DNMT3a contributes to the development and maintenance of bone cancer pain by silencing Kv1.2 expression in spinal cord dorsal horn

Abstract

Metastatic bone tumor-induced changes in gene transcription and translation in pain-related regions of the nervous system may participate in the development and maintenance of bone cancer pain. Epigenetic modifications including DNA methylation regulate gene transcription. Here, we report that intrathecal injection of decitabine, a DNA methyltransferase (DNMT) inhibitor, dose dependently attenuated the development and maintenance of bone cancer pain induced by injecting prostate cancer cells into the tibia. The level of the de novo DNMT3a, but not DNMT3b, time dependently increased in the ipsilateral L4/5 dorsal horn (not L4/5 dorsal root ganglion) after prostate cancer cells injection. Blocking this increase through microinjection of recombinant adeno-associated virus 5 (AAV5) expressing Dnmt3a shRNA into dorsal horn rescued prostate cancer cells-induced downregulation of dorsal horn Kv1.2 expression and impaired prostate cancer cells-induced pain hypersensitivity. In turn, mimicking this increase through microinjection of AAV5 expressing full-length Dnmt3a into dorsal horn reduced dorsal horn Kv1.2 expression and produced pain hypersensitivity in the absence of prostate cancer cells injection. Administration of neither decitabine nor virus affected locomotor function and acute responses to mechanical, thermal, or cold stimuli. Given that Dnmt3a mRNA is co-expressed with Kcna2 mRNA (encoding Kv1.2) in individual dorsal horn neurons, our findings suggest that increased dorsal horn DNMT3a contributes to bone cancer pain through silencing dorsal horn Kv1.2 expression. DNMT3a may represent a potential new target for cancer pain management.

Figure 1.

Effect of intrathecal administration of decitabine (Dec) on the development of mechanical allodynia, thermal hyperalgesia, and cold allodynia induced by injecting prostate cancer cells (PCC) into the tibia. Decitabine given once daily for seven days dose dependently blocked PCC-induced decreases in paw withdrawal threshold (PWT) to mechanical stimulation (a) and paw withdrawal latency (PWL) to thermal (b) or cold (c) stimulation on the ipsilateral side. Decitabine at 20 µg alone did not affect basal responses to mechanical, thermal, or cold stimuli on the ipsilateral side in rats injected with Hank’s balance salt solution (HSBB, (a) to (c)). Decitabine at the doses used did not affect basal responses to mechanical (d) and thermal (e) stimuli on the contralateral side. n = 5/group. Two-way ANOVA followed by post hoc Tukey’s test. *P < 0.05, **P < 0.01 versus the vehicle plus PCC group at the corresponding time points.

Figure 2.

Effect of intrathecal administration of decitabine on the maintenance of mechanical allodynia, thermal hyperalgesia, and cold allodynia induced by injecting prostate cancer cells (PCC) into the tibia. Decitabine given once daily for five days starting at day 7 post-PCC injection dose dependently attenuated PCC-induced decreases in paw withdrawal threshold (PWT) to mechanical stimulation (a) and paw withdrawal latency (PWL) to thermal (b) or cold (c) stimulation on the ipsilateral side. Post-treatment with decitabine at the doses used did not affect basal responses to mechanical (d) and thermal (e) stimuli on the contralateral side of PCC rats. n = 5/group. Two-way ANOVA followed by post hoc Tukey’s test. **P < 0.01 versus the vehicle plus PCC group at the corresponding time points.

Figure 3.

Time-dependent increase of DNMT3a in the ipsilateral L4/5 spinal dorsal horn, but not L4/5 DRG, after prostate cancer cell (PCC) injection. (a) The amounts of DNMT3a protein, but not DNMT3b protein, were significantly increased in the ipsilateral L4/5 dorsal horn on days 3, 5, 7, and 12 after PCC injection. n = 3/time point. One-way ANOVA followed by post hoc Tukey’s test. **P < 0.01 versus the corresponding naive rats (0 day). (b) PCC injection did not lead to changes in basal expression of DNMT3a and DNMT3b in the contralateral L4/5 dorsal horn during the observation period. n = 3/time point. One-way ANOVA followed by post hoc Tukey’s test. (c) Basal expression of both DNMT3a and DNMT3b in the ipsilateral L4/5 dorsal horn was not altered after Hank’s balanced salt solution (HBSS) injection. n = 3/time point. One-way ANOVA followed by post hoc Tukey’s test. (d) PCC injection did not lead to changes in basal expression of DNMT3a and DNMT3b in the ipsilateral L4/5 dorsal root ganglion during the observation period. n = 3 /time point. One-way ANOVA followed by post hoc Tukey’s test.

Figure 4.

Effect of dorsal horn DNMT3a knockdown on the development of mechanical allodynia, thermal hyperalgesia, and cold allodynia induced by injecting prostate cancer cells (PCC) into the tibia. The level of DNMT3a, but not DNMT3b, increased in the ipsilateral L4/5 dorsal horn of rats microinjected with AAV5-GFP (GFP) or AAV5-Dnmt3a scrambled shRNA (Scram) on day 7 after PCC injection. This increase was abolished in the PCC rats microinjected with AAV5-Dnmt3a shRNA (shRNA). (a) Representative Western blots. (b) A summary of densitometric analysis. n = 3/group. One-way ANOVA followed by post hoc Tukey’s test. **P < 0.01 versus the corresponding AAV5-GFP plus HBSS group. ##P < 0.01 versus the corresponding AAV5-GFP plus PCC group. Microinjection of AAV5-Dnmt3a shRNA, but not AAV5-Dnmt3a scrambled shRNA and AAV5-GFP, into unilateral L4/5 dorsal horn 30 days before PCC injection blocked PCC-induced decreases in paw withdrawal threshold (PWT) to mechanical stimulation (c) and paw withdrawal latency (PWL) to thermal (d) and cold (e) stimuli on the ipsilateral side. n = 5/group. Two-way ANOVA followed by post hoc Tukey’s test. *P < 0.05, **P < 0.01 versus the AAV5-GFP plus PCC group at the corresponding time points.

Figure 5.

Dorsal horn DNMT3a overexpression led to mechanical allodynia, thermal hyperalgesia, and cold allodynia in naive rats. (a) The level of DNMT3a protein increased in the ipsilateral L4/5 dorsal horn 6 weeks after microinjection of AAV5-Dnmt3a (DNMT3a), but not AAV5-GFP (GFP), into unilateral L4/5 dorsal horn. Top: representative Western blots. Bottom: a summary of densitometric analysis. n = 3/group. One-way ANOVA followed by post hoc Tukey’s test. *P < 0.05 versus naive rats. Microinjection of AAV5-Dnmt3a, but not AAV5-GFP, produced decreases in paw withdrawal threshold (PWT) to mechanical stimulation (b) and paw withdrawal latency to thermal (c) and cold (d) stimuli on the ipsilateral (Ipsi) side. Neither virus altered basal responses to mechanical (b) and thermal (c) stimuli on the contralateral (Cont) side. n = 5/group. Two-way ANOVA followed by post hoc Tukey’s test. *P < 0.05, **P < 0.01 versus the AAV5-GFP group on the contralateral side at the corresponding time points.

Figure 6.

DNMT3a mediates PCC-induced Kv1.2 downregulation in dorsal horn. (a) The level of Kcna2 mRNA, but not Kcna1 mRNA, Kcna4 mRNA, Oprd1 mRNA, Oprm1 mRNA, Oprk1 mRNA, Gad1 mRNA, and Gad2 mRNA, decreased in the ipsilateral L4/5 dorsal horn on day 7 after PCC (not HBSS) injection. n = 3/group. One-way ANOVA followed by post hoc Tukey’s test. **P < 0.01 versus naive rats. (b) Time-dependent reduction of Kv1.2 in the ipsilateral L4/5 dorsal horn post-PCC injection. Top: representative Western blots. Bottom: a summary of densitometric analysis. n = 3/time point. One-way ANOVA followed by post hoc Tukey’s test. **P < 0.01 versus naive rats (0 day). (c) Microinjection of AAV5-Dnmt3a shRNA (shRNA), but not AAV5-GFP (GFP) and HBSS, rescued the expression of Kv1.2 in the ipsilateral L4/5 dorsal horn on day 7 post-PCC injection. Top: representative Western blots. Bottom: a summary of densitometric analysis. n = 3/time point. One-way ANOVA followed by post hoc Tukey’s test. **P < 0.01 versus the AAV-GFP plus HBSS group. (d) Microinjection of AAV5-Dnmt3a (DNMT3a), but not AAV5-GFP (GFP), reduced the expression of Kv1.2 in the ipsilateral L4/5 dorsal horn 6 weeks after microinjection. Top: representative Western blots. Bottom: a summary of densitometric analysis. n = 3/time point. One-way ANOVA followed by post hoc Tukey’s test. *P < 0.05 versus naive rats. (e) Co-expression of Kcna2 mRNA with Dnmt3a mRNA in individual dorsal horn neurons from naive adult rats. Gapdh was used as a positive control. M: ladder marker. n = 3 repeats.