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MicroRNA cluster miR-17-92 regulates multiple functionally related voltage-gated potassium channels in chronic neuropathic pain

Sakai, Atsushi, Saitow, Fumihito, Maruyama, Motoyo, Miyake, Noriko, Miyake, Koichi, Shimada, Takashi, Okada, Takashi, Suzuki, Hidenori·2017·Nature Communications
DOI 10.1038/ncomms16079PMC PMC5504285
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Abstract

miR-17-92 is a microRNA cluster with six distinct members. Here, we show that the miR-17-92 cluster and its individual members modulate chronic neuropathic pain. All cluster members are persistently upregulated in primary sensory neurons after nerve injury. Overexpression of miR-18a, miR-19a, miR-19b and miR-92a cluster members elicits mechanical allodynia in rats, while their blockade alleviates mechanical allodynia in a rat model of neuropathic pain. Plausible targets for the miR-17-92 cluster include genes encoding numerous voltage-gated potassium channels and their modulatory subunits. Single-cell analysis reveals extensive co-expression of miR-17-92 cluster and its predicted targets in primary sensory neurons. miR-17-92 downregulates the expression of potassium channels, and reduced outward potassium currents, in particular A-type currents. Combined application of potassium channel modulators synergistically alleviates mechanical allodynia induced by nerve injury or miR-17-92 overexpression. miR-17-92 cluster appears to cooperatively regulate the function of multiple voltage-gated potassium channel subunits, perpetuating mechanical allodynia.

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Figure 1 miR-17-92 cluster upregulation in L5 DRG neurons after nerve injury. (a,c) Change in expression of mature miRNAs of the miR-17-92 cluster (a) and primary transcripts of miR-17-92 (c) in the L5 DRG over time after SNL. Numbers of samples are indicated at the base of each bar. *P<0.05, **P<0.01 and ***P<0.001 (miR-17, P=0.002 for 1d, P=0.010 for 3d, P=0.017 for 7d, P=0.004 for 14d and P<0.001 for 28d; miR-18a, P=0.006 for 1d, P=0.011 for 3d, P=0.005 for 7d, P<0.001 for 14d and P<0.001 for 28d; miR-19a, P=0.017 for 1d, P=0.033 for 3d, P=0.022 for 7d, P<0.001 for 14d and P<0.001 for 28d; miR-19b, P=0.002 for 1d, P=0.049 for 3d, P=0.005 for 7d, P=0.002 for 14d and P<0.001 for 28d; miR-20a, P=0.008 for 1d, P=0.015 for 3d, P=0.020 for 7d, P<0.001 for 14d and P<0.001 for 28d; miR-92a, P=0.006 for 1d, P=0.016 for 3d, P=0.011 for 7d, P=0.002 for 14d and P<0.001 for 28d; pri-miR-17-92, P=0.043 for 1d, P=0.005 for 3d, P=0.048 for 7d, P=0.022 for 14d and P=0.011 for 28d) compared with the contralateral intact side, paired t-test. (b) Paw withdrawal responses to mechanical stimuli evaluated on the SNL and contralateral sides. Error bars are s.e.m. *P<0.05 (P=0.043 for 3d, P=0.042 for 7d, P=0.041 for 14d and P=0.039 for 28d) compared with the contralateral side, Mann–Whitney U test.
Figure 1 miR-17-92 cluster upregulation in L5 DRG neurons after nerve injury. (a,c) Change in expression of mature miRNAs of the miR-17-92 cluster (a) and primary transcripts of miR-17-92 (c) in the L5 DRG over time after SNL. Numbers of samples are indicated at the base of each bar. *P<0.05, **P<0.01 and ***P<0.001 (miR-17, P=0.002 for 1d, P=0.010 for 3d, P=0.017 for 7d, P=0.004 for 14d and P<0.001 for 28d; miR-18a, P=0.006 for 1d, P=0.011 for 3d, P=0.005 for 7d, P<0.001 for 14d and P<0.001 for 28d; miR-19a, P=0.017 for 1d, P=0.033 for 3d, P=0.022 for 7d, P<0.001 for 14d and P<0.001 for 28d; miR-19b, P=0.002 for 1d, P=0.049 for 3d, P=0.005 for 7d, P=0.002 for 14d and P<0.001 for 28d; miR-20a, P=0.008 for 1d, P=0.015 for 3d, P=0.020 for 7d, P<0.001 for 14d and P<0.001 for 28d; miR-92a, P=0.006 for 1d, P=0.016 for 3d, P=0.011 for 7d, P=0.002 for 14d and P<0.001 for 28d; pri-miR-17-92, P=0.043 for 1d, P=0.005 for 3d, P=0.048 for 7d, P=0.022 for 14d and P=0.011 for 28d) compared with the contralateral intact side, paired t-test. (b) Paw withdrawal responses to mechanical stimuli evaluated on the SNL and contralateral sides. Error bars are s.e.m. *P<0.05 (P=0.043 for 3d, P=0.042 for 7d, P=0.041 for 14d and P=0.039 for 28d) compared with the contralateral side, Mann–Whitney U test.CC BY · PubMed Central
Figure 2 miR-17-92 cluster overexpression in DRG neurons causes mechanical allodynia. (a) Representative image of EGFP immunofluorescence in the L5 DRG 7 days after injection of the AAV vector encoding miR-17-92 and EGFP. Scale bar, 100 μm. (b) Expression levels of miR-17-92 primary transcript in L5 DRGs 7 days after control or miR-17-92 AAV vector injection. Numbers of samples are indicated at the base of each bar. *P=0.031 by Welch’s test. (c–e) Paw withdrawal threshold (c,e) and latencies (d) to mechanical and thermal stimuli, respectively, were evaluated on the injected sides before and 7 days after the injection of AAV vector encoding the whole miR-17-92 cluster (c,d) or individual miR-17-92 cluster members (e). Error bars are s.e.m. *P<0.05 and **P<0.01 (P=0.004 for miR-17-92; P=0.023 for miR-18a; P=0.004 for miR-19a; P=0.008 for miR-19b; P=0.014 for miR-92) compared with the control injection, Mann–Whitney U test.
Figure 2 miR-17-92 cluster overexpression in DRG neurons causes mechanical allodynia. (a) Representative image of EGFP immunofluorescence in the L5 DRG 7 days after injection of the AAV vector encoding miR-17-92 and EGFP. Scale bar, 100 μm. (b) Expression levels of miR-17-92 primary transcript in L5 DRGs 7 days after control or miR-17-92 AAV vector injection. Numbers of samples are indicated at the base of each bar. *P=0.031 by Welch’s test. (c–e) Paw withdrawal threshold (c,e) and latencies (d) to mechanical and thermal stimuli, respectively, were evaluated on the injected sides before and 7 days after the injection of AAV vector encoding the whole miR-17-92 cluster (c,d) or individual miR-17-92 cluster members (e). Error bars are s.e.m. *P<0.05 and **P<0.01 (P=0.004 for miR-17-92; P=0.023 for miR-18a; P=0.004 for miR-19a; P=0.008 for miR-19b; P=0.014 for miR-92) compared with the control injection, Mann–Whitney U test.CC BY · PubMed Central
Figure 3 Blockade of miR-17-92 cluster members suppresses mechanical allodynia in SNL. Paw withdrawal responses to mechanical stimuli were evaluated on the AAV-injected/SNL side. Numbers of animals are indicated to the right of the plots. (a) AAV vector encoding control TuD antisense RNA or TuD antisense RNA against each miR-17-92 cluster member was administered to the L5 DRG 7 days before SNL (indicated by arrows). (b) Mixture of AAV vectors encoding TuD antisense RNAs against miR-18a, miR-19a, miR-19b and miR-92a was administered to the L5 DRG 7 days after SNL (indicated by arrows). Error bars are s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (miR-18a, P=0.011 for 3d, P=0.030 for 14d and P=0.030 for 28d; miR-19a, P=0.008 for 3d, P=0.002 for 7d; P=0.014 for 21d and P=0.035 for 28d; miR-19b, P=0.004 for 7d, P=0.002 for 14d, P=0.002 for 21d and P=0.002 for 28d; miR-92a, P=0.002 for 3d, P=0.002 for 7d, P=0.009 for 14d, P=0.002 for 21d and P=0.004 for 28d; antisense RNA mixture, P=0.003 for 14d, P<0.001 for 21d and P<0.001 for 28d) compared with the control injection, Mann–Whitney U test.
Figure 3 Blockade of miR-17-92 cluster members suppresses mechanical allodynia in SNL. Paw withdrawal responses to mechanical stimuli were evaluated on the AAV-injected/SNL side. Numbers of animals are indicated to the right of the plots. (a) AAV vector encoding control TuD antisense RNA or TuD antisense RNA against each miR-17-92 cluster member was administered to the L5 DRG 7 days before SNL (indicated by arrows). (b) Mixture of AAV vectors encoding TuD antisense RNAs against miR-18a, miR-19a, miR-19b and miR-92a was administered to the L5 DRG 7 days after SNL (indicated by arrows). Error bars are s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (miR-18a, P=0.011 for 3d, P=0.030 for 14d and P=0.030 for 28d; miR-19a, P=0.008 for 3d, P=0.002 for 7d; P=0.014 for 21d and P=0.035 for 28d; miR-19b, P=0.004 for 7d, P=0.002 for 14d, P=0.002 for 21d and P=0.002 for 28d; miR-92a, P=0.002 for 3d, P=0.002 for 7d, P=0.009 for 14d, P=0.002 for 21d and P=0.004 for 28d; antisense RNA mixture, P=0.003 for 14d, P<0.001 for 21d and P<0.001 for 28d) compared with the control injection, Mann–Whitney U test.CC BY · PubMed Central
Figure 4 miR-17-92 cluster members differentially target the 3′-UTR sequences of voltage-gated potassium channels and modulatory subunits. (a) List of voltage-gated potassium channel subunits predicted as miR-17-92 target genes; these subunits were reportedly downregulated in a previous microarray study of SNL20. Parenthesized targeting miRNAs represent predicted miRNAs not validated by a luciferase assay. (b) Activities of luciferase with voltage-gated potassium channel subunit 3′-UTRs or mutated 3′-UTRs in HEK293 cells co-transfected with control or miR-17-92 cluster member-expressing plasmid vector. Numbers of cell cultures are indicated at the base of each bar. Error bars are s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (Kcna1, P<0.001 for miR-18a and P=0.004 for miR-92a; Kcna4, P<0.001 for miR-19a and P<0.001 for miR-19b; Kcnc4, P<0.001 for miR-19a, P<0.001 for miR-19b and P<0.001 for miR-92a; Kcnd3, P=0.021 for miR-18a; Kcnq5, P=0.008 for miR-19a and P=0.008 for miR-19b; Dpp10, P=0.006 for miR-92a; Scn1b, P<0.001 for miR-19a and P<0.001 for miR-19b) compared with control plasmid, unpaired t-test or Dunnett’s test.
Figure 4 miR-17-92 cluster members differentially target the 3′-UTR sequences of voltage-gated potassium channels and modulatory subunits. (a) List of voltage-gated potassium channel subunits predicted as miR-17-92 target genes; these subunits were reportedly downregulated in a previous microarray study of SNL20. Parenthesized targeting miRNAs represent predicted miRNAs not validated by a luciferase assay. (b) Activities of luciferase with voltage-gated potassium channel subunit 3′-UTRs or mutated 3′-UTRs in HEK293 cells co-transfected with control or miR-17-92 cluster member-expressing plasmid vector. Numbers of cell cultures are indicated at the base of each bar. Error bars are s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (Kcna1, P<0.001 for miR-18a and P=0.004 for miR-92a; Kcna4, P<0.001 for miR-19a and P<0.001 for miR-19b; Kcnc4, P<0.001 for miR-19a, P<0.001 for miR-19b and P<0.001 for miR-92a; Kcnd3, P=0.021 for miR-18a; Kcnq5, P=0.008 for miR-19a and P=0.008 for miR-19b; Dpp10, P=0.006 for miR-92a; Scn1b, P<0.001 for miR-19a and P<0.001 for miR-19b) compared with control plasmid, unpaired t-test or Dunnett’s test.CC BY · PubMed Central
Figure 5 miR-17-92 cluster members differentially inhibit expression of voltage-gated potassium channel subunits in vivo. (a,b) Representative images of L5 DRG stained with toluidine blue before (a) and after (b) laser-captured microdissection. Scale bar, 50 μm. (c) Expression profile of pri-miR-17-92 and voltage-gated potassium channel subunits in a single DRG neuron of indicated cell size; DRG neurons<600 μm2 and >600 μm2 were considered to be small and medium/large neurons, respectively. n.d.; not determined. (d) Expression of voltage-gated potassium channel subunit mRNAs examined in the L5 DRG 7 days after control or miR-17-92 AAV vector administration. Numbers of samples are indicated at the base of each bar. Error bars are s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (KV1.1, P=0.014 for miR-17-92 and P=0.034 for miR-18a; KV1.4, P<0.001 for miR-17-92; KV3.4, P<0.001 for miR-17-92, P=0.047 for miR-19b and P=0.009 for miR-92a; KV4.3, P=0.031 for miR-17-92; KV7.5, P=0.002 for miR-17-92, P=0.048 for miR-19a and P=0.013 for miR-19b; DPP10, P=0.003 for miR-17-92 and P=0.043 for miR-92a; NaVβ1, P=0.002 for miR-17-92 and P=0.030 for miR-19b), Dunnett’s test.
Figure 5 miR-17-92 cluster members differentially inhibit expression of voltage-gated potassium channel subunits in vivo. (a,b) Representative images of L5 DRG stained with toluidine blue before (a) and after (b) laser-captured microdissection. Scale bar, 50 μm. (c) Expression profile of pri-miR-17-92 and voltage-gated potassium channel subunits in a single DRG neuron of indicated cell size; DRG neurons<600 μm2 and >600 μm2 were considered to be small and medium/large neurons, respectively. n.d.; not determined. (d) Expression of voltage-gated potassium channel subunit mRNAs examined in the L5 DRG 7 days after control or miR-17-92 AAV vector administration. Numbers of samples are indicated at the base of each bar. Error bars are s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (KV1.1, P=0.014 for miR-17-92 and P=0.034 for miR-18a; KV1.4, P<0.001 for miR-17-92; KV3.4, P<0.001 for miR-17-92, P=0.047 for miR-19b and P=0.009 for miR-92a; KV4.3, P=0.031 for miR-17-92; KV7.5, P=0.002 for miR-17-92, P=0.048 for miR-19a and P=0.013 for miR-19b; DPP10, P=0.003 for miR-17-92 and P=0.043 for miR-92a; NaVβ1, P=0.002 for miR-17-92 and P=0.030 for miR-19b), Dunnett’s test.CC BY · PubMed Central
Figure 6 miR-17-92 reduces potassium currents in DRG neurons. Potassium currents were recorded in acutely-dissociated DRG neurons 7 days after control or miR-17-92 AAV vector injection. Only EGFP-fluorescent DRG neurons were recorded. (a,b) Representative traces of total, non-A-type and A-type potassium currents elicited by stepwise depolarization from −70 to 60 mV at holding potential of −80 mV in small (a) and medium/large (b) DRG neurons. (c,d) Current density of each potassium current component plotted against voltage in small (c) and medium/large (d) DRG neurons obtained from three rats. Error bars are s.e.m. *P=0.016 and **P=0.003, two-way repeated-measures ANOVA.
Figure 6 miR-17-92 reduces potassium currents in DRG neurons. Potassium currents were recorded in acutely-dissociated DRG neurons 7 days after control or miR-17-92 AAV vector injection. Only EGFP-fluorescent DRG neurons were recorded. (a,b) Representative traces of total, non-A-type and A-type potassium currents elicited by stepwise depolarization from −70 to 60 mV at holding potential of −80 mV in small (a) and medium/large (b) DRG neurons. (c,d) Current density of each potassium current component plotted against voltage in small (c) and medium/large (d) DRG neurons obtained from three rats. Error bars are s.e.m. *P=0.016 and **P=0.003, two-way repeated-measures ANOVA.CC BY · PubMed Central
Figure 7 miR-17-92 inhibition rescues potassium channel expressions and potassium currents in DRG neurons in the neuropathic pain state. (a) Representative images of immunoblotting for voltage-gated potassium channel α subunits in the L5 DRG obtained from SNL rats at day 28. Full blots can be found in the Supplementary Fig. 12. AAV vectors expressing either a control AAV vector or mixture of AAV vectors encoding TuD antisense RNAs against miR-18a, miR-19a, miR-19b and miR-92a were administered 7 days after SNL. GAPDH was used as a loading standard. (b) Expression levels of voltage-gated potassium channel protein, as a percentage of expression level in intact L5 DRG. Numbers of samples are indicated at the base of each bar. *P<0.05 and **P<0.01 (P=0.004 for KV1.1; P=0.038 for KV1.4; P=0.015 for KV3.4; P=0.028 for KV4.3; P=0.003 for KV7.5), Welch’s test. (c,d) Potassium currents were recorded in acutely-dissociated DRG neurons 7 days after SNL. Control AAV vector or AAV vector mixture was injected 7 days before SNL. Only EGFP-fluorescent DRG neurons were recorded. (c) Representative traces of total, non-A-type and A-type potassium currents elicited by stepwise depolarization from −70 to 60 mV at a holding potential of −80 mV in small DRG neurons. (d) Current density of each potassium current component plotted against voltage in small DRG neurons obtained from three rats. Error bars are s.e.m. *P<0.05 (P=0.045 for total current; P=0.046 for non-A-type current; P=0.015 for A-type current), two-way repeated-measures ANOVA.
Figure 7 miR-17-92 inhibition rescues potassium channel expressions and potassium currents in DRG neurons in the neuropathic pain state. (a) Representative images of immunoblotting for voltage-gated potassium channel α subunits in the L5 DRG obtained from SNL rats at day 28. Full blots can be found in the Supplementary Fig. 12. AAV vectors expressing either a control AAV vector or mixture of AAV vectors encoding TuD antisense RNAs against miR-18a, miR-19a, miR-19b and miR-92a were administered 7 days after SNL. GAPDH was used as a loading standard. (b) Expression levels of voltage-gated potassium channel protein, as a percentage of expression level in intact L5 DRG. Numbers of samples are indicated at the base of each bar. *P<0.05 and **P<0.01 (P=0.004 for KV1.1; P=0.038 for KV1.4; P=0.015 for KV3.4; P=0.028 for KV4.3; P=0.003 for KV7.5), Welch’s test. (c,d) Potassium currents were recorded in acutely-dissociated DRG neurons 7 days after SNL. Control AAV vector or AAV vector mixture was injected 7 days before SNL. Only EGFP-fluorescent DRG neurons were recorded. (c) Representative traces of total, non-A-type and A-type potassium currents elicited by stepwise depolarization from −70 to 60 mV at a holding potential of −80 mV in small DRG neurons. (d) Current density of each potassium current component plotted against voltage in small DRG neurons obtained from three rats. Error bars are s.e.m. *P<0.05 (P=0.045 for total current; P=0.046 for non-A-type current; P=0.015 for A-type current), two-way repeated-measures ANOVA.CC BY · PubMed Central
Figure 8 Combined administration of potassium channel modulators potently alleviated mechanical allodynia induced by miR-17-92 or SNL. Paw withdrawal responses to mechanical stimuli were evaluated on the SNL (a) and AAV-injected (b) sides and the contralateral sides (n=4). NS-5806, flupirtine or both were administered intraperitoneally 7 days after SNL; the same volume of vehicle was injected as a control. Rats were examined before SNL or AAV injection (BL), and before and after drug administration. Error bars are s.e.m. *P<0.05 compared with vehicle, Steel test.
Figure 8 Combined administration of potassium channel modulators potently alleviated mechanical allodynia induced by miR-17-92 or SNL. Paw withdrawal responses to mechanical stimuli were evaluated on the SNL (a) and AAV-injected (b) sides and the contralateral sides (n=4). NS-5806, flupirtine or both were administered intraperitoneally 7 days after SNL; the same volume of vehicle was injected as a control. Rats were examined before SNL or AAV injection (BL), and before and after drug administration. Error bars are s.e.m. *P<0.05 compared with vehicle, Steel test.CC BY · PubMed Central

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Authors
Sakai, A., Saitow, F., Maruyama, M., Miyake, N., Miyake, K., Shimada, T., Okada, T., & Suzuki, H.
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-07-05
Volume
8
Issue
1
ISSN
2041-1723
DOI
10.1038/ncomms16079
PMC
PMC5504285
Cited by
108
References
55
Language
en
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