caspase 3 的蛋白表达水平。
生物信息学分析表明，Bax mRNA 的 3'非翻译区（UTR）包含 hsa-miR-573 的“种子匹配序列”，该序列通过报告基因检测得到验证。miR-573 被 HBO 诱导，同时观察到 NP 细胞中 Bax 的表达下调。HBO 处理细胞中 miR-573 的敲低导致 Bax 表达上调。此外，HBO 通过过表达 miR-573 增加细胞增殖，并伴有细胞凋亡抑制。HBO 处理细胞中 pro-caspase 9 和 pro-caspase 3 的裂解被抑制，而 cleaved caspase 9 和 caspase 3 的水平降低。转染抗 miR-573 部分抑制了 HBO 的作用。
Mir-573 通过靶向 Bax 调节 HBO 处理后人退变 NP 细胞的增殖和凋亡。
