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. 2021 Aug 20:12:697139.
doi: 10.3389/fphys.2021.697139. eCollection 2021.

Downregulation of let-7 by Electrical Acupuncture Increases Protein Synthesis in Mice

Ying Huang  1   2 , Manshu Yu  1   3 , Akihiro Kuma  1   4 , Janet D Klein  1 , Yanhua Wang  1 , Faten Hassounah  1 , Hui Cai  1   5 , Xiaonan H Wang  1
Affiliations

Downregulation of let-7 by Electrical Acupuncture Increases Protein Synthesis in Mice

Ying Huang et al. Front Physiol. .

Abstract

Background: Our previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) prevents muscle atrophy by attenuation of protein degradation in mice. The current study examines the impact of Acu/LFES on protein synthesis.

Method: C57/BL6 mice received Acu/LFES treatment on hindlimb for 30 min once. Acu/LFES points were selected by WHO Standard Acupuncture Nomenclature and electric stimulation applied using an SDZ-II Electronic acupuncture instrument. Muscle protein synthesis was measured by the surface-sensing of translation (SUnSET) assay. Exosomes were isolated using serial centrifugation and concentration and size of the collected exosomes were measured using a NanoSight instrument. The mature microRNA library in serum exosomes was validated using a High Sensitivity DNA chip.

Results: Protein synthesis was enhanced in the both hindlimb and forelimb muscles. Blocking exosome secretion with GW4869 decreased the Acu/LFES-induced increases in protein synthesis. MicroRNA-deep sequencing demonstrated that four members of the Let-7 miRNA family were significantly decreased in serum exosomes. Real time qPCR further verified Acu/LFES-mediated decreases of let-7c-5p in serum exosomes and skeletal muscles. In cultured C2C12 myotubes, inhibition of let-7c not only increased protein synthesis, but also enhanced protein abundance of Igf1 and Igf1 receptors. Using a luciferase reporter assay, we demonstrated that let-7 directly inhibits Igf1.

Conclusion: Acu/LFES on hindlimb decreases let-7-5p leading to upregulation of the Igf1 signaling and increasing protein synthesis in both hindlimb and forelimb skeletal muscles. This provides a new understanding of how the electrical acupuncture treatment can positively influence muscle health.

Keywords: Acu/LFES; IGF-1 signaling; exosome; mTOR; microRNA; skeletal muscle.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Acu/LFES in hindlimb increased protein synthesis in both gastrocnemius and triceps brachii muscles. Experiments were performed in the sham and Acu/LFES treated mice. Puromycin was injected 30 min before harvest. Protein was isolated from the muscle of mice immediately (0), and 6–, 24–, 48– and 72-h after Acu/LFES. Puromycin in tissue lysates of gastrocnemius muscle (A) and triceps brachii muscle (C) was measured by Western blots. The point graphs show the change in incorporated puromycin normalized to their corresponding GAPDH protein (n = 6/group; *p < 0.05 vs. sham). The proteins mTORC1, 4EBP1 and P70S6 were measured by Western blotting in gastrocnemius muscle (B) and triceps brachii muscle (D) of sham and Acu/LFES mice. The bottom point graphs show the ratio of phosphorylated protein to total protein, each normalized to the GAPDH from the same sample. Data is provided in arbitrary units (n = 6/group; *p < 0.05 vs. sham).
FIGURE 2
FIGURE 2
GW4869 limited exosome secretion. Exosomes were isolated from sham, Acu/LFES, GW4869 and Acu/LFES+GW4869 treated mice. The exosome distribution (A), concentration (B), mode size (C) and mean size (D) were measured using a NanoSight instrument (means ± SE; n = 3/group; ∗p < 0.05 vs. sham).
FIGURE 3
FIGURE 3
Blocking exosome secretion limited the Acu/LFES-induced increase in protein synthesis. Experiments were performed in the sham and Acu/LFES treated mice. GW4869 was injected 16 h before Acu/LFES and puromycin was injected 30 min before harvest. Protein was isolated from the muscle of mice immediately (0), 6–, 24, 48 and 72 h after Acu/LFES. Puromycin in tissue lysates was measured by Western blots. The point graph of incorporate puromycin into gastrocnemius muscle (A) and triceps brachii muscle (B) show the change of the density of puromycin protein normalized to their corresponding GAPDH protein (n = 6/group; *p < 0.05 vs. sham). The proteins mTORC1, 4EBP1 and P70S6 were measured by Western blotting in gastrocnemius muscle (C) of sham and Acu/LFES mice. The bottom point graphs show the ration of phosphate protein to total protein normalized to the same sample GAPDH. Data is provided in arbitrary units (n = 6/group; *p < 0.05 vs. sham).
FIGURE 4
FIGURE 4
Acu/LFES decreased let-7 in serum exosome and skeletal muscles. Total RNA was isolated from serum exosomes (A), gastrocnemius muscle (B) and triceps brachii muscles (C) of sham and Acu/LFES treated mice. The expression of let-7c-5p was assayed by real time qPCR. The bar graph shows microRNA from the exosomes of Acu/LFES mice compared with levels in shams (defined as onefold). Results are normalized to miR-103a for serum and U6 for muscle (Bars: mean ± SE.; n = 6/group; *p < 0.05 vs. sham). (D) Protein was isolated from gastrocnemius muscle. Igf1, Igfrα and Igfrβ measured by Western blots. The point graph of protein showed the change of the density of proteins normalized to their corresponding GAPDH protein Data is provided in arbitrary units. (n = 6/group; *p < 0.05 vs. sham).
FIGURE 5
FIGURE 5
Provision of let-7 inhibited Igf-1, Igfrα and Igfrβ in cultured C2C12 myotubes. (A) Total RNA was isolated from C2C12 myotubes transfected with control/mimic, let-7-5p/mimic and let-7/inhibitor. The expression of let-7-5p was assayed by real time qPCR. The bar graph shows let-7-5p from different group compared with levels in control/mimic (defined as onefold). Results are normalized to miR103 (Bars: mean ± SE; n = 4/group; *p < 0.05 vs. control/mimic). (B) Protein was isolated from the C2C12 myotubes transfected with control/mimic, let-7-5p/mimic and let-7/inhibitor. Puromycin was added into cell culture medium (1 μg final concentration) exactly 30 min before harvesting the cells. Puromycin in cell lysates was measured by Western blots. The point graph of incorporate puromycin into myotube shows the change of the density of puromycin protein normalized to their corresponding GAPDH protein (n = 4/group; *p < 0.05 vs. sham). (C) Protein was isolated from the C2C12 myotubes transfected with control/mimic, let-7-5p/mimic and let-7/inhibitor. Igf-1, Igfrα and Igfrβ in myotube lysates was measured by Western blots. The point graph showed the change of the density of each protein normalized to their corresponding GAPDH protein. Data is provided in arbitrary units. (n = 6/group; *p < 0.05 vs. control/mimic). (D) C2C12 cells were transfected with luciferase pMIR–ctrl vector (white bars) or the vector containing the 3′-UTR of Igf1 (pMIR-Igf: black bars). Cells were co-transfected with control mimic (miR-ctrl), let-7c-5p or let-7 inhibitor. Luciferase activity in cells that received the pMIR–ctrl with miR-ctrl was designated as the 100% (far left white bar). The other bars show the response to let-7 expressed as a percent of this control. Triplicate determinations were made in each condition and each experiment was repeated twice; the firefly luciferase (FFL) results were normalized by renilla luciferase (RL) activity. Data: mean ± SE; n = 9; * = p < 0.05 vs. pMIR-ctrl + miR-ctrl.
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