BACKGROUND Intervertebral disc degeneration (IVDD) is a well-known cause of lower back pain, which is induced by multiple factors including increased apoptosis and decreased survival of nucleus pulposus cells. In this study, we evaluate the effect and potential mechanism of miR-660 on the nucleus pulposus cells apoptosis induced by TNF-α. METHODS First, we collected tissue of nucleus pulposus from IVDD and healthy controls. General characteristic of the IVDD and healthy control was also collected. And, we also collected nucleus pulposus cells that stimulated by TNF-α or control. miRNA microarray was performed to identify the differentially expressed miRNAs. Apoptosis rate and miR-660 relative expression was measured after stimulated with different concentration of TNF-α to identify the optimal concentration of TNF-α. Second, we successfully constructed antigomiR-660 to block the miR-660 expression in nucleus pulposus cells and then stimulated with TNF-α (100 ng/ml, 12 h). The apoptosis rates and relative protein expression were then measured again. The target association between miR-660 and SAA1 was confirmed by dual-luciferase reporter. RESULTS There was no significant difference between the age (IVDD: 39 ± 10 years, healthy controls: 36 ± 7 years), BMI and sex between IVDD and healthy controls. Microarray analysis found that miR-660 was significantly up-regulated in IVDD and TNF-α treated groups, which was further identified by PCR. We found that the rate of apoptosis and miR-660 expression increased with TNF-α concentration increased. Finally, TNF-a with 100 ng/ml was used for further experiment. Compared with TNF-α group, TNF-α + antigomiR-660 could significantly down-regulated the apoptosis rate and relative protein (c-Caspase3 and c-Caspase7). Dual-luciferase reporter revealed that miR-660 could directly binding to the SAA1 at 80-87 sites. Compared with TNF-α alone group, TNF-α + antigomiR-660 significantly up-regulated the SAA1 expression (P < 0.05). CONCLUSION These results indicated that knockdown of miR-660 protected the nucleus pulposus from apoptosis that induced TNF-α via up-regulation of SAA1. Further studies should focus on the role of miR-660 in protecting IVDD in vivo.

中文翻译：

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敲低miR-660可通过靶向血清淀粉样蛋白A1保护髓核细胞免受TNF-a诱导的细胞凋亡。

背景技术椎间盘退变（IVDD）是下背部疼痛的众所周知的原因，其由多种因素诱导，包括增加的凋亡和髓核细胞存活的降低。在这项研究中，我们评估了miR-660对TNF-α诱导的髓核细胞凋亡的影响及其潜在机制。方法首先，我们从IVDD和健康对照中收集髓核组织。还收集了IVDD和健康对照的一般特征。并且，我们还收集了由TNF-α或对照刺激的髓核细胞。进行miRNA微阵列鉴定差异表达的miRNA。用不同浓度的TNF-α刺激后，测定其凋亡率和miR-660的相对表达，以确定最佳的TNF-α浓度。第二，我们成功构建了antigomiR-660来阻断miR-660在髓核细胞中的表达，然后用TNF-α（100 ng / ml，12 h）刺激。然后再次测量凋亡率和相对蛋白表达。双荧光素酶报告基因证实了miR-660与SAA1之间的靶标关联。结果IVDD与健康对照者的年龄（IVDD：39±10岁，健康对照者：36±7岁），BMI和性别之间无显着差异。芯片分析发现，miR-660在IVDD和TNF-α治疗组中明显上调，并通过PCR进一步鉴定。我们发现凋亡率和miR-660表达随TNF-α浓度的增加而增加。最后，将100 ng / ml的TNF-α用于进一步的实验。与TNF-α组相比，TNF-α+ antigomiR-660可以显着下调细胞凋亡率和相关蛋白（c-Caspase3和c-Caspase7）。双重荧光素酶报道基因显示，miR-660可以直接在80-87位与SAA1结合。与单独使用TNF-α组相比，TNF-α+ antigomiR-660显着上调了SAA1表达（P <0.05）。结论这些结果表明，敲低miR-660可以保护髓核免受SAA1上调引起的TNF-α诱导的细胞凋亡。进一步的研究应集中在miR-660在体内保护IVDD方面的作用。TNF-α+ antigomiR-660显着上调SAA1表达（P <0.05）。结论这些结果表明，敲低miR-660可以保护髓核免受SAA1上调引起的TNF-α诱导的细胞凋亡。进一步的研究应集中在miR-660在体内保护IVDD方面的作用。TNF-α+ antigomiR-660显着上调SAA1表达（P <0.05）。结论这些结果表明，敲低miR-660可以保护髓核免受SAA1上调引起的TNF-α诱导的细胞凋亡。进一步的研究应集中在miR-660在体内保护IVDD方面的作用。