梁文權(quán)，侯豫，趙存友

精神分裂癥相關(guān)單核苷酸多態(tài)性調(diào)控microRNA功能研究進展

梁文權(quán)1，侯豫2，趙存友1

1. 南方醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院醫(yī)學(xué)遺傳學(xué)教研室，廣州 510515 2. 陸軍總醫(yī)院附屬八一兒童醫(yī)院神經(jīng)發(fā)育科，北京 100700

MicroRNA (miRNAs)是一類長約22nt且對基因表達(dá)具有廣泛的調(diào)控作用的非編碼RNA，并在神經(jīng)元增殖、分化和發(fā)育成熟的過程中扮演重要角色。近年來全基因組關(guān)聯(lián)研究(genome-wide association studies, GWAS)發(fā)現(xiàn)了眾多的精神分裂癥相關(guān)單核苷酸多態(tài)性(single nucleotide polymorphisms, SNPs)位點多位于非編碼區(qū)，表明miRNA在精神分裂癥的發(fā)病過程中存在重要作用。本文綜述了精神分裂癥相關(guān)SNP與miRNA可能發(fā)生相互作用的4種機制(SNP位于miRNA基因、SNP位于miRNA宿主基因、SNP位于miRNA種子序列和SNP位于miRNA結(jié)合位點)，為研究miRNA在精神分裂癥發(fā)生發(fā)展中的作用提供參考。

全基因組關(guān)聯(lián)研究；單核苷酸多態(tài)性；微小RNA；精神分裂癥

精神分裂癥是多基因變異引發(fā)的復(fù)雜性遺傳病，也是最嚴(yán)重的精神類疾病之一[1]。該疾病通常在青壯年時期發(fā)病，致殘率高，醫(yī)療資源消耗大，不僅影響患者的勞動能力，而且給其家屬及社會造成嚴(yán)重負(fù)擔(dān)，因此探索行之有效的預(yù)防和治療措施，已經(jīng)成為一項全球性的社會和經(jīng)濟問題。既往的遺傳學(xué)研究發(fā)現(xiàn)了若干與精神分裂癥相關(guān)的基因或位點，尤其近年來的全基因組關(guān)聯(lián)研究(genome-wide association study, GWAS)手段更是發(fā)現(xiàn)了大量與該疾病相關(guān)的單核苷酸多態(tài)性(single nucleotide poly-morphisms, SNPs)。然而，這些遺傳變異只有少數(shù)位于蛋白編碼區(qū)，更多的位于非編碼區(qū)，這使得研究者從基因表達(dá)調(diào)控角度研究疾病相關(guān)非編碼區(qū)的變異成為可能。已有多項報道發(fā)現(xiàn)與精神分裂癥相關(guān)的SNP位于microRNA (miRNA)的靶基因或結(jié)合位點，miRNA通過對上述基因或位點的負(fù)調(diào)控作用介導(dǎo)了精神分裂癥的發(fā)生和發(fā)展。

miRNA是一類長約22 nt的非編碼RNA，通過與靶基因mRNA 3?UTR區(qū)特異位點的結(jié)合使靶基因mRNA發(fā)生降解或翻譯阻遏[2,3]。當(dāng)位于靶基因3?UTR區(qū)的SNP位于miRNA的結(jié)合位點時，這會使miRNA與靶基因的結(jié)合能力發(fā)生改變，從而造成靶基因的表達(dá)或翻譯發(fā)生改變[4]。人類mRNA 3?UTR擁有超過45 000個保守的miRNA結(jié)合位點，約60%的人類蛋白質(zhì)編碼基因擁有至少一個保守的miRNA結(jié)合位點[5], 這些保守位點的堿基變異會影響到miRNA的調(diào)控功能。近年來采用GWAS手段發(fā)現(xiàn)的大量與精神分裂癥相關(guān)的SNPs多位于非編碼區(qū)，為從miRNA角度研究精神分裂癥的發(fā)病機制提供了線索。自2008年第1篇關(guān)于精神分裂癥GWAS分析的文章發(fā)表以來[6]，通過GWAS分析已發(fā)現(xiàn)許多與精神分裂癥相關(guān)的SNP[7~10]。對歐洲人群通過GWAS研究，發(fā)現(xiàn)基因內(nèi)含子區(qū)的SNP rs1625579與精神分裂癥相關(guān)[11]，在漢族人群中也得到了驗證[12]。之后的研究支持為精神分裂癥危險因素[13]，并且基因上的GWAS SNP rs1625579導(dǎo)致的等位基因表達(dá)不均一性[14]。Ning等[15]通過連鎖不平衡分析發(fā)現(xiàn)在中國漢族核心家系中基因座上的SNPs (rs1877670，rs3750192和rs7009708)構(gòu)建的單倍型與精神分裂癥顯著性關(guān)聯(lián)。越來越多的研究發(fā)現(xiàn)miRNA在神經(jīng)發(fā)育過程中調(diào)控基因表達(dá)，表明miRNA的表達(dá)改變可能會導(dǎo)致諸如精神分裂癥等精神疾病的發(fā)生。Perkins等[16]發(fā)現(xiàn)在精神分裂癥病人前額葉皮質(zhì)表達(dá)下降，基因位于22q11.2微缺失上，攜帶該微缺失的病人有1/4被確診為精神分裂癥患 者[17]?；蛐酒芯堪l(fā)現(xiàn)在精神分裂癥患者的尸檢組織中顳上回和背外側(cè)前額葉中miRNAs的表達(dá)水平普遍增加[18]。遭遇童年創(chuàng)傷的精神分裂癥患者血液樣本中顯著下降，該miRNA在產(chǎn)前應(yīng)激的大鼠海馬組織中同樣發(fā)現(xiàn)表達(dá)顯著下降在[19]。研究發(fā)現(xiàn)和或、和對精神分裂癥具有較好的預(yù)測作用[20]。這些證據(jù)表明，精神分裂癥患者的miRNA表達(dá)譜發(fā)生改變是精神分裂癥發(fā)生發(fā)展的一個重要因素。

非編碼miRNA的調(diào)控功能為人們研究GWAS中發(fā)現(xiàn)的眾多非編碼區(qū)SNP的可能調(diào)控機制提供了新的線索。本文將從以下4個方面闡述與精神分裂癥相關(guān)的SNP通過miRNA實現(xiàn)對基因表達(dá)的調(diào)控作用(圖1)：(1) SNP位于miRNA基因：通過影響miRNA的表達(dá)量從而使下游的靶基因的表達(dá)量發(fā)生改變；(2) SNP位于miRNA的宿主基因：通過調(diào)控宿主基因的表達(dá)從而調(diào)控miRNA的表達(dá)量；(3) SNP位于miRNA的種子序列內(nèi)：影響miRNA與mRNA結(jié)合的穩(wěn)定性，使mRNA的穩(wěn)定性或翻譯受阻；(4) SNP位于miRNA靶基因的結(jié)合位點內(nèi)：通過改變miRNA與靶基因的結(jié)合能力從而調(diào)控靶基因的表達(dá)。

1 SNP位于miRNA基因

Ripke等[11]發(fā)現(xiàn)位于下游8 kb的rs1625579與精神分裂癥相關(guān)(=1.6×10–11)。另有研究發(fā)現(xiàn)在北美人群中攜帶TT基因型的精神分裂癥發(fā)病年齡較攜帶G基因的發(fā)病年齡提前，影像學(xué)研究發(fā)現(xiàn)攜帶TT基因型的人群腦白質(zhì)密度減少、海馬體積縮小以及側(cè)腦室增大[14]。研究表明，在腦組織中表達(dá)豐富[21]，與神經(jīng)元的分化與突觸可塑性相關(guān)，rs1625579及其連鎖的SNP (rs1198588、rs2660304和rs2802535)調(diào)控的表達(dá)量。Siegert等[22]研究發(fā)現(xiàn)擁有上述SNPs的minor-allele的神經(jīng)類似細(xì)胞(neuron-like cells)的表達(dá)量上升，的上調(diào)使和的表達(dá)量下調(diào)導(dǎo)致突觸囊泡釋放功能異常[22]。

圖1 SNP通過miRNA實現(xiàn)對基因表達(dá)的調(diào)控

miRNA加工通路首先在細(xì)胞核內(nèi)經(jīng)RNA聚合酶Ⅱ或Ⅲ作用下產(chǎn)生初級miRNA轉(zhuǎn)錄本(pri-miRNA)，之后被Drosha-DGCR8 復(fù)合物裂解形成具有發(fā)夾結(jié)構(gòu)的前體miRNA(pre-miRNA)，并由Exportin-5-Ran-GTP復(fù)合物從細(xì)胞核中輸出。在細(xì)胞質(zhì)中，RNase Dicer和雙鏈RNA結(jié)合蛋白TRBP復(fù)合物共同作用將pre-miRNA發(fā)夾切割到成熟長度。成熟miRNA的功能鏈與Argonaute (Ago2)蛋白一起加載到RNA誘導(dǎo)沉默復(fù)合物(RISC)中，引導(dǎo)RISC到靶向mRNA，進而通過mRNA裂解、翻譯抑制或脫腺苷化沉默靶mRNA。在這一過程中，SNP通過miRNA實現(xiàn)對基因表達(dá)調(diào)控作用的4種主要機制是：(1) SNP位于miRNA基因；(2) SNP位于miRNA宿主基因；(3) SNP位于miRNA種子區(qū)域；(4) SNP位于miRNA結(jié)合位點。根據(jù)參考文獻(xiàn)[27]修改繪制。

Budach等[23]研究發(fā)現(xiàn)SNP位于miRNA的啟動子中，提示SNP基因型的變化將對miRNA的表達(dá)量產(chǎn)生影響[23]。miRNA-eQTL多富集于miRNA的前體、啟動子、增強子和轉(zhuǎn)錄因子結(jié)合區(qū)域，并且與其宿主基因的mRNA的eQTL存在大量交集。目前只有少量幾篇miRNA的eQTL數(shù)據(jù)發(fā)表，包括Parts等[24]發(fā)表的脂肪細(xì)胞的miRNA-eQTL、Siddle等[25]發(fā)表的樹突狀細(xì)胞的miRNA-eQTL和Huan 等[26]發(fā)現(xiàn)在全血中的miRNA-eQTL。上述研究表明位于miRNA的SNP可以調(diào)控miRNA的表達(dá)量，從而影響miRNA的下游調(diào)控通路，而使罹患精神分裂癥的風(fēng)險上升。

2 SNP位于miRNA宿主基因

約40%的哺乳動物miRNA位于其宿主基因的內(nèi)含子區(qū)[28]，有證據(jù)表明位于宿主基因內(nèi)含子區(qū)的miRNA的成熟并不需要Drosha酶的參與[29]。有研究表明被miRNA沉默的基因與miRNA的宿主基因在功能上具有拮抗作用，而miRNA的表達(dá)量與宿主基因的表達(dá)量呈正相關(guān)[30]；該研究還發(fā)現(xiàn)通過沉默與其宿主基因具有拮抗功能的和從而使對軸突生長的刺激作用增強[30]。另外有研究檢測了和與其宿主基因在海馬神經(jīng)元成熟過程中的動態(tài)表達(dá)變化，發(fā)現(xiàn)mRNA的3?UTR具有的結(jié)合位點，mimics轉(zhuǎn)染大鼠B35成神經(jīng)細(xì)胞瘤細(xì)胞發(fā)現(xiàn)的表達(dá)水平顯著降低，而轉(zhuǎn)染觀察不到該種現(xiàn) 象[31]。亦有研究報道多巴胺受體基因也是靶向基因，的缺失或的下調(diào)可導(dǎo)致22q11.2微缺失小鼠出現(xiàn)精神病性癥狀[32]。利用序列特異性miRNA海綿在發(fā)育期小鼠皮層中阻遏作用發(fā)現(xiàn)神經(jīng)元極性的丟失和皮層內(nèi)神經(jīng)元數(shù)目的減少，相反的，在皮層中過表達(dá)使神經(jīng)元的數(shù)目增加并且呈現(xiàn)多極性[33]。Ferrari等[34]發(fā)現(xiàn)6個SNPs (rs906175、rs2725391、rs969413、rs2659030、rs9319617和rs1048775)的致病基因型能降低的表達(dá)量。上述研究提示，位于miRNA宿主基因的SNP可能影響宿主基因的表達(dá)，從而使miRNA的表達(dá)異常；或者位于宿主基因的SNP影響miRNA的剪切成熟過程，從而使miRNA的表達(dá)異常；又或者位于宿主基因的SNP會使miRNA的轉(zhuǎn)錄調(diào)控機制異常，導(dǎo)致miRNA表達(dá)異常。不管如何，miRNA的表達(dá)異常，都有可能使罹患疾病的風(fēng)險率上升。

3 SNP位于miRNA的種子序列內(nèi)

在成熟miRNA序列中5?端2~8位的堿基序列被稱為miRNA的種子序列，這一種子序列在miRNA與mRNA的識別與互補配對中扮演重要角色[35]。據(jù)統(tǒng)計，超過500個SNPs位于miRNA的種子序列，研究人員發(fā)現(xiàn)位于miRNA種子序列中心位置的SNP比位于種子序列外圍區(qū)域的少，因此miRNA種子序列中心區(qū)域在進化上更保守[36]。研究表明即使是在miRNA種子序列的單個SNP變異也將導(dǎo)致大于50%的該miRNA靶基因頻譜變化[36]。目前還缺乏SNP位于miRNA種子序列導(dǎo)致精神分裂癥的直接證據(jù)，然而已有文章報道了這種SNP在其他疾病中的重要作用。Menc??a等[37]發(fā)現(xiàn)在內(nèi)耳毛細(xì)胞表達(dá)的種子序列的點突變將導(dǎo)致常染色體顯性遺傳的漸進性失聰，種子序列的SNP (+13 G>A)將使下游靶基因、、、和表達(dá)發(fā)生改變。有研究表明在種子序列的SNP (+57C>T)能夠?qū)е翬DICT (endoth-elial dystrophy, congenital cataract, and stromal thinning)綜合征[38]。Jiang等[39]研究發(fā)現(xiàn)有617個SNPs位于牛源的331個成熟miRNAs的種子序列內(nèi)，位于種子序列的SNP (rs109462250)因為結(jié)合位點的改變影響的表達(dá)，通過雙熒光驗證了rs109462250的42198 087 G>A突變使與的結(jié)合能力降低。Chai等[40]發(fā)現(xiàn)位于種子序列的SNP(rs331295049 A17G)使的表達(dá)上調(diào)(miR-378/G)，并且該SNP改變了的二級結(jié)構(gòu)，使其對原有85%靶基因失去調(diào)控作用，但同時又產(chǎn)生了700多個新的靶基因。上述研究表明，位于miRNA種子序列的SNP具有十分重要的生物學(xué)作用，然而現(xiàn)在缺乏SNP位于miRNA種子序列內(nèi)導(dǎo)致精神分裂癥患病風(fēng)險上升的相關(guān)報道。

4 SNP位于miRNA結(jié)合位點內(nèi)

據(jù)報道，大約5%的miRNA識別元件(miR recognition element, MRE)中具有SNP，約3%的miRNA識別元件種子序列((miR recognition element seed sites, MRESS)具有SNP，同時研究表明約38%的MRESS-SNP位于8mer MRESSs[41]。

位于靶基因mRNA 3?UTR區(qū)miRNA結(jié)合位點內(nèi)的SNP將會影響miRNA與mRNA的結(jié)合，從而使miRNA對mRNA的調(diào)控過程受阻。近年來，人們已經(jīng)發(fā)現(xiàn)若干個位于miRNA結(jié)合位點的SNP對精神分裂癥的發(fā)生發(fā)展具有影響。Hou等[42]通過對GWAS精神分裂癥相關(guān)變異位點的檢索發(fā)現(xiàn)，位于基因3?UTR區(qū)rs4702-G可通過增加的結(jié)合能力而參與FURIN的表達(dá)下調(diào)，而FURIN的表達(dá)量降低將導(dǎo)致成熟腦源性神經(jīng)營養(yǎng)因子(brain derived neurotrophic factor，BDNF)的生成減少，后者的降低已經(jīng)報道與精神分裂癥的發(fā)生相關(guān)。Gong等[4]發(fā)現(xiàn)在精神分裂癥患者中rs10759-C將降低miR-124與RGS4 mRNA結(jié)合的最小自由能(minimum free energy, MFE)，從而使與mRNA的結(jié)合更加穩(wěn)定，顯著降低RGS4的表達(dá)量。Rossi等[43]發(fā)現(xiàn)rs11122396位于3? UTR區(qū)，且位于的結(jié)合位點，通過雙熒光素酶報告載體證實了rs11122396對結(jié)合mRNA的影響，且他們認(rèn)為SNP對miRNA的影響是有等位基因差異的，即SNP只對miRNA結(jié)合其中一條染色體轉(zhuǎn)錄的mRNA有影響，對另外一條染色體轉(zhuǎn)錄的mRNA無影響，簡稱等位基因差異性調(diào)控。Hauberg等[23]發(fā)現(xiàn)rs11191548能影響與mRNA的結(jié)合，從而影響NT5C2的表達(dá)，且通過雙熒光素酶報告載體驗證; 值得注意的是是所有保守miRNA中對精神分裂癥易感基因有明顯調(diào)節(jié)作用的miRNA之一[44]。研究證明位于3?-UTR的SNP(rs1060120)將影響對H3F3B的轉(zhuǎn)錄后調(diào)控，并且可能參與的精神分裂癥的發(fā)生[45]。John等[46]在精神分裂癥病人樣本中驗證了35個位于精神分裂癥相關(guān)基因3?-UTR miRNA結(jié)合位點上的SNPs，研究發(fā)現(xiàn)位于3?-UTR的rs7430與精神分裂癥具有顯著相關(guān)性(= 0.01; OR (95%) 1.24 (1.04~1.48))。位于3?-UTR的rs550067317將影響miR-137對EFNB2的調(diào)控能力[47]。上述研究表明位于miRNA結(jié)合區(qū)域的SNP將會使miRNA與mRNA的結(jié)合能發(fā)生變化，從而影響miRNA與RNA的結(jié)合，使miRNA對mRNA的調(diào)控發(fā)生異常。

5 SNP對miRNA調(diào)控過程影響的預(yù)測方法

研究SNP與miRNA的相互作用，可能需要利用生物信息學(xué)方法對miRNA的靶基因以及SNP與miRNA的關(guān)系進行預(yù)測。因為miRNA能調(diào)控數(shù)百個靶基因，所以研究miRNA最大的困難是預(yù)測miRNA的靶基因，現(xiàn)有的miRNA靶基因預(yù)測軟件或網(wǎng)站如表1。

現(xiàn)有的預(yù)測軟件或網(wǎng)站只能從生物信息學(xué)方面預(yù)測位于miRNA結(jié)合位點或種子序列的SNP對miRNA結(jié)合mRNA的影響，具體的功能還需要構(gòu)建熒光報告載體驗證。同樣，人們也可以通過過表達(dá)miRNA結(jié)合ChIP-seq的方式獲得miRNA結(jié)合的mRNA的序列信息，然而其生物學(xué)功能仍需構(gòu)建熒光報告載體和基因敲降實驗進行驗證。還可以通過CRISPR/Cas9獲得不同基因型的細(xì)胞，從而驗證miRNA對位于mRNA 3? UTR的不同基因型SNP的作用或驗證不同基因型對miRNA表達(dá)或剪切成熟過程的影響或位于miRNA種子序列不同基因型SNP對其下游通路的影響。最后可以構(gòu)建轉(zhuǎn)基因動物模型從而驗證不同基因型SNP罹患精神分裂癥的風(fēng)險。

表1 miRNA靶基因預(yù)測軟件或網(wǎng)站

6 結(jié)語與展望

miRNA在大腦中的廣泛性表達(dá)和在精神分裂癥中的表達(dá)紊亂，提示miRNA與精神分裂癥存在相關(guān)性。近年來多篇GWAS的研究結(jié)果為人們解析精神分裂癥的致病機制提供了許多可能的具有生物學(xué)功能的遺傳變異資源，然而在后GWAS時代如何去驗證GWAS報道的SNP卻十分困難，因為雖然有部分SNP位于蛋白質(zhì)編碼區(qū)，然而也有一部分位于非編碼區(qū)，如何去驗證位于非編碼區(qū)的SNP的生物學(xué)功能至今是一個難題。已有研究證明miRNA與GWAS-SNPs存在相互作用，提示位于非編碼區(qū)域的GWAS-SNPs可能在轉(zhuǎn)錄調(diào)控或轉(zhuǎn)錄后調(diào)控的過程中具有重要的生物學(xué)作用，例如位于宿主基因的SNPs可能影響miRNA的表達(dá)從而改變下游靶基因的表達(dá)，位于miRNA基因上的SNPs直接導(dǎo)致miRNA表達(dá)異常參與了精神分裂癥的發(fā)生與發(fā)展，位于種子序列的SNPs可能導(dǎo)致mRNA表達(dá)譜的改變從而導(dǎo)致精神分裂癥的發(fā)生，位于結(jié)合位點的SNPs可能是miRNA與靶基因的轉(zhuǎn)錄后調(diào)控過程發(fā)生異常導(dǎo)致精神分裂癥的發(fā)生。

精神分裂癥作為一種重性精神疾病，在我國“十三五”腦計劃提出的大背景下必將受到更多的關(guān)注，精神分裂癥作為環(huán)境與遺傳因素共同作用的多基因遺傳病，或許miRNA結(jié)合SNP的研究是闡明精神分裂癥病理過程的更好的切入點，為以后臨床診斷和治療精神分裂癥提供理論基礎(chǔ)。盡管GWAS研究已經(jīng)發(fā)現(xiàn)許多與精神分裂癥相關(guān)的SNP，但如何驗證其功能仍將是未來的一個研究熱點，此外將miRNA與精神分裂癥相關(guān)SNP結(jié)合在一起，從遺傳學(xué)和表觀遺傳學(xué)角度闡明精神分裂癥的發(fā)病機制也將會是未來研究的一個重要發(fā)展方向。

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Schizophrenia-associated single nucleotide polymorphisms affecting microRNA function

Wenquan Liang1, Yu Hou2, Cunyou Zhao1

MicroRNAs (miRNAs) compose a class of non-coding transcripts with a mean length of 22 nucleotides, and play critical roles in regulating gene expression in the process of development, proliferation and differentiation of neurons. Recent genome-wide association studies (GWAS) find most of schizophrenia-associated single nucleotide polymorphisms (SNPs) locating in the non-coding regions, providing functional implications of miRNAs in the development of schizophrenia. In this review, we highlight the interplays between GWAS-SNPs and miRNAs in four perspectives: SNP in miRNA gene; miRNA located in the host gene; SNP located in the miRNA’s seed sequence; SNP located in the miRNA’s binding site. We also speculate on the future research on the role of miRNA in the development of schizophrenia.

GWAS; SNP; miRNA; schizophrenia

2019-05-08;

2019-07-07

廣東省科技計劃項目(編號：2019B030316032)，廣州市科技計劃項目(編號：201804010259)和國家自然科學(xué)基金項目(編號：81601175，81671333)資助[Supported by the Guangdong Science and Technology Foundation (No. 2019B030316032), the Guangzhou Science and Technology Foundation (No. 201804010259) and the National Natural Science Foundation of China (Nos. 81601175, 81671333)]

梁文權(quán)，碩士在讀研究生，專業(yè)方向：表觀遺傳。E-mail: wen1390229749@163.com

趙存友，博士，教授，研究方向：表觀遺傳。E-mail: cyzhao@smu.edu.cn

10.16288/j.yczz.19-126

2019/8/5 20:59:49

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20190805.2059.003.html

(責(zé)任編委: 夏昆)