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, 30 (18), 3830-41

Bidirectional Integrative Regulation of Cav1.2 Calcium Channel by microRNA miR-103: Role in Pain

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Bidirectional Integrative Regulation of Cav1.2 Calcium Channel by microRNA miR-103: Role in Pain

Alexandre Favereaux et al. EMBO J.

Abstract

Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Integrative regulation of Cav1.2-LTC by miR-103. (A) Bioinformatics analyses revealed that all Cav1.2-LTC subunit mRNA contain a target site for miR-103. Cacna1c and Cacna2d1 each contain one miR-103 site while Cacnb1 has two miR-103 sites. Hybridization to Cacna1c, Cacna2d1 and Cacnb1 3′UTR, yellow indicates perfect base pairing, green indicates wobble base pairing and grey indicates no match. (B) Luciferase reporters consisted of a Renilla luciferase coding sequence under CMV promoter control that was fused on its 3′ end to the 3′UTR of the different Cav1.2-LTC subunits. (C) Compared with control conditions, Cacna2d1 3′UTR mediated significant inhibition of Renilla luciferase activity at the lowest miR-103 dose (150 ng, −24%, P<0.001) whereas Cacna1c and Cacnb1 3′UTRs reporter activities were still unaffected. At higher miR-103 dose (500 ng), all Cav1.2-LTC subunit reporter activities were significantly repressed: −40.51%, P<0.001, −56.45%, P<0.001 and −31.51%, P<0.001 for Cacna1c, Cacna2d1 and Cacnb1, respectively. At the highest miR-103 dose (750 ng), inhibition reached a plateau for Cacna1c and Cacna2d1 (−31.59%, P<0.001 and −53.94%, P<0.001, respectively) whereas Cacnb1 reporter was more efficiently inhibited (−53.07%, P<0.001). Mutation of miR-103 seed region (seed-mut-miR-103) or reporter 3′UTR seed region (seed-mut-3′UTR) resulted in a complete abolition of miR-103 inhibition. Endogenous miR-103 knockdown resulted in a moderate but significant increase of all Cav1.2-LTC subunit reporter activities (miR-103-KD). #P<0.001, ANOVA, Holm–Sidak post hoc test. Data are expressed as mean values±s.e.m. n=6. (D) Mutation of miR-103 (seed-mut-miR-103) at nucleotide positions 2–8 (antisense of original sequence, highlighted in red). (E) qRT–PCR analysis of luciferase mRNA levels showed that miR-103 over-expression induced reporter mRNA decay for all Cav1.2-LTC subunits (Cacna1c −36.88%, P=0.021; Cacna2d1 −28.94 %, P=0.013; Cacnb1 −36.74%, P=0.004). *P<0.05, **P<0.01. Data are expressed as mean values±s.e.m. n=3.
Figure 2
Figure 2
MiR-103 bidirectionally regulates Cav1.2-LTC expression in spinal cord neurons. To assess all Cav1.2-LTC subunit expressions, we performed a specific labelling for Cav1.2-α1, α2δ1 and β1 subunits (A, C, E, respectively). In non-transfected neurons (a, b), labelling appears more intense, particularly at the plasma membrane (arrows), than in miR-103 over-expressing cells (star in c, d). Quantification of immunostaining intensity on confocal images demonstrated that miR-103 over-expression induced a significant decrease in Cav1.2-α1, α2δ1 and β1 labelling intensity compared with control conditions (−23.47% (B), −32.75% (D) and −25.66% (F), respectively, ***P<0.001). In miR-103 knockdown experiment, Cav1.2-α1, α2δ1 and β1 expressions were significantly increased (+59.64% (B), +57.62% (D) and +64.20% (F), respectively, P<0.001). Transfection with seed-mut-miR-103 or scrambled miRNA inhibitor had no effect on Cav1.2-LTC subunit labelling intensities. Data are expressed as mean values±s.e.m. of four experiments with a number of quantified neurons ranging from 11 to 19 for each condition, bar=10 μm.
Figure 3
Figure 3
MiR-103 does not affect Cav1.2, Cav2.2 and Cav3.2 trafficking. We assessed Cav1.2, Cav2.2 and Cav3.2 trafficking by measuring the ratio between membrane and cytoplasm immunostaining with and without miR-103 application. Membrane is identified with a lipophilic dye, DiD (blue in A, C, E). (A, B) miR-103 over-expressing neurons (red, A, star) showed an overall decrease in Cav1.2 labelling (green, B, arrow and arrowhead) but trafficking was not affected. In contrast, miR-103 over-expression (red, star in C, E) did not alter Cav2.2 nor Cav3.2 labelling (green; D, F). (G) Statistical analysis showed no difference in Cav1.2, Cav2.2 and Cav3.2 trafficking, data are expressed as mean values±s.e.m. n=10 for each condition, bar=20 μm.
Figure 4
Figure 4
MiR-103 regulation modulates neuronal calcium transients in vitro. (A) Spinal cord neurons were loaded with Fluo4-AM calcium indicator and depolarization was induced by KCl bath application (25 mM). (B) MiR-103 over-expression strongly reduced calcium responses compared with control (−58.44%, P<0.001) while miR-103 knockdown increased calcium transients (+22.23%, P<0.01). Like miR-103, anti-Cav1.2 siRNA application significantly reduced calcium signals compared with control, (−55.69%, P<0.001). Seed-mut-miR-103 and scrambled miRNA inhibitor did not change calcium transients. **P<0.01, ***P<0.001, ANOVA, Holm–Sidak post hoc test. Data are expressed as mean values±s.e.m. n=16/7, 21/7, 30/8, 46/8, 44/9 and 18/7 (first and second numbers indicate the number of tested cells and cultures, respectively) for control, miR-103, miR-103-KD, seed-mut-miR-103, siRNAs and scrambled miRNA inhibitor experiments, respectively.
Figure 5
Figure 5
MiR-103 knockdown in naive animals induces hypersensitivity to pain. Naive animals were subjected to four daily intrathecal injections of miR-103-KD and their sensitivity to pain was measured with mechanical stimulations. Compared with control animals who received scrambled miRNA inhibitor, miR-103-KD-injected rats demonstrated a slight but significant hypersensitivity to pain (−14.57% in mechanical threshold after treatment, P<0.01). Data are expressed as mean values±s.e.m. n=6 for miR-103-KD and scrambled miRNA inhibitor injected rats, **P<0.01, Wilcoxon signed rank test.
Figure 6
Figure 6
MiR-103 expression in the spinal cord is modulated in neuropathic pain conditions. (A) MiR-103 was detected in SNL rats by in situ hybridization with a double-digoxigenin-labelled probe. MiR-103 expression is enriched in superficial laminae of the dorsal horn and in ventral motoneurons. The signal intensity is weaker in the dorsal horn ipsilateral to the peripheral nerve injury (red mask). Bar: 500 μm. (B) Quantification of miR-103 labelling intensity showed a significant decrease in miR-103 expression in the ipsilateral dorsal horn of SNL animals (−15.92% compared with controlateral side, P=0.006). Data are expressed as mean values±s.e.m. n=5. (CE) Double detection of miR-103 (green) and MAP2 (red) shows neuronal expression of the miRNA. MiR-103 is expressed as discrete clusters throughout cell bodies and dendritic processes (arrows). n=nucleus; bar=10 μm. (F, G) Ultrastructural localization of miR-103 in dorsal horn neurons by in situ hybridization, double-digoxigenin-labelled probe was visualized with silver-enhanced ultra-small gold particles. The expression of miR-103 is restricted to subdomains of the soma (arrowheads in F). It is also present in the nucleus, near the nuclear envelope (arrows in F). In processes, miR-103 is detected in both axons (A) and dendrites (D) (double arrowheads in F and G). N=nucleus, star indicates dentritic process; bar=2 μm in (F) and bar=200 nm in (G).
Figure 7
Figure 7
MiR-103 dynamically regulates all Cav1.2-LTC subunits in vivo and is involved in pain sensitization. (A) In SNL animals, all three Cav1.2 subunit mRNAs were over-expressed as compared with sham (Cacna1c +138.57%, P<0.001; Cacna2d1 +47.09%, P<0.001; Cacnb1 +84.44%, P=0.005) while miR-103 was significantly decreased (−28.3%, P<0.001). ††P<0.01, †††P<0.001, ANOVA, Holm–Sidak post hoc test. MiR-103 intrathecal application significantly reduced Cacna1c, Cacna2d1 and Cacnb1 expressions (as compared with SNL level, −78.62%, P<0.001, −58.72%, P<0.001, −42.93%, P=0.004, respectively). In contrast, seed-mut-miR-103 did not change Cav1.2-LTC subunit expression. **P<0.01, ***P<0.001, ANOVA, Holm–Sidak post hoc test. Data are expressed as mean values±s.e.m. n=6, 6 and 3 for SNL, miR-103 and seed-mut-miR-103, respectively. (B) Rats were tested for pain sensitization using an electronic von Frey device: 7 days after surgery, they showed a decrease in paw withdrawal threshold in response to mechanical stimulation (40.70% of the pre-SNL threshold). Four daily intrathecal applications of miR-103 (days 7–10) alleviated mechanical allodynia (76.14% of pre-SNL threshold, P=0,031). Seed-mut-miR-103-injected animals showed no response to treatment (42.41% of pre-SNL threshold, P=0.219). *P<0.05, Wilcoxon signed rank test. Data are expressed as mean values±s.e.m. n=4, 4 and 6 for sham, miR-103 and seed-mut-miR-103-injected rats, respectively. (C) Cold allodynia was evaluated with a cold-plate device where animals are placed on a thermostatic steel plate set to +4°C. Elevation of the operated hind limb latency is measured and compared with the latency of the same animal just before surgery. Seven days after surgery (nerve ligation), SNL animals showed hypersensitivity to cold (23.53% of pre-surgery latency). Intrathecal miR-103 injections significantly reduced cold allodynia in SNL rats (58.75% of pre-surgery latency) whereas seed-mut-miR-103-injected animals showed no response to treatment. Data are expressed as mean values±s.e.m. n=6 and 4 for miR-103 and seed-mut-miR-103-injected rats, respectively.

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