楊俊娜，何麗，徐陶，許阿香，徐瑞，楊帆，曾俊偉

(遵義醫(yī)學(xué)院生理學(xué)教研室 貴州省麻醉與器官功能保護重點實驗室，貴州遵義 563000)

微小RNA (miRNA) 是一類多功能的內(nèi)源性非編碼小分子RNA，長度為18～25個核苷酸序列，與靶mRNA分子的3′非翻譯區(qū)(3′-UTR)不完全性互補結(jié)合，在轉(zhuǎn)錄水平調(diào)控基因表達與蛋白合成，參與細胞增殖、分化、凋亡等生命活動過程[1]。早在2002年，Lagos-Quintana等[2]首次發(fā)現(xiàn)miR-124高表達于小鼠腦組織。miR-124具有三條未成熟的前體序列，分別為miR-124-1、miR-124-2和miR-124-3，分別定位于染色體8p23.1、8q12.3和20q13.33[3]，Dicer酶剪切后成為成熟的miR-124序列，與下游靶基因序列結(jié)合，發(fā)揮生物學(xué)效應(yīng)[4]。在大鼠、小鼠、果蠅以及非洲爪蟾等多物種組織中均有miR-124表達，其基因結(jié)構(gòu)和功能在物種進化的過程中具有高度保守性。使用miRNA分子靶基因預(yù)測數(shù)據(jù)庫miRbase、Target scan和miRDB對人類miR-124靶基因進行預(yù)測并結(jié)合韋恩分析，在不同數(shù)據(jù)庫中共同預(yù)測到256個靶分子，其中NR4A1、SERP1、ROCK1等在腦發(fā)育以及神經(jīng)損傷與修復(fù)中均有非常重要的作用[5～7]。提示miR-124可能與神經(jīng)系統(tǒng)發(fā)育、損傷與修復(fù)密切相關(guān)。近年來，圍繞這一方面的研究取得了一些進展，現(xiàn)綜述如下。

1 miR-124在神經(jīng)系統(tǒng)組織中的表達

在外周神經(jīng)系統(tǒng)，miR-124表達于Corti′s器、嗅神經(jīng)、背根神經(jīng)節(jié)、交感神經(jīng)節(jié)以及視網(wǎng)膜等部位[3,8]；在小鼠中樞神經(jīng)系統(tǒng)中miR-124的表達量是肝、腎、肺、脾等器官的100倍以上。miR-124在人腦組織中表達水平較高，占成年人大腦中總miRNA的25%～48%，在大腦皮層含量最多；與大腦皮質(zhì)相比，miRNA在小腦中所占的比例約為60%，在脊髓的比例約為35%[9]。其次，在海馬、枕葉、顳葉、中腦黑質(zhì)、腦室下區(qū)和脊髓背角等部位miR-124表達亦較高。miR-124在腦區(qū)高水平表達提示其與神經(jīng)系統(tǒng)的發(fā)育密切相關(guān)。形態(tài)學(xué)觀察結(jié)果顯示，小鼠內(nèi)耳神經(jīng)干細胞、前額葉皮質(zhì)神經(jīng)元、皮層星形膠質(zhì)細胞、海馬神經(jīng)元、脊髓的神經(jīng)元和小膠質(zhì)細胞、背根神經(jīng)節(jié)的神經(jīng)元和小鼠中腦小膠質(zhì)細胞表達miR-124[10]。在離體條件下，培養(yǎng)的小鼠交感神經(jīng)元、CX3CR1-GFP小鼠骨髓來源巨噬細胞誘導(dǎo)的小膠質(zhì)細胞，以及肌萎縮側(cè)索硬化小鼠的脊髓和腦干部位的神經(jīng)干細胞中均觀察到miR-124表達[11～13]。在BV-2小膠質(zhì)細胞、SH-SY5Y細胞、U87神經(jīng)膠質(zhì)瘤細胞以及MN9D多巴胺神經(jīng)元等細胞系也觀察到miR-124表達[14]。

2 miR-124在神經(jīng)元發(fā)育過程中的作用

在小鼠胚胎腦發(fā)育過程中，miR-124表達與大腦神經(jīng)元發(fā)育成熟趨勢相一致，在出生后仍然高表達。將秀麗隱桿線蟲和果蠅腦組織的神經(jīng)元和星形膠質(zhì)細胞共培養(yǎng)發(fā)現(xiàn)，miR-124優(yōu)先表達于神經(jīng)元。Baek等[15]研究發(fā)現(xiàn)，將miR-124轉(zhuǎn)染到HeLa細胞之后，可抑制數(shù)百種非神經(jīng)元基因表達。由此初步推測，miR-124可能在神經(jīng)元的發(fā)育成熟階段發(fā)揮作用，對于非神經(jīng)元細胞的生長發(fā)育亦具有一定調(diào)控作用。近年研究表明，miR-124不僅可促進干細胞向神經(jīng)元方向分化，而且對于神經(jīng)元突起的延伸具有促進作用。

2.1 促進干細胞向神經(jīng)元分化 神經(jīng)干細胞(NSCs)具有增殖能力與分化能力，在神經(jīng)系統(tǒng)發(fā)育過程中起著至關(guān)重要的作用[16]。miR-124可作用于以下下游靶點，促進多種干細胞或祖細胞向神經(jīng)元方向分化：①性別決定區(qū)Y框蛋白9(SOX9)： miR-124和sox9共表達于室管膜室下區(qū)(SVZ)干細胞。在成年小鼠的腦室下區(qū)，miR-124過表達可顯著降低轉(zhuǎn)錄子SOX9表達，促進SVZ干細胞向神經(jīng)元發(fā)育，而不是向星形膠質(zhì)細胞發(fā)育[17]。②原肌球蛋白相關(guān)激酶B(TrkB)和細胞分裂周期蛋白42(Cdc42)：在新生C57BL/6小鼠的螺旋神經(jīng)節(jié)，miR-124可促進TrkB和Cdc42表達，促進內(nèi)耳神經(jīng)干細胞向神經(jīng)元分化，神經(jīng)元特異性Ⅲ類β-微管蛋白(Tuj1)的表達在第3天開始升高，在第14天達到頂峰。相反，miR-124表達下調(diào)導(dǎo)致TrkB和Cdc42蛋白表達降低，神經(jīng)元分化減慢，突起生長停滯[18]。③RNA聚合酶Ⅱ的C端結(jié)構(gòu)域多肽的小磷酸酯酶1(SCP1)：在雞脊髓神經(jīng)祖細胞，miR-124與SCP1的3′-UTR端結(jié)合，抑制SCP1表達，導(dǎo)致REST/SCP1信號通路受到抑制，從而解除SCP1抑制神經(jīng)祖細胞向神經(jīng)元分化的效應(yīng)[19]；⑤特異性蛋白1(SP1)：SP1是一種轉(zhuǎn)錄因子，在調(diào)控細胞周期中具有關(guān)鍵作用。脂肪來源的間充質(zhì)干細胞向神經(jīng)元發(fā)育過程中，miR-124能夠與SP1結(jié)合，促進間充質(zhì)干細胞向神經(jīng)元分化[20]；⑥促紅細胞產(chǎn)生肝細胞受體B1(Ephrin B1)：miR-124能夠通過與Ephrin B1結(jié)合，降低Ephrin B1表達，促進神經(jīng)干細胞向神經(jīng)元分化[21]；相反，在Ephrin B1基因敲除小鼠神經(jīng)干細胞向神經(jīng)元分化增加。

在研究神經(jīng)發(fā)育的分子機制時發(fā)現(xiàn)，miR-124過表達可作用于M17細胞的下游靶點Rho相關(guān)卷曲螺旋形成蛋白激酶1(ROCK1)，顯著下調(diào)ROCK1表達，減弱其對PI3K/Akt信號通路的抑制作用，促進M17細胞向神經(jīng)元分化[22]；而在P19細胞中，miR-124下游靶點有多聚嘧啶序列結(jié)合蛋白1(PTBP1)和Ⅱa類組蛋白去乙?；?HDACs)。miR-124可下調(diào)PTBP1和HDACs表達，促進PTBP2以及轉(zhuǎn)錄因子MEF2C表達，靶向M6a轉(zhuǎn)錄起始位點，促進M6a表達，進而促進P19細胞向神經(jīng)元分化[23]。

2.2 促進神經(jīng)元突起延伸 神經(jīng)元的突起不僅能夠?qū)w發(fā)出的沖動通過突起傳遞給另外一個神經(jīng)元，而且也是神經(jīng)元接受信號的重要入口。在神經(jīng)系統(tǒng)發(fā)育過程中，miR-124可能是通過作用于下游靶點促進了神經(jīng)元突起的延伸：①HDAC5和Ras家族同源性生長相關(guān)基因(RhoG)：HDAC5可抑制C57/BL小鼠皮層神經(jīng)元突觸的生長和延伸；而miR-124能下調(diào)HDAC5表達，促進神經(jīng)元突觸和軸突生長[24]。在大鼠海馬神經(jīng)元，RhoG活性增強，可通過ELMO/Dock180/Rac1通路抑制突觸形成與突起延伸；miR-124與RhoG的3′-UTR結(jié)合，下調(diào)RhoG表達，促進突觸形成與突起延伸[25]。②Ras家族相關(guān)蛋白Rap2a：Rap2a能夠顯著抑制神經(jīng)元樹突形成。小鼠大腦神經(jīng)元中，miR-124結(jié)合Rap2a的3′-UTR來抑制Rap2a表達，負性調(diào)節(jié)AKT/GSK3β信號，解除Rap2a對神經(jīng)元突起生長的抑制效應(yīng)，從而促進神經(jīng)元突起延伸[26]。③氧甾醇結(jié)合蛋白(OSBP)：OSBP的3′-UTR存在4個保守的miR-124靶基因位點。在C57BL/6小鼠腦發(fā)育過程中，miR-124高表達引起OSBP表達下降。在N2a神經(jīng)瘤母細胞，miR-124通過與OSBP結(jié)合，降低其表達，可促進N2a細胞中神經(jīng)突觸的生長和延伸[27]。④cAMP反應(yīng)元件結(jié)合蛋白1 (CREB1)：海兔感覺神經(jīng)元和運動神經(jīng)元共培養(yǎng)，miR-124與CREB1的3′-UTR相結(jié)合，抑制CREB1 表達；miR-124表達降低時CREB1表達上調(diào)，突觸可塑性增強[28,29]。

2.3 參與膠質(zhì)細胞發(fā)育成熟 神經(jīng)膠質(zhì)細胞包括星形膠質(zhì)細胞、少突膠質(zhì)細胞、施萬細胞和衛(wèi)星細胞。神經(jīng)膠質(zhì)細胞調(diào)節(jié)突觸的生長和興奮性，對神經(jīng)系統(tǒng)發(fā)育有重要影響。放射狀膠質(zhì)細胞轉(zhuǎn)化為星形膠質(zhì)細胞是皮層發(fā)育過程中的一個重要事件，而miR-124在此過程中發(fā)揮重要作用。miR-124可與Jagged2(Jag2，Notch1的配體)結(jié)合，使Jag2表達顯著下調(diào)，進而抑制Notch1信號通路，促進皮層放射狀膠質(zhì)細胞向星形膠質(zhì)細胞的轉(zhuǎn)化[30]。敲除Dicer1基因小鼠大腦皮層Jag2表達增強，導(dǎo)致放射狀膠質(zhì)細胞轉(zhuǎn)化為星形膠質(zhì)細胞出現(xiàn)延遲，大腦皮層中易位的放射狀膠質(zhì)細胞數(shù)目明顯減少。此外，miR-124在少突膠質(zhì)細胞的發(fā)育以及髓鞘形成過程中也發(fā)揮了重要作用。原位雜交技術(shù)觀察顯示，miR-124通過抑制其靶基因rabgef1表達，導(dǎo)致包裹在軸突外側(cè)的少突膠質(zhì)細胞數(shù)目減少。斑馬魚胚胎注射嗎啉后腹側(cè)后腦miR-124表達降低，神經(jīng)元軸突的發(fā)育不良，尾巴形態(tài)異常彎曲[31]。

3 miR-124在神經(jīng)損傷與修復(fù)過程中的作用

研究發(fā)現(xiàn)，小鼠大腦皮層撞擊損傷3天后，損傷區(qū)域小膠質(zhì)細胞顯著活化，miR-124表達同步增加；將損傷后小鼠腦組織提取物與BV2細胞共培養(yǎng)，發(fā)現(xiàn)miR-124可促進BV2細胞向M2型極化，從而抑制神經(jīng)炎癥發(fā)生。隨后研究證實，miR-124作用于靶基因PDE4B，抑制PDE4B和后續(xù)的mTOR信號通路，導(dǎo)致促炎性細胞因子IL-1β、IL-6和TNF-α下調(diào)，而抗炎因子IL-10上調(diào)[32]。Yu等[33]檢測到腦出血小鼠腦損傷區(qū)域miR-124表達顯著降低；腦室注射miR-124模擬物后，miR-124模擬物作用于下游靶點C/EBP-α，抑制C/EBP-α表達，導(dǎo)致促炎性細胞因子IL-1β和TNF-α表達顯著下降，而抗炎因子IL-10表達上調(diào)，細胞凋亡減少，M2型小膠質(zhì)細胞標(biāo)記物Arg-1表達也顯著上調(diào)。證實miR-124能促進小膠質(zhì)細胞向M2型極化，發(fā)揮抗炎作用，減輕神經(jīng)系統(tǒng)損傷。

C57BL/6小鼠脊髓灰質(zhì)損傷后周圍神經(jīng)元中miR-124表達逐漸降低，損傷7天時miR-124表達為未損傷時的1/6[34]。Louw等[35]在離體實驗和在體實驗均觀察到，大鼠脊髓小膠質(zhì)細胞miR-124過表達可降低骨髓來源的小膠質(zhì)細胞MHC-Ⅱ表達，并可抑制TNF-α以及ROS產(chǎn)生。Song等[36]研究發(fā)現(xiàn)，miR-124a可抑制脊髓損傷大鼠吡哆醛激酶的活性，導(dǎo)致磷酸吡哆醛代謝紊亂，進而引起氨基酸代謝紊亂以及神經(jīng)系統(tǒng)繼發(fā)性損傷。這些研究說明，miR-124不僅能夠特異性反映脊髓的損傷程度，且有可能成為藥物治療促進神經(jīng)損傷后修復(fù)的作用靶點之一。Zhao等[34]將小鼠BMMSCs誘導(dǎo)分化成神經(jīng)元細胞并過表達miR-124后移置入損傷脊髓的小鼠體內(nèi)，小鼠脊髓損傷癥狀得到顯著緩解；轉(zhuǎn)染miR-124的大鼠NSCs經(jīng)過靜脈注射進入脊髓損傷大鼠體內(nèi)，同樣能促進NSCs分化成神經(jīng)元和星形膠質(zhì)細胞，促進脊髓損傷后的恢復(fù)。

綜上所述，miR-124作為一個重要的、多效能的miRNA，廣泛表達于外周和中樞神經(jīng)系統(tǒng)，不僅可促進神經(jīng)系統(tǒng)的發(fā)育成熟，而且參與神經(jīng)損傷后的修復(fù)。miR-124在神經(jīng)系統(tǒng)中表達下降往往是神經(jīng)系統(tǒng)退行性變以及膠質(zhì)瘤病變的一個重要環(huán)節(jié)。miR-124相關(guān)的信號通路機制雖已被初步被闡明或證實，但可能也有許多潛在的機制還沒被發(fā)現(xiàn)。對此進行深入研究將使miR-124在神經(jīng)系統(tǒng)的發(fā)育或病變過程中的作用更加明確，也可為神經(jīng)系統(tǒng)疾病的治療提供新思路。在神經(jīng)系統(tǒng)發(fā)育及損傷修復(fù)過程中，miR-124表達呈動態(tài)變化，提示miR-124表達可反映神經(jīng)發(fā)育或損傷或病變的程度，可能成為藥物治療的作用靶點之一。血清miR-124表達與神經(jīng)系統(tǒng)發(fā)育或疾病關(guān)系的研究可能在未來受到重視。

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