MiR-135-5p Alleviates Bone Cancer Pain by Regulating Astrocyte-Mediated Neuroinflammation in Spinal Cord through JAK2/STAT3 Signaling Pathway

Abstract

Bone cancer pain (BCP) was associated with microRNA dysregulation. In this study, we intended to clarify the potential role of miR-135-5p in a BCP mouse model, which was established by tumor cell implantation (TCI) in the medullary cavity of the mouse femur. The BCP related behaviors were tested, including the paw withdrawal mechanical threshold (PWMT) and number of spontaneous flinches (NSF). The miRNA expression profiles in astrocytes of the sham and tumor groups were compared, and miRNA microarray and quantitative real-time PCR (qRT-PCR) assays confirmed that the amount of expression of miR-135-5p was significantly decreased in astrocytes of the tumor group. Gain-and loss-of-function studies showed that miR-135-5p could inhibit astrocytes activation and inflammation cytokines (TNF-α and IL-1β) expression. The relation between miR-135-5p and JAK2 was detected by bioinformatic analysis and dual luciferase reporter gene assay. By conducting in vitro experiments, it was shown that the miR-135-5P mimics lowered the level of JAK2/STAT3 proteins and inflammatory factors in astrocytes. Moreover, in vivo analysis on BCP mice model indicated that the miR-135-5p agonist could sufficiently increase PWMT and decrease NSF. Meanwhile, reduced activation of astrocytes in the spinal cord, as well as decreased expression of JAK2/STAT3 and inflammatory mediators, were found after miR-135-5p agonist treatment. Collectively, the results showed that miR-135-5p could potentially reduce BCP in mice through inhibiting astrocyte-mediated neuroinflammation and blocking of the JAK2/STAT3 signaling pathway, indicating that the upregulation of miR-135-5P could be a therapeutic focus in BCP treatment.

The role of astrocytes in neuropathic pain

“…Some experiments have demonstrated that the proliferative capacity of astrocytes intervened by AG490 or with STAT3 defects is reduced to a certain extent ( Washburn and Neary, 2006 ; Sarafian et al, 2010 ). Liu et al (2021) demonstrated that not only were the activation levels of JAK2 and STAT3 significantly reduced by miR-135-5p mimics intervention but also GFAP, IL-1β, and TNF-α expression, which was consistently decreased. Moreover, functional in vitro studies have also confirmed that miR-135-5p potentially inhibits the JAK2-STAT3 signaling pathway in bone cancer pain, and histological staining has proven that miR-135-5p can directly interrupt the activity process of JAK2 and its upstream and downstream molecules in astrocytes ( Liu et al, 2021 ).…”

“… Liu et al (2021) demonstrated that not only were the activation levels of JAK2 and STAT3 significantly reduced by miR-135-5p mimics intervention but also GFAP, IL-1β, and TNF-α expression, which was consistently decreased. Moreover, functional in vitro studies have also confirmed that miR-135-5p potentially inhibits the JAK2-STAT3 signaling pathway in bone cancer pain, and histological staining has proven that miR-135-5p can directly interrupt the activity process of JAK2 and its upstream and downstream molecules in astrocytes ( Liu et al, 2021 ). These studies demonstrate that JAK-STAT3 signaling has a key contribution in the occurrence and progression of neuropathic pain via the modulation of astrocyte activation.…”

DNA Methylation and Non-Coding RNAs during Tissue-Injury Associated Pain

“…In addition, miR-34c-5p has recently been identified in DRG neurons as pronociceptive by regulating the transcription of Cav2.3 , P2rx6 , Oprd1 , and Oprm1 [ 84 ]. In the spinal cord, following the induction of various models of cancer-induced pain, imbalances on the levels of miRNAs have been found: down-regulation of miR-124, which negatively regulated synaptopodin ( Synpo ), a key component in synaptic transmission [ 65 ]; up-regulation of miRNA-330, which seems to modulate the expression of GABA B R2 [ 83 ]; and decreases in miR-135-5p, which regulated JAKs/Stat3 signalling, in spinal astrocytes [ 70 ]. Furthermore, changes in miRNAs have also been associated with certain drug-induced pain disorders, such as that induced by the use of oxaliplatin to treat advanced colorectal cancer [ 150 ].…”

“…However, microarrays use already known sequences, thus making them unsuitable for the discovery of novel ncRNAs. Nonetheless, microarray-based methods can be used as cost-effective procedures to validate the presence of ncRNA from a known or predicted transcript and have proven useful in pain research [ 66 , 70 ].…”

CXCR1 participates in bone cancer pain induced by Walker 256 breast cancer cells in female rats

“…JAK2/STAT3 pathway is implicated in inflammatory pain, 11 neuropathic pain, 12 and BCP. 13 NLRP3 inflammasome is also involved in the development and progression of BCP. 14 , 15 In addition, the activation of NLRP3 inflammasome is inhibited after the suppression of JAK2/STAT3 pathway.…”