循環miRNA生物標記物的運動生理研究進展

陳淑婷,晁天樂,劉文萍

循環miRNA生物標記物的運動生理研究進展

陳淑婷1,晁天樂2,劉文萍3

1. 山東農業大學體育學院, 山東 泰安 271018 2. 山東農業大學動物科技學院, 山東 泰安 271018 3. 泰安市精神病醫院, 山東 泰安 271018

運動是維持健康和預防疾病的最佳方法之一，而miRNA被認為是運動后體增益相關分子機制的關鍵調控因子。循環miRNA因其穩定性、分布廣泛性、以及易檢測性，特別有望成為未來的運動相關非侵入性生物標記物。基于循環miRNA的優點，構建比目前更易用、更精準的運動效果、運動能力評估方法，將積極影響當前的運動評估方法，并更好地監測運動反應。這篇綜述總結了循環miRNA的當前知識，與運動有關的循環miRNA的最新研究進展，并論述了它們作為運動相關生物標記物的作用和前景。

循環miRNA; 運動生理; 生物標記物

生物標記物是對正常生物學過程、致病過程或對治療干預的藥理反應的客觀測量和評估指標，可以提供生物體當前生理狀態的準確信息[1]。臨床表型測定、代謝產物和蛋白質是生物標記物主要來源[2]。近年來，循環miRNA越來越多的被推薦作為診斷和預后不同生理、病理狀態的生物標記物，如糖尿病[3]，肌肉損傷[4]和癌癥[5,6]。

miRNA是短序列的非編碼調控RNA，主要通過轉錄后負調控發揮功能，在廣泛的細胞過程中起重要調節作用。運動訓練有助于維持和改善健康，是許多疾病的有效非藥物治療方法[7]。早在2007年[8]和2011年[9]就有報道，miRNA能響應運動訓練發生變化。從那時起，關于運動與miRNA之間關聯的研究不斷開展。這些研究表明，miRNA參與肌肉的發育、恢復和損傷，并與有氧運動能力直接相關。短期或長期運動會大幅改變肌肉和循環系統中miRNA的表達量。其中，循環系統miRNA因其分布特點和穩定性，成為了運動相關非侵入性生物標記物的候選分子[10,11]。但是，由于運動的復雜性及干擾因素的多樣性，至今對于循環miRNA與運動間的具體關系所知甚少。此外，由于miRNA作用多樣，很難理解不同生理狀態下miRNA表達譜變化的實際意義。不同研究間的miRNA檢測結果往往存在矛盾，這可能是研究的試驗模型、運動條件、樣品來源或分析方法差異等因素造成的。盡管如此，仍有跡象表明miRNA與特定運動類型能夠對應，這增強了miRNA可用作運動相關生物標記物的可能性。本綜述旨在總結運動相關循環miRNA的最新研究進展，探討循環miRNA作為運動相關生物標記物的作用和面臨的問題。

1 運動與miRNA

1.1 運動與健康

眾所周知，堅持運動對維持心血管健康，提高認知和免疫功能，改善能量代謝均起到極大積極作用，也是改善生活質量的最好且最有效的方法之一[12]。定期運動可降低50％心血管疾病患病風險、29％癌癥死亡率，以及52％的全因死亡率[13,14]。長期參與體育運動不但可改善生活質量，還能降低抑郁癥和腎臟疾病風險[15-17]。由于體育運動能大幅提高人體健康，堅持運動還等于節省了醫療保健開支[18]。根據類型、強度和頻率不同，運動可引發身體的急性或慢性變化，有氧/阻力運動、急性/慢性運動引起的生理變化各不相同[19]。在肌肉纖維中，運動通過機械刺激和代謝激活等途徑調節蛋白編碼基因和非編碼基因的表達，其中包括miRNA[2,20]。

1.2 miRNA的生物學功能

當前在人類基因組中，有2600多條miRNA獲得正式命名。MicroRNAs是小分子的內源性非編碼RNA，廣泛參與基因轉錄后調控[21]。編碼miRNA的基因通過RNA聚合酶II或III激活，轉錄到細胞核中，并通過一系列復雜生物學發生過程形成[22]。

在基因表達轉錄后調控中，miRNA能夠通過靶向mRNA的3'-非翻譯區或其他區域發生針對性結合[23]。如果靶標區域完全互補，則蛋白Ago2可以裂解靶標mRNA分子，致其降解；在部分靶向互補情況下，相互作用往往為翻譯抑制[24]。因此，miRNA實際作用為引發靶標mRNA降解或翻譯抑制，或兩種過程組合發生[25]。

1.3 miRNA與運動

在已命名miRNA中，很大一部分被報道能通過多種生物學途徑對各種細胞過程（包括發育、增殖和凋亡）起調節作用[25,26]。當前，越來越多的miRNA被發現參與肌肉發育調控，同時這些miRNA的表達受運動的調節。如miR-1，miR-133和miR-206三種miRNA在人體肌肉組織中高表達，且其表達受到運動的影響[27]。

在運動生理活動中，miRNA能夠以組織特異性或時間特異性模式釋放到體循環中，而機體中普遍的免疫反應以及活動中骨骼肌和心肌組織的適應性調節進一步影響了這種反應[28]。近年來，有多項研究指出，體循環中miRNA表達譜的改變可以作為某些生理適應或病理變化的分子標記[29-31]。

2 循環miRNA作為生物標記物的有利特征

2.1 循環miRNA的生成途徑

近年來發現，在血清、血漿、唾液、尿液、乳汁中穩定存在大量miRNA[32-35]，這些miRNA被稱作循環miRNA（circulating miRNA，ci-miRNA）。根據來源，可將循環miRNA分為三類：（一）遭受損傷、慢性炎癥、細胞凋亡的細胞、或半衰期短的細胞（如血小板）發生被動泄漏產生的循環miRNA；（二）通過細胞衍生膜囊泡（如微粒，外泌體，脫落的囊泡和凋亡小體）主動分泌的循環miRNA；（三）通過蛋白質-miRNA復合物主動分泌產生的循環miRNA[31,36,37]。以上三種途徑產生的miRNA均可進入循環系統或體液，并在運輸至受體細胞位置后被吸收，吸收方式可以為內吞、膜融合或者配體-受體結合等多種方式[36]。

2.2 循環miRNA的穩定性

循環miRNA穩定性較高，其原因是：（1）部分循環miRNA被囊泡包裹，如外泌體或凋亡小體，避免了與核糖核酸酶的接觸[38]；（2）miRNA本身分子較小及特殊結構帶來抗降解能力；（3）miRNA與蛋白質、核磷脂或高密度脂蛋白形成復合物提高了自身抗降解能力[38-40]。目前認為miRNA穩定性主要源自其對核糖核酸酶作用的抗性[41-43]，這種穩定性意味著miRNA在各種體液中具備較長壽命，因此有成為標記物的潛力[31,40,44]。此外，由于循環miRNA可通過血液或其他體液采集，使得其在針對活體、尤其是人體的研究中應用更為簡便，這也表明它們是生物標記物的理想來源[45]。內源性循環miRNA對嚴酷脅迫（如高溫，反復凍融循環）、pH值過低或過高，和長存儲時間等條件均有高抗性[40,46,47]，但游離循環miRNA相對穩定性弱于囊泡和復合體循環miRNA[30]。此外，已進行穩定性測試的miRNA數量相當有限，是否所有miRNA均能在循環狀態下保持高穩定性仍為未知[48]。綜上而言，至少一部分循環miRNA已被發現具備較好的穩定性，符合生物標記物基本要求。

2.3 循環miRNA的易采集性

循環miRNA能夠通過無創或微創方法進行樣品收集[49-51]，這一特征使循環miRNA具備以下優點：（1）避免了其他標記物樣品可能涉及的侵入性手術；（2）為某些不能采集組織的重要器官（如心肌），提供了信息采集的可能；（3）避免了目標組織太小或難以取得帶來的采樣限制[31,52]。

循環miRNA的便捷性使其能夠替代部分目前繁瑣昂貴的檢測方法，如X射線、計算機斷層掃描（CT）、正電子發射斷層掃描（PET）-CT、磁共振成像、超聲檢查等[31,53]。而根據目前病理學方面的研究結果，循環miRNA作為生物標記物的特異性和靈敏度相對較高。例如，循環miRNA表達量變化能夠區分乍看相似，卻需要不同治療方案的疾病[31,53]。

如上所述，miRNA存在于幾乎所有體液，完全滿足生物標記物的相對可及性和最小侵入性原則。然而，與體組織相比，循環系統中的miRNA整體表達量偏低，因此循環miRNA的實際應用仍有許多問題需要注意[54]。

3 與運動相關的循環miRNA

在運動引起的轉錄后水平分子調控過程中，循環miRNA被認為是重要的調控因子。多種miRNA已被確認在肌肉或心臟中特異性表達，可參與調節肌肉生長發育、代謝適應、肌肉損傷修復等生物學過程。健康狀態下，這些miRNA在循環系統中表達量極低。因此，可通過檢測表達量來將它們用作生物標記以測試特定運動方案效果或及早發現被測者對高運動量的異常反應（如心肌梗塞）[8,55,56]。

根據被測者、鍛煉類型、持續時間和強度的不同，發生上調和下調的循環miRNA也會發生相應的變化[10,57]。例如，在急性耐力訓練后，血清中miR-21、miR-221、miR-222和miR-146a的表達水平被發現瞬時升高，進一步分析發現它們主要與炎癥、缺氧、血管生成和肌肉分化等生物學途徑有關[9]。而其它研究中，在相同類型的鍛煉后，miR-146a和miR-221的血清表達水平卻非常低[58,59]。

miR-486在骨骼肌和心肌組織高表達，以70％最大攝氧量進行60 min穩態自行車運動后，發現miR-486表達量顯著下降，且在24 h內恢復到基線水平。該研究也發現循環miR-486的變化率與最大攝氧量顯著相關，表明循環系統miR-486表達量可能是有氧運動能力的生物標記物[58]。

循環系統中miR-1、miR-133a、miR-206、miR-208a和miR-499被發現在馬拉松比賽后顯著增加，且這些miRNA被證明與肌肉分化增殖有關。有觀點認為，這種循環miRNA表達量增加是由于組織破壞和細胞凋亡引起的，或是由于骨骼肌細胞持續收縮，導致細胞內miRNA代謝和分泌增強引發的[60;61]。然而在同一項研究中，65％最大攝氧量下運動1 h未能影響循環miRNA表達水平，但這項研究還是發現循環miR-1，miR-133a，miR-206與運動員的有氧運動能力之間的相關性，并推斷出它們作為有氧運動能力生物標記物的價值[60]。

近幾年，循環miRNA在運動生理中的實際功能逐漸揭示。大鼠和人類志愿者運動試驗表明，外泌體循環miRNA miR-342-5p是重要的心肌保護分子。miR-342-5p被發現能夠通過靶向Caspase 9和Jnk2抑制缺氧、復氧誘導的心肌細胞凋亡，還通過靶向磷酸酶基因Ppm1f增強了細胞生存率，并在人體內源性心肌保護機制中起關鍵作用[62]。總的來看，循環miRNA在運動生理中確有實際調控作用，具有很高的生物標記物價值，但仍有待更加廣泛和徹底的研究理解。

4 循環miRNA實際應用中的問題

4.1 試驗設計

在運動生理學和運動醫學中，試驗設計對影響因素的錯估或忽視往往是造成結果謬誤的主要原因，而循環miRNA的研究應用也必須注意這一問題[63-65]。運動量的差異、被測者運動能力、被測者運動適應性、運動前或運動中飲食飲水等因素均可能影響分析結果[66,67]。

血樣采集本身也是引入變異的原因之一。采集血樣時的肌肉狀態與多種物質含量相關[68]。采集血樣時，使用止血帶輔助采集會引起靜脈淤滯，進而導致穿刺部位血液分析物濃度增加[69]。此外，其他常見誤差源有：樣品基質、抽血與樣品處理之間的時間、樣品存儲時間、樣品存儲溫度等[70-74]。在多因素影響的外界環境下和機體內不穩定的生化環境下，循環miRNA的準確測量頗為困難[30]。

目前，在不同類型循環miRNA中，最常用的當屬血清miRNA。但采血、運輸和存儲過程可能污染樣品[75]，采血針頭中的少量組織細胞、或血樣溶血都可導致血清miRNA污染[75,76]。miRNA可以在凝血過程中由血小板釋放，也可從溶血性紅細胞中釋放出來[76,77]。在后一種情況下，使用miR-23a與miR-451的ΔCT比可用于檢查微溶血事件（如果為N 7則為溶血），進而判斷樣品污染程度[78]。

4.2 樣品基質與處理方式的選擇

在開發新型生物標記物時，正確的樣品基質至關重要，其對最終研究結果影響極大[79,80]。有報道稱相同物種個體之間血清miRNA表達模式穩定，測定結果可重復且一致性較好[32]，其他研究證明血漿來源miRNA也有類似性質[33]。

部分抗凝劑對血樣中miRNA濃度能夠造成影響，因此應慎重選擇。肝素和檸檬酸均為常見血液抗凝劑，但它們均能干擾PCR反應酶活性，因此在miRNA的qPCR定量中，不建議將這兩種抗凝劑處理過的血樣與其它類型樣品混用[81,82]。與肝素不同，EDTA可從PCR反應混合物中去除，因此被認為是基于PCR的miRNA分析樣品最佳抗凝劑[83]。NaF/KOx抗凝血漿常被認為是EDTA的合適替代品，但可能影響miRNA檢測結果[84]。

血樣的離心時間和離心速度也會嚴重影響EDTA血漿樣品中的最終miRNA濃度，因為它可能會導致血小板衍生的miRNA污染[48]。血清miRNA則對這種預處理變異不太敏感[85]。

4.3 循環miRNA檢測方法的選擇

目前，測量循環miRNA的主要方法是qRT-PCR、芯片檢測和二代測序技術（NGS）。

qRT-PCR技術可應用于各種不同類型RNA分子的定量檢測與比較，在循環miRNA檢測中始終起到重要的作用。miRNA的qRT-PCR檢測主要有莖環引物法和多聚腺苷酸尾PCR法[86]。qRT-PCR技術特異性好、檢測快、成本低，但亦有試驗操作繁瑣、單次檢測miRNA數量少等劣勢。

芯片檢測也稱微陣列，是檢測核酸突變和RNA表達量的常用技術。核酸芯片基于qPCR或雜交技術，通過探針一次測定大量靶RNA的表達量或堿基突變信息[5,87]。芯片的局限性包括酶標記中的序列特異性標記偏倚，缺乏絕對定量能力以及相對較低的動態范圍。此外，miRNA芯片的另一缺陷是對探針設計的依賴性，無法用于檢測和發現新miRNA[5]。

二代測序技術具有很高的動態檢測范圍、靈敏度和可重復性，在擴大檢測范圍，提高結果準確性方面均具有優勢。該技術既能檢測已知miRNA，也可以鑒定新型miRNA。二代測序的局限性主要涉及技術問題，如酶促連接導致的序列特異性偏倚。值得注意的是，盡管二代測序技術成本快速下降，miRNA測序的價格卻始終變化不大，因此循環miRNA的檢測仍需注意多方法結合使用。

5 結語

近年來，循環miRNA作為運動相關生物標記物的潛力越來越重要。通過檢測各種體液中大量存在的循環miRNA，可實現對各種類型運動適應癥狀或運動能力的快速無創或微創檢測分析。此外，低成本、易檢測使循環miRNA成為運動相關分子機制研究中的潛在生物標記。當前，循環miRNA廣泛應用的主要障礙是難以保證樣本質量和完整性。盡管目前仍有方法學上的問題存在，但只要正確使用試劑、選擇適合的RNA提取方法、以及避免樣本污染，循環miRNAs仍可為運動生理學的檢測、診斷和分析提供準確的信息。總的來說，循環miRNAs作為運動相關生物標記物的臨床應用潛力極大，但還需通過更多研究來進一步驗證。

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Research Progress of Circulating miRNA as Biomarker in Exercise Physiology

CHEN Shu-ting1, CHAO Tian-le2, LIU WEN-ping3

1.271018,2.271018,3.271000,

Exercise is one of the best ways to maintain health and prevent disease, and miRNA is considered to be a key regulatory factor related to exercise benefits. Because of its stability, wide distribution and easy detection, circulating miRNA is expected to become a non-invasive exercise related biomarker in the future. Based on the advantages of circulating miRNA, the construction of a more easy-to-use and more accurate method for the evaluation of motor effect and motor ability will positively affect the current method of motor evaluation and better monitor the exercise response. This review summarizes the current knowledge of circulating miRNAs, the latest research progress of miRNAs related to exercise, and discusses their functions and prospects as biomarkers related to exercise.

Circulating miRNA; exercise physiology; biomarker

G804.23

A

1000-2324(2022)01-0163-08

10.3969/j.issn.1000-2324.2022.01.025

2021-02-10

2021-03-21

陳淑婷(1975-),女,碩士,副教授,長期從事高校體育教育與運動生理學研究. E-mail:wfdzz@sdau.edu.cn