miR-195 及其靶基因 Bcl-2 在人椎间盘退变中的表达及其对髓核细胞凋亡的影响。
Expression of miR-195 and its target gene Bcl-2 in human intervertebral disc degeneration and their effects on nucleus pulposus cell apoptosis.
机构信息
Second Department of Spinal Surgery, The Second Hospital of Liaocheng Affiliated to Shandong First Medical University, Linqing, 252600, Shandong, China.
出版信息
J Orthop Surg Res. 2021 Jun 28;16(1):412. doi: 10.1186/s13018-021-02538-8.
OBJECTIVE
To investigate the expression of miR-195 and its target gene Bcl-2 in intervertebral disc degeneration (IVDD) and its effect on nucleus pulposus (NP) cell apoptosis.
METHODS
The expressions of miR-195 and Bcl-2 in NP tissues of IVDD patients were quantified by qRT-PCR and western blotting, respectively. NP cells were divided into blank group, TNF-α group, TNF-α + miR-NC group, TNF-α + siBcl-2 group, and TNF-α + miR-195 inhibitors + siBcl-2 group. Cell proliferation was detected by MTT assay, cell apoptosis evaluated by flow cytometry, and mitochondrial membrane potential (MMP) tested by JC-1 staining. Moreover, the function of miR-195 on IVDD in vivo was investigated using a puncture-induced IVDD rat model.
RESULTS
IVDD patients had significantly increased miR-195 expression and decreased Bcl-2 protein expression in NP tissues. The expression of miR-195 was negatively correlated with the expression of Bcl-2 in IVDD patients. Dual-luciferase reporter gene assay indicated that Bcl-2 was a target gene of miR-195. In comparison with blank group, TNF-α group showed decreased cell proliferation and MMP, increased cell apoptosis, upregulated expression of miR-195, Bax, and cleaved caspase 3, and downregulated Bcl-2 protein, while these changes were attenuated by miR-195 inhibitors. Additionally, siBcl-2 can reverse the protective effect of miR-195 inhibitors on TNF-α-induced NP cells. Besides, inhibition of miR-195 alleviated IVDD degeneration and NP cell apoptosis in the rat model.
CONCLUSION
MiR-195 was significantly upregulated in NP tissues of IVDD patients, and inhibition of miR-195 could protect human NP cells from TNF-α-induced apoptosis via upregulation of Bcl-2.
目的
研究 miR-195 及其靶基因 Bcl-2 在椎间盘退变（IVDD）中的表达及其对髓核（NP）细胞凋亡的影响。
方法
通过 qRT-PCR 和 Western blot 分别定量检测 IVDD 患者 NP 组织中 miR-195 和 Bcl-2 的表达。将 NP 细胞分为空白组、TNF-α 组、TNF-α+miR-NC 组、TNF-α+siBcl-2 组和 TNF-α+miR-195 抑制剂+siBcl-2 组。通过 MTT 检测细胞增殖，通过流式细胞术评估细胞凋亡，通过 JC-1 染色检测线粒体膜电位（MMP）。此外，通过穿刺诱导的 IVDD 大鼠模型研究 miR-195 对 IVDD 的体内作用。
结果
IVDD 患者 NP 组织中 miR-195 表达显著升高，Bcl-2 蛋白表达降低。IVDD 患者 miR-195 表达与 Bcl-2 表达呈负相关。双荧光素酶报告基因实验表明，Bcl-2 是 miR-195 的靶基因。与空白组相比，TNF-α 组细胞增殖和 MMP 降低，细胞凋亡增加，miR-195、Bax 和 cleaved caspase 3 表达上调，Bcl-2 蛋白表达下调，而 miR-195 抑制剂可减弱这些变化。此外，siBcl-2 可逆转 miR-195 抑制剂对 TNF-α 诱导的 NP 细胞的保护作用。此外，抑制 miR-195 可减轻大鼠模型中 IVDD 退变和 NP 细胞凋亡。
结论
miR-195 在 IVDD 患者的 NP 组织中显著上调，抑制 miR-195 可通过上调 Bcl-2 来保护人 NP 细胞免受 TNF-α 诱导的凋亡。
Zotero 插件