背景:非编码microRNAs已成为神经系统基因表达的关键参与者,它们有助于调节神经疾病。如先前的研究所述,miR-155-5p上调发生在脊髓处于伤害性状态。目前尚不清楚miR-155-5p是否与骨癌疼痛(BCP)有关。在这里,我们旨在研究miR-155-5p在BCP过程中的功能调节功能,并描述其潜在机制.
方法:通过RNA测序确定miRNA-155-5p水平和细胞分布,荧光原位杂交(FISH),和定量实时PCR(qPCR)。免疫印迹,qPCR,双荧光素酶报告基因测定,免疫荧光,重组过表达腺相关病毒,小干扰RNA,椎管内给药,和行为测试用于探索下游信号通路。
结果:脊髓神经元中miR-155-5p的高表达有助于BCP的维持。通过鞘内注射miR-155-5pantagomir阻断miR-155-5p缓解疼痛行为;相反,通过阿戈米尔诱导的疼痛超敏反应上调miR-155-5p。miR-155-5p直接绑定到TCF4mRNA的3UTR。与Sham组相比,BCP显著降低TCF4的蛋白表达。抑制miR-155-5p可缓解脊髓TCF4蛋白的低表达水平,而miR-155-5p的上调通过miR-155-5p阿戈米尔鞘内注射降低了原初大鼠的TCF4蛋白表达。此外,BCP大鼠TCF4过表达可增加Kv1。此外,TCF4敲低抑制BCP大鼠Kv1.1的表达。的确,TCF4和Kv1.1在BCP脊髓神经元中共表达。
结论:研究发现表明miR-155-5p通过直接靶向脊髓神经元中的TCF4在调节BCP中的关键作用,并表明miR-155-5p可能是治疗BCP的有希望的靶标。
BACKGROUND: Noncoding microRNAs have emerged as critical players of gene expression in the nervous system, where they contribute to regulating nervous disease. As stated in previous research, the miR-155-5p upregulation happens in the spinal cord at the nociceptive state. It was unclear if miR-155-5p is linked to bone cancer pain (BCP). Herein, we aimed at investigating the miR-155-5p functional regulatory function in BCP process and delineating the underlying mechanism.
METHODS: The miRNA-155-5p levels and cellular distribution were determined by RNA sequencing, fluorescent in situ hybridization (FISH), and quantitative real-time PCR (qPCR). Immunoblotting, qPCR, dual-luciferase reporter gene assays, immunofluorescence, recombinant overexpression adeno-associated virus, small interfering RNA, intraspinal administration, and behavioral tests were utilized for exploring the downstream signaling pathway.
RESULTS: The miR-155-5p high expression in spinal neurons contributes to BCP maintenance. The miR-155-5p blockage via the intrathecal injection of miR-155-5p antagomir alleviated the pain behavior; in contrast, upregulating miR-155-5p by agomir induced pain hypersensitivity. The miR-155-5p bounds directly to TCF4 mRNA\'s 3\' UTR. BCP significantly reduced protein expression of TCF4 versus the Sham group. The miR-155-5p inhibition relieved the spinal TCF4 protein\'s down-expression level, while miR-155-5p upregulation by miR-155-5p agomir intrathecal injection decreased TCF4 protein expression in naïve rats. Additionally, TCF4 overexpression in BCP rats could increase
Kv1.1. Moreover, TCF4 knockdown inhibited
Kv1.1 expression in BCP rats. Indeed, TCF4 and
Kv1.1 were co-expressed in BCP spinal cord neurons.
CONCLUSIONS: The study findings stated the miR-155-5p pivotal role in regulating BCP by directly targeting TCF4 in spinal neurons and suggested that miR-155-5p could be a promising target in treating BCP.