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Functional analysis of miR-23−27−24 cluster miRNAs in skeletal muscle plasticity

李, 玟姃 東京大学 DOI:10.15083/0002002346

2021.10.13

概要

[背景と目的]
骨格筋はヒト生体内で最も大きい器官であり,身体の構造維持や動きを生み出すのみならずエネルギー代謝にも重要であることが知られている.骨格筋は様々な外部刺激に応答し,その機能や量を変化させる高い可塑性を示す.

骨格筋は収縮・代謝特性の異なるいくつかの筋線維によって構成されている.それぞれの筋線維に発現するミオシン重鎖(MyHC)により収縮特性が決定され,MyHC I は収縮速度が遅い遅筋型で,ミトコンドリアの量が多く酸化的代謝に優れており,筋線維を取り巻く毛細血管が多く,疲労耐性が高い.MyHC IIa は速筋型でありながら疲労耐性が高く, MyHC I と類似した酸化的代謝特性を有する.一方,MyHC IId/x と IIb は速筋型で,ミトコンドリア量や毛細血管が少なく解糖的代謝に優れており,疲労耐性が低い.

持久性運動は,速筋において疲労耐性の高い筋線維を増加させ,骨格筋ミトコンドリアや毛細血管の新生を亢進させることが知られている.これは,継続的な筋収縮活動に対して骨格筋が酸化的代謝能や筋疲労耐性を向上させるために,機能的可塑性を図った結果である.一方,レジスタンス運動のような負荷の高い筋収縮活動は骨格筋を肥大させ,ベッドレストやギブス固定などの筋活動の低下は筋を萎縮させる.このような骨格筋の量的可塑性は骨格筋内のタンパク質の合成と分解のバランスにより調節される.

近年,約 20 塩基長の短いノンコーディング RNA であるマイクロ RNA(miRNA)が遺伝子発現を転写後レベルで制御することが明らかになり,様々な生命現象への関与が報告されてきた.miRNA は,自身の 5’末端側にある 2 から 8 番目の塩基で構成された「Seed」配列により,部分的な相補性を介して標的 mRNA を認識し,その発現を負に調節する.多くの miRNA がバクテリアのオペロンのように,遺伝子クラスターを形成してゲノム上にコードされている.クラスターを形成する miRNA は単一の polycistronic な転写物として転写され,その後のプロセシングを経て成熟型 miRNA として機能する.同じクラスターから産生された miRNA は組織において類似した発現パターンを示し,標的とする遺伝子が類似したシグナル経路に関わっていることから,同じクラスターに属する miRNA は同じ生命現象を協調して制御することが示唆されている.

ヒトゲノム上にコードされている約60%以上の遺伝子がmiRNA の標的であると予測され,miRNA の標的 mRNA への制御は進化的に保存されていることから,その機能的重要性が示唆されている.骨格筋においても,これまでにいくつかの miRNA が骨格筋の発生や可塑性の制御に関与することが報告されている.

ある細胞や組織において高い発現を示すmiRNA はその組織内の遺伝子発現調節に大きな影響を及ぼすと考えられる.miR-23−27−24 クラスターを形成する miR-23,miR-27,miR-24は骨格筋に多く発現する miRNA であり,骨格筋可塑性の制御において中心となるいくつかの分子を標的とすることが報告されている.これらのことから,骨格筋の可塑性制御に miR-23−27−24 クラスターmiRNA が重要な役割を担っている可能性が考えられる.しかし,骨格筋における miR-23−27−24 クラスターmiRNA の機能については十分な検討が行われて いない.

本研究では,筋特異的 miR-23−27−24 クラスター欠損マウスを作製し,このマウスの骨格筋可塑性について検討することで,骨格筋における miR-23−27−24 クラスターmiRNA の機能を明らかにすることを目的とした.

[結果と考察]
筋特異的 miR-23− 27− 24 クラスター欠損マウスの作製
筋特異的に miR-23−27−24 クラスターmiRNA を欠損するマウスを得るために,Cre-loxP システムを利用した.miR-23−27−24 クラスターには 2 種のパラログ(miR-23a−27a−24-2; miR-23a クラスターと miR-23b−27b−24-1; miR-23b クラスター)が存在するため,両クラスター間の代償機能が考えられた.このため,miR-23a−27a−24-2 と miR-23b−27b−24-1 領域の各両端に loxP 配列が挿入された miR-23a クラスターflox(flanked by loxP sequences)マウス及び miR-23b クラスターflox マウスと,Ckmm(Mck; Muscle creatinine kinase promoter)-Creマウスを交配し,筋特異的 miR-23−27−24 クラスター欠損マウス(dKO マウス)を作製した. dKO マウスはメンデルの法則に沿って生まれ,コントロール(Con)マウスと同様な成長を示した.Ckmm-Cre は未分化な筋芽細胞ではなく分化が完了した多核の筋線維でのみで活性化することが知られており,dKO マウスにおいて miR-23a//b クラスターに属する全ての miRNA(miR-23a,miR-23b,miR-27a,miR-27b,miR-24)は単離した筋線維特異的に顕著に減少していた.ところが,Con と dKO マウスのヒラメ筋(遅筋)と足底筋(速筋)では, MyHC やミトコンドリア新生を調節する PGC-1α,ミトコンドリア量に有意な差が認められなかった.dKO マウスにおいて MyHC I と IIa 線維の肥大が認められたが,筋力や筋重量に有意な変化をもたらすことのない微細な変化であった.これらのことから,Ckmm-Cre による筋特異的な miR-23−27−24 クラスターmiRNA の欠損は骨格筋の発生や成長において大きな影響を及ぼさないことが示唆された.

骨格筋の運動適応における miR-23a/b クラスターmiRNA の機能
骨格筋の持久性運動適応におけるmiR-23a/b クラスターmiRNA の機能を検討するために,4 週間の自発走行運動を実施した.その結果,マウスの走行距離,持久性運動による疲労耐性の高い筋線維(主に MyHC IIa 線維)の増加,SDH(Succinate dehydrogenase; コハク酸脱水素酵素)活性の増加,ミトコンドリア新生,毛細血管新生について,Con と dKO マウスの間に有意な差は認められなかった.

miR-23a/b クラスターmiRNA の発現量が運動によって変化することで,Ckmm-cre による miR-23a/b クラスターの欠損を補償し,dKO マウスの骨格筋運動適応に差がなくなった可能性が考えられたため,運動による miR-23a/b クラスターmiRNA の発現量を定量した.運動後の dKO マウスの骨格筋 miR-23a/b クラスターmiRNA の発現量は運動後でも,Con マウスに比べて顕著に低かったため,Ckmm-cre による欠損が補償された可能性は低いと考えられた.これらのことから,筋線維由来の miR-23a/b クラスターmiRNA は運動による骨格筋の収縮・代謝特性の適応に影響しないことが示唆された.

一方,4 週間の自発性走行運動は Con と dKO マウスにおいて心臓と骨格筋の肥大を誘導し,特に dKO マウスにおいて有意にヒラメ筋が肥大した.このことから,miR-23a/b クラスターmiRNA が遅筋の量的可塑性に関与する可能性が示唆された.

骨格筋の萎縮における miR-23a/b クラスターの機能
除神経による筋委縮モデルを用いて,骨格筋の量的可塑性における miR-23a/b クラスター miRNA の機能を検討した.マウスの片脚の坐骨神経を大腿部で切除し(dnv 脚),下腿骨格筋への神経入力を除去することで筋萎縮を誘導し,非手術脚をコントロール(Contralateral, CL 脚)として比較した.除神経から 1 週と 2 週間後の足底筋及びヒラメ筋の重量変化とタンパク合成と分解に関わるシグナル経路の変化を検討した.

除神経から 1 週間後において,Con と dKO マウスの足底筋とヒラメ筋の重量は除神経により有意に減少した.Con マウスに比べ dKO マウスの足底筋とヒラメ筋の重量が有意に大きかったが,筋萎縮率には 2 群間で有意な差を認めなかった.タンパク合成シグナルである Akt と S6K のリン酸化,タンパク分解系では筋特異的ユビキチンリガーゼの Atrogin-1 と MuRF1 の発現量にも 2 群間で有意な差を認めなかった.

除神経から 2 週間後でも,Con と dKO マウスの足底筋とヒラメ筋の重量が除神経により有意に減少した.足底筋重量についてはジェノタイプ間の差は認められなかったが,ヒラメ筋ではdKO マウスの筋重量が有意に大きかった.筋萎縮率に関しても,ヒラメ筋では dKOマウスで筋委縮率が有意に低かった.Atrogin-1 と MuRF1 は Con と dKO マウスとも除神経 2 週間後には検出されなかった.一方,足底筋の Akt と S6K のリン酸化においては有意な差が認められなかったが,ヒラメ筋では dKO マウスにおいて有意な Akt のリン酸化の亢進が認められ,S6K のリン酸化も亢進する傾向を示した(P=0.185).これらのことから, miR-23a/b クラスターmiRNA の欠損がタンパク質合成シグナルを亢進し,除神経による遅筋の萎縮を抑制することが示唆された.

一方,片脚の除神経手術により移動の際の体重負荷が CL 脚に集中するため,CL 脚に通常より高い負荷がかかっている状態となる.前述のように,4 週間の自発走行運動によっても dKO マウスにおいてヒラメ筋特異的に筋肥大の増加が認められたことから,dKO マウスの遅筋ではメカニカルストレス増大による筋肥大に対して感受性が増加している可能性が考えられた.

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参考文献

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