Spinal miRNA-124 regulates synaptopodin and nociception in an animal model of bone cancer pain

Abstract

Strong breakthrough pain is one of the most disabling symptoms of cancer since it affects up to 90% of cancer patients and is often refractory to treatments. Alteration in gene expression is a known mechanism of cancer pain in which microRNAs (miRNAs), a class of non-coding regulatory RNAs, play a crucial role. Here, in a mouse model of cancer pain, we show that miR-124 is down-regulated in the spinal cord, the first relay of the pain signal to the brain. Using in vitro and in vivo approaches, we demonstrate that miR-124 is an endogenous and specific inhibitor of synaptopodin (Synpo), a key protein for synaptic transmission. In addition, we demonstrate that Synpo is a key component of the nociceptive pathways. Interestingly, miR-124 was down-regulated in the spinal cord in cancer pain conditions, leading to an up-regulation of Synpo. Furthermore, intrathecal injections of miR-124 mimics in cancerous mice normalized Synpo expression and completely alleviated cancer pain in the early phase of the cancer. Finally, miR-124 was also down-regulated in the cerebrospinal fluid of cancer patients who developed pain, suggesting that miR-124 could be an efficient analgesic drug to treat cancer pain patients.

Figure 1

Quantification of bone destruction and assessment of nociceptive state. (A) Radiographic images of intact femur 21 days after saline (left) or sarcoma cells injection (right), revealing destruction of bone upon tumor development. (B) Bone volume quantification shows significant loss of bone density in tumor-injected group at day 21 after surgery (***P < 0.001, Mann-Whitney). (C) Hematoxylin-eosin-safran staining in control animals clearly shows normal structure of bone and bone marrow cells. (D) In contrast, in mice injected with NCTC 2472 sarcoma cells, there is a clear degradation of bone and replacement of bone marrow by sarcomatous cells. (E) Tumor-injected mice show a significant decrease in weight borne by ipsilateral paw compared to control group at days 7, 14 and 21 after surgery. Contralateral paws of tumor-injected group show a significant increase in weight borne at days 7, 14 and 21 after surgery in comparison to control group. Statistical comparison of tumor and control groups performed with two-way ANOVA with repeated measures followed by Bonferroni post-test, *P < 0.05, ***P < 0.001 compared to day 0.

Figure 2

Profiling of mRNAs and miRNAs in the spinal cord of cancerous mice. (A) Schematic representation of RNA screening strategy. (B) MA plot representing differential expression of mRNAs in bone-cancer-pain condition compared to control condition: 3909 mRNAs differentially expressed. (C) Heatmap representation of miRNA expression in cancerous versus naive mice: out of 742 miRNAs tested, 525 miRNAs were differentially expressed with 175 up-regulated (fold change > 2) and 350 down-regulated (fold change < 0.5).

Figure 3

miR-124 is specific regulator of synaptopodin. (A) Down-regulation of miR-124 expression in cancer mice (**P < 0.01, Student’s t test) and over-expression of different predicted target genes: Capn1, Nxph4, Synpo, and Tpm4 (*P < 0.05, Mann-Whitney). (B) To test Synpo-miR-124 interaction, a luciferase reporter was designed by fusing Synpo 3′UTR downstream of Renilla luciferase sequence: seed region is highlighted in red. HEK-293 cells were transfected with luciferase reporter together with control miRNA (miR-Ctl) or miR-124. miR-124 induced decrease in luciferase expression compared to miR-Ctl (***P < 0.001, one-way ANOVA followed by Bonferroni post-test). To check for binding specificity, we also used a mutated 3′UTR where seed region was deleted. Mutated 3′UTR was not able to mediate luciferase regulation (***P < 0.001, one-way ANOVA followed by Bonferroni post-test). (C and D) Immunostaining of synpo in spinal cord after miR-124 intrathecal injections: only the dorsal horn which receive nociceptive information was quantified (white dash area). Measurement of synaptopodin stained area reveals ability of miR-124 to inhibit endogenous Synpo expression (20/3 and 17/3 denotes number of sections/animals for control and miR-124-injected mice, respectively, ***P < 0.001, Mann-Whitney).

Figure 4

Synaptopodin is key element of nociceptive pathways. (A) To inhibit Synpo expression in spinal cord, we transfected a plasmid encoding both an ShRNA against Synpo and a GFP to track transfected cells; control plasmid only expressed GFP. Synpo expression assessed by immunolabeling and quantified in transfected cells only (white circles). (B) Quantification of Synpo immunolabeling confirmed that ShRNA strategy efficiently reduced Synpo expression by 57.15% (*P < 0.05, Mann-Whitney). (C) Synpo knockdown induced increase in paw withdrawal threshold as assessed with von Frey hairs (*P < 0.05, two-way ANOVA repeated measures followed by Bonferroni post-test).

Figure 5

Regulation of synaptopodin by miR-124 has analgesic properties. (A) RT-qPCR analysis of spinal cord from intrathecally injected mice (day 14) shows up-regulation of miR-124 in miR-124 mimics-injected group (n = 5, *P < 0.05, Mann-Whitney), associated with down-regulation of Synpo when compared to Cel-miR-67-injected mice (n = 5, **P < 0.01, Mann-Whitney). (B) DWB measurements of cancer mice subjected to miR-124 or Cel-miR-67 intrathecal injections. Cel-miR-67 animals show an altered weight bearing in tumor-bearing hindpaw from day 1 until day 14 indicative of nociceptive state (two-way ANOVA with repeated measure followed by Bonferroni post-test, * P < 0.05, **P < 0.01, ***P < 0.001 compared to Day 0). In contrast, miR-124 alleviated nociceptive behavior in early phase of cancer pain (day 1, 3, 5 and 7, two-way ANOVA with repeated measure followed by Bonferroni post-test, *P < 0.05, ***P < 0.001 compared to Day 0). (C) miR-124 expression in CSF samples from bone-cancer patients (n = 24) significantly lower than in controls (n = 20) (*P < 0.05, Mann-Whitney).