LncRNA 51325 Alleviates Bone Cancer Induced Hyperalgesia Through Inhibition of Pum2

Abstract

Background: Bone cancer pain (BCP) represents one of the most challenging comorbidities associated with cancer metastasis. Long non-coding RNAs (lncRNAs) have garnered attention as potential therapeutic agents in managing neuropathic pain. However, their role in the regulation of nociceptive information processing remains poorly understood. In this study, we observed a significant down-regulation of the spinal lncRNA ENSRNOG00000051325 (lncRNA51325) in a rat model of bone cancer pain. Our study sought to elucidate the potential involvement of lncRNA51325 in the development of BCP by modulating the expression of molecules associated with pain modulation.

Methods: We established the BCP model by injecting Walker 256 cells into the tibial plateau of rats. We conducted tests on the pain behaviors and anxiety-like responses of rats through von-Frey test, Gait analysis, and Open Field Test. Spinal lumbar expansion was harvested for molecular biology experiments to explore the relationship between lncRNA51325 and Pumilio RNA binding family member 2 (Pum2).

Results: Notably, the overexpression of lncRNA51325 effectively attenuated mechanical allodynia in rats afflicted with BCP, whereas the knockdown of lncRNA51325 induced pain behaviors and anxiety-like responses in naïve rats. Additionally, we observed a time-dependent increase in the expression of Pum2 in BCP-afflicted rats, and intrathecal injection of Pum2-siRNA alleviated hyperalgesia. Furthermore, our investigations revealed that lncRNA51325 exerts a negative modulatory effect on Pum2 expression. The overexpression of lncRNA51325 significantly suppressed Pum2 expression in BCP rats, while the knockdown of lncRNA51325 led to elevated Pum2 protein levels in the spinal cord of naïve rats. Subsequent treatment with Pum2-siRNA mitigated the downregulation of lncRNA51325-induced mechanical allodynia in naïve rats.

Conclusion: Our findings indicate that lncRNA51325 plays a role in regulating bone cancer pain by inhibiting Pum2 expression, offering a promising avenue for novel treatments targeting nociceptive hypersensitivity induced by bone metastatic cancer.

Keywords: Pum2; bone cancer pain; long non-coding RNA; spinal cord.

Figure 1

Intratibial inoculation of Walker 256 cells produces bone cancer pain. (A) Timeline of von-Frey pain measurement. (B) The paw withdrawal threshold (PWT) of the ipsilateral hind paw was significantly reduced from 6 to 18 days after Walker 256 cell injection. ***p<0.001, vs sham group; n=8, two-way repeated measures ANOVA. (C and D) Quantification of behavioral parameters from the sham and BCP groups in open fields. ***p<0.001, vs sham group; n=8, Unpaired t test. (E-H) Representative Catwalk gait, including Print view, Timing view and Print intensity. Statistical changes of gait parameters between the sham and BCP groups. ***p<0.001, vs sham group; n=9 or 10, Unpaired t test. (I) 3D reconstruction of CT scan showing significant bone destruction of the left tibia 12 days after tumor inoculation in the sham and BCP groups. Active cancer cells invading the bone medullary cavity have destroyed the normal structure. (J)Representative images of H&E staining showed that the bone marrow cavity of rats in the sham-operated group was filled with distinct bone trabeculae (Shown by a yellow five-pointed star). Numerous tumor cells with darkly stained nuclei (Shown by a yellow dashed box) and bone resorption pits (Shown by a yellow arrow) appeared in the bone marrow cavity of the tibia 18 days after tumor cell inoculation. n=4, Scale bar: 50μm.

Figure 2

LncRNA51325 is downregulated in the spinal cord of the BCP rat. (A) The expression of lncRNA51325 is down-regulated in the spinal cord after the injection of Walker 256 cell. ***p<0.001, vs sham group; n=11 or 12, Unpaired t test. (B) The genome locations and species conservation of lncRNA51325. (C) Distribution of lncRNA51325 in the nuclear and cytoplasmic compartments of the spinal cord. n=3. (D-F) The colocalization of lncRNA51325 with NeuN, Iba1 and GFAP was detected by a double-label immunofluorescence assay. Immunofluorescence data show that lncRNA51325 (red) was predominantly expressed in neurons (green), and only a small number of lncRNA51325 was co-labeled with astrocytes (green) and microglia (green). All sections were counterstained with DAPI (blue) to show the nucleus. The white arrows indicate possible colocalization sites. Scale bar = 50μm. The image on the right is an enlargement of the image on the left. (G) Data analysis showed that lncRNA51325 was primarily present in NeuN positive neurons with a small amount in GFAP-positive astrocytes and Iba1-positive microglial cells in BCP. Statistical analysis showed that the percentage of lncRNA51325-positive neurons was markedly reduced in BCP rats when compared with sham rats. ***p<0.001, vs sham group; n=3, two-way ANOVA. (H) FISH of lncRNA51325 in the spinal neurons cultured in vitro. The pictures in the right panel showed the extent of co-localization of lncRNA51325 with NeuN. The yellow staining indicated the co-localization. Nucleus stained with DAPI (blue). Scale bar = 10μm.

Figure 3

The overexpression of lncRNA51325 repressed neuropathic pain development. (A) Timeline of lncRNA51325-LV spinal injection and von-Frey pain measurement. (B) Overexpression of lncRNA51325 alleviated mechanical hyperalgesia in the BCP rats. *p<0.05, **p<0.01, ***p<0.001, vs BCP+LV-NC group; n=8, two-way ANOVA. (C and D) Quantification of behavioral parameters from the BCP+LV-NC and BCP+LV groups in open fields. ***p<0.001, vs BCP+LV-NC group; n=8, Unpaired t test. (E) Schematic illustration of a lentiviral vector injection into the spinal cord of rats. (F) Lentiviral vectors express green fluorescent protein. Scale bar = 50μm. (G) The validation of lncRNA51325 lentivirus transfection efficiency in the spinal cord of BCP rats. ***p<0.001, vs BCP+LV-NC group; n=8, Ordinary one-way ANOVA. (H and I) c-Fos expression in the ipsilateral spinal cord after the dorsal horn injection of lncRNA51325-LV in BCP rats. *p<0.05, **p<0.01, vs BCP+LV-NC group; n=3, Ordinary one-way ANOVA. Scale bar = 50μm.

Figure 4

Effect of the overexpression of lncRNA51325 on the Catwalk gait parameters in BCP rats. (A-C) Representative Catwalk gait, including Print view, Timing view and Print intensity. Statistical changes of gait parameters between the BCP+LV-NC and BCP+LV groups. **p<0.01, ***p<0.001, vs BCP+LV-NC group; n=8, Unpaired t test.

Figure 5

Knockdown of lncRNA51325 induced neuropathic pain-like hypersensitivity in naïve rats. (A) Schematic diagram of cellular experiments for screening lncRNA51325 siRNAs with PC12 cell. (B) The validation of siRNA (lncRNA51325) transfection efficiency in PC12 cell. ***p<0.001, vs scr-siRNA group; n=3, Ordinary one-way ANOVA. (C and D) Timeline of lncRNA51325-siRNA intrathecal injection and von-Frey pain measurement. Knockdown of lncRNA51325 produced mechanical hyperalgesia in the naïve rats. **p<0.01, ***p<0.001, vs naïve+scr-siRNA group; n=8, two-way ANOVA. (E and F) Quantification of behavioral parameters from the naïve, naïve+scr-siRNA, and naïve+siRNA groups in open fields. **p<0.01, ***p<0.001, vs naïve+scr-siRNA group; n=8, Ordinary one-way ANOVA. (G) The relative expression of lncRNA51325 in the ipsilateral spinal cord in the naïve, naïve+scr-siRNA, and naïve+siRNA groups. ***p<0.001, vs naïve+scr-siRNA group; n=8, Ordinary one-way ANOVA. (H and I) Ipsilateral spinal c-Fos expression after continuous posttreatment with lncRNA51325-siRNA in naïve rats. *p<0.05, vs naïve+scr-siRNA group; n=3, Ordinary one-way ANOVA. Scale bar = 50μm.

Figure 6

Effect of the knockdown of lncRNA51325 on the Catwalk gait parameters in naïve rats. (A-D) Representative Catwalk gait, including Print view, Timing view and Print intensity. Statistical changes of gait parameters between the naïve, naïve+scr-siRNA, and naïve+siRNA groups. *p<0.05, vs naïve+scr-siRNA group; n=8, Ordinary one-way ANOVA.

Figure 7

Pum2 expression is increased in the spinal cord and contributes to the BCP. (A) Predicted target proteins that may bind to lncRNA51325 can be found at the RBPDB website (the database of RNA-binding protein specificities, http://rbpdb.ccbr.utoronto.ca/). (B) The expression of Pum2 in the spinal cord was detected by Q-PCR after the injection of Walker 256 cell. ***p<0.001, vs sham group; n=8, Unpaired t test. (C)The informatics analysis of lncRNA51325 binding to the bases in Pum2 mRNA. (D and E) The expression of Pum2 in the ipsilateral dorsal horn after the injection of Walker 256 cell. *p<0.05, **p<0.01, vs sham group; n=4, Ordinary one-way ANOVA. (F) The validation of siRNA (Pum2) transfection efficiency in the spinal cord of the naïve rats. **p<0.01 vs Pum2-scr-siRNA group; n=3, Ordinary one-way ANOVA. (G) Timeline of Pum2-siRNA intrathecal injection and von-Frey pain measurement. (H) RT-PCR data showed that the expression of Pum2 in the BCP spinal cord treated with siRNA was significantly lower than in the BCP+Pum2-scr-siRNA group. ***p<0.001, vs BCP+Pum2-scr-siRNA group; n=8, Ordinary one-way ANOVA. (I) siRNA silencing of Pum2 alleviated mechanical hyperalgesia in the BCP rats. ***p<0.001, vs BCP+Pum2-scr-siRNA group; n=8, two-way ANOVA. (J and K) Quantification of behavioral parameters from the BCP+Pum2-scr-siRNA, and BCP+Pum2-siRNA groups in open fields. **p<0.01, ***p<0.001, vs BCP+scr-siRNA group; n=8, Unpaired t test.

Figure 8

LncRNA51325 regulates BCP by targeting Pum2. (A-C) The colocalization of Pum2 with NeuN, Iba1 and GFAP was detected by a double-label immunofluorescence assay. All sections were counterstained with DAPI (blue) to show the nucleus. The white arrows indicate possible colocalization sites. Scale bar = 50μm. The image on the right is an enlargement of the image on the left. (D) Immunofluorescence data show that Pum2 was predominantly expressed in neurons, and a small amount in GFAP and Iba1. Data analysis showed that Pum2 was primarily present in NeuN positive neurons with a small amount in GFAP-positive astrocytes and Iba1-positive microglial cells in BCP. The percentage of Pum2-positive neurons was markedly increased in BCP rats when compared to that of sham rats. ***p<0.001, vs sham group; n=3, two-way ANOVA. (E) FISH-immunofluorescence double staining of lncRNA51325 and Pum2. The pictures in the right panel showed the extent of co-localization of lncRNA51325 with Pum2. The position indicated by the arrow indicated the co-localization. (F) The mRNA expression of Pum2 in the BCP, BCP+LV-NC, and BCP+LV group. ***p<0.001, vs BCP+LV-NC groups; n=8, Ordinary one-way ANOVA. (G and H) The relative protein expression of Pum2 in BCP rats. **p<0.01, vs BCP+LV-NC group; n=4, Ordinary one-way ANOVA. (I) The mRNA expression of Pum2 in the naïve, naïve+Pum2-scr-siRNA, and naïve+Pum2-siRNA groups. **p<0.01, vs naïve+Pum2-scr-siRNA group; n=8, Ordinary one-way ANOVA. (I and J) The relative protein expression of Pum2 in naïve rats. ***p<0.001, vs naïve+Pum2-scr-siRNA group; n=4, Ordinary one-way ANOVA. (J and K) Timeline of Pum2 and lncRNA51325-siRNA intrathecal injection and von-Frey pain measurement in the naïve rats. Treatment with Pum2-siRNA alleviated the downregulation of lncRNA51325 induced mechanical allodynia effects in naïve rats. ***p<0.001, vs naïve+Pum2-scr-siRNA group; n=8, two-way ANOVA. (L and M) Quantification of behavioral parameters from the naïve+Pum rats. *p<0.05, **p<0.01, ***p<0.001, vs naïve+Pum2-siRNA+lncRNA51325-siRNA group; n=8, two-way ANOVA.