circRNA及其調(diào)控動(dòng)物骨骼肌發(fā)育研究進(jìn)展

鄭婷，甘麥鄰，沈林園，牛麗莉，郭宗義，王金勇，張順華，朱礪

circRNA及其調(diào)控動(dòng)物骨骼肌發(fā)育研究進(jìn)展

鄭婷1，甘麥鄰1，沈林園1，牛麗莉1，郭宗義2，王金勇2，張順華1，朱礪1

1. 四川農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，成都 611130 2. 重慶市畜牧科學(xué)院，榮昌 402460

環(huán)狀RNA (circular RNA, circRNA)是一類反向剪接形成的閉合環(huán)狀RNA分子，廣泛存在于生物體內(nèi)，近年來(lái)成為非編碼RNA中的研究熱點(diǎn)。骨骼肌在生物體中起到協(xié)調(diào)運(yùn)動(dòng)的作用，是維持體內(nèi)正常代謝和內(nèi)分泌的器官之一。隨著測(cè)序技術(shù)和生物信息學(xué)分析技術(shù)的發(fā)展，circRNA在動(dòng)物骨骼肌發(fā)育中的功能和調(diào)控機(jī)制逐漸被揭示。本文就當(dāng)前circRNA的分子調(diào)控機(jī)制類型、circRNA經(jīng)典研究思路及功能研究方法以及circRNA參與骨骼肌正常發(fā)育與骨骼肌疾病發(fā)生調(diào)控的研究進(jìn)展進(jìn)行了綜述，以期為進(jìn)一步深入研究circRNA參與骨骼肌生長(zhǎng)發(fā)育調(diào)控的遺傳機(jī)制以及circRNA相關(guān)研究方法提供參考。

環(huán)狀RNA；骨骼肌發(fā)育；骨骼肌疾病；RNA測(cè)序

骨骼肌是動(dòng)物體執(zhí)行運(yùn)動(dòng)功能的重要器官，同時(shí)也是體內(nèi)重要的內(nèi)分泌和代謝器官，與動(dòng)物有機(jī)體的正常生長(zhǎng)發(fā)育密切相關(guān)[1,2]。骨骼肌中肌纖維是由大量單核成肌細(xì)胞融合成多核肌管，進(jìn)一步分化成熟而來(lái)。肌纖維聚集形成肌束，大量肌束和結(jié)締組織、毛細(xì)血管等共同構(gòu)成了肌肉組織[3,4]。研究表明，MRFs (myogenic regulator factors)[5,6]、MEF2 (myocyte en-hance factor 2)[7]、MSTN (myostatin)[8]和Pax基因家族[9]參與調(diào)控骨骼肌發(fā)育的基本過程。除了這些編碼基因，越來(lái)越多的非編碼RNA也參與調(diào)控骨骼肌生長(zhǎng)發(fā)育過程，主要包括微小RNA (microRNA, miRNA)、長(zhǎng)鏈非編碼RNA (long non-coding RNA, lncRNA)和環(huán)狀RNA (circular RNA, circRNA)[10~12]。

近年來(lái)，circRNA的相關(guān)研究日益增多，這種非編碼RNA具有表達(dá)特異性，且在生物體內(nèi)不易被降解，在生物體發(fā)育和腫瘤發(fā)生過程中的生物學(xué)功能被逐漸揭示。本文歸納了circRNA的分子調(diào)控機(jī)制，分析了circRNA經(jīng)典研究方法，并結(jié)合circRNA在骨骼肌發(fā)育調(diào)控方面的最新研究，對(duì)circRNA在骨骼肌正常生長(zhǎng)發(fā)育與相關(guān)疾病發(fā)生中的調(diào)控作用進(jìn)行了總結(jié)，以期為進(jìn)一步闡明circRNA參與骨骼肌發(fā)育的遺傳調(diào)控機(jī)制提供參考，為探究circRNA生物學(xué)功能提供研究思路。

1 circRNA的產(chǎn)生及作用機(jī)制

1.1 circRNA產(chǎn)生方式及特征

circRNA通過反向剪接形成閉合環(huán)狀RNA分子。目前發(fā)現(xiàn)的circRNA生物形成方式包括直接反向剪切、外顯子跳躍、內(nèi)含子的反義互補(bǔ)序列配對(duì)模式、依賴于RNA結(jié)合蛋白(RNA-binding proteins, RBPs)的環(huán)化模式以及類似于可變剪切的可變環(huán)化模式等。Gao等[13]對(duì)circRNA外顯子結(jié)構(gòu)和可變剪接事件進(jìn)行了研究，發(fā)現(xiàn)circRNA普遍是通過可變剪接形成的，并且大部分circRNA由外顯子剪接形成(ecirc-RNAs)，主要存在于細(xì)胞質(zhì)(圖1A)；由內(nèi)含子剪接環(huán)化的circRNA (ciRNAs)主要存在于細(xì)胞核中(圖1C)；一小部分既含外顯子也含內(nèi)含子的circRNA (EIciRNAs)，主要也存在于細(xì)胞核中(圖1B)[14~16]。circRNA因其閉合環(huán)狀的結(jié)構(gòu)，在細(xì)胞中不易被RNA核酸外切酶降解，穩(wěn)定性好。此外，大部分circRNA在不同物種間是進(jìn)化保守的，circRNA在不同組織或發(fā)育階段的表達(dá)模式具有較強(qiáng)的組織時(shí)空特異性[17]。另外，已經(jīng)有大量研究發(fā)現(xiàn)circRNA與許多疾病的發(fā)生和發(fā)展有關(guān)[18,19]，有潛力成為疾病診斷的生物標(biāo)志物。

1.2 circRNA功能調(diào)控機(jī)制

目前普遍認(rèn)可的circRNA分子調(diào)控機(jī)制包括：競(jìng)爭(zhēng)性結(jié)合miRNA分子；調(diào)控基因的轉(zhuǎn)錄；與RBPs互作間接調(diào)控RNA或DNA；參與蛋白質(zhì)翻譯過程；作為蛋白質(zhì)支架促進(jìn)蛋白酶化學(xué)修飾(圖1)[12,20~22]。

1.2.1 circRNA競(jìng)爭(zhēng)性吸附miRNA

這類circRNA含有miRNA結(jié)合位點(diǎn)，能競(jìng)爭(zhēng)性吸附miRNA進(jìn)而調(diào)控miRNA下游靶mRNA表達(dá)(圖1E)。在生物體中這類circRNA大多是由外顯子剪接而成的ecircRNA。現(xiàn)已發(fā)現(xiàn)相當(dāng)數(shù)量的競(jìng)爭(zhēng)性內(nèi)源circRNA，如Hansen等[23]在人()和小鼠()腦組織中發(fā)現(xiàn)一種高表達(dá)的，含有至少70個(gè)miR-7的結(jié)合位點(diǎn)，間接調(diào)控相關(guān)靶基因表達(dá)；由性別決定基因(sex- determining region Y)剪接形成的含有至少16個(gè)miR-138結(jié)合位點(diǎn)，作為miR-138分子海綿發(fā)揮調(diào)控作用[23]。

1.2.2 circRNA調(diào)控親本基因轉(zhuǎn)錄

細(xì)胞核中的ciRNAs能夠與RNA聚合酶II (RNA polymerase II, Pol II)相互作用，從而促進(jìn)其自身編碼基因的轉(zhuǎn)錄(圖1D)，如在人細(xì)胞中首次發(fā)現(xiàn)的ciRNA ()，在親本基因轉(zhuǎn)錄位點(diǎn)上大量積累，發(fā)揮順式調(diào)控作用[15]。此外，EIciRNAs能夠與Pol II和U1小核核糖核蛋白(U1 small nuclear ribonucleo-pro-teins, snRNP)結(jié)合，促進(jìn)其親本基因的轉(zhuǎn)錄(圖1D)，如Li等[16]發(fā)現(xiàn)的一種EIciRNA ()。

圖1 circRNA產(chǎn)生的方式及生物學(xué)功能

A：外顯子剪接形成的ecircRNA；B：既含外顯子又含內(nèi)含子的EIciRNA；C：內(nèi)含子剪接形成的ciRNA；D：circRNA調(diào)控親本基因轉(zhuǎn)錄；E：circRNA競(jìng)爭(zhēng)性結(jié)合miRNA；F：circRNA與RBPs互作；G：circRNA自身翻譯蛋白質(zhì)；H：circRNA作為蛋白質(zhì)支架，促進(jìn)酶及其底物的共域化。參考文獻(xiàn)[12,20~22]總結(jié)繪制。

1.2.3 circRNA與RBPs結(jié)合互作

circRNA可以直接與蛋白質(zhì)相互作用形成RNA-蛋白質(zhì)復(fù)合體(RNA-protein complex, RPC)，進(jìn)一步與RNA或DNA結(jié)合發(fā)揮調(diào)控作用(圖1F)[24]，如能與miRNA效應(yīng)因子AGO蛋白相結(jié)合[25]。另外，Ashwal-Fluss等[26]發(fā)現(xiàn)在人和果蠅()盲肌基因(muscle-blind)第二個(gè)外顯子環(huán)化形成的，與MBL蛋白特異性結(jié)合，平衡體內(nèi)和mRNA的水平進(jìn)而發(fā)揮調(diào)控作用。

1.2.4 circRNA參與蛋白質(zhì)翻譯

有研究發(fā)現(xiàn)，具有內(nèi)部核糖體進(jìn)入位點(diǎn)(internal ribosome entry site，IRES)的circRNA可以招募核糖體啟動(dòng)翻譯機(jī)制(圖1G)，如編碼蛋白質(zhì)FBXW7-185aa抑制了膠質(zhì)瘤細(xì)胞的增殖[27]；作為一種可以翻譯蛋白質(zhì)的circRNA，在肌生成過程中也發(fā)揮調(diào)控作用[28]。

1.2.5 circRNA作為蛋白質(zhì)支架促進(jìn)酶及其底物的共域化

與AKT1 (protein kinase B)和PDK1 (phosphoinositide dependent protein kinase 1)結(jié)合，促進(jìn)AKT1的PDK1依賴性磷酸化，激活A(yù)KT1在小鼠模型中起到心臟保護(hù)作用[29]；可以促進(jìn)小鼠雙微粒體基因(murine double minute 2)介導(dǎo)的p53突變體泛素化，促使其靶向蛋白酶體降解[30](圖1H)。

2 circRNA研究策略

總結(jié)現(xiàn)有研究報(bào)道，circRNA的經(jīng)典研究思路是基于生物學(xué)問題，通過轉(zhuǎn)錄組測(cè)序技術(shù)(RNA-seq)或微陣列芯片檢測(cè)(Microarray)得到circRNA數(shù)據(jù)集，篩選出兩種或多種差異表型樣本間的差異circRNA，接著對(duì)差異circRNA進(jìn)行鑒定和軟件分析，補(bǔ)充相應(yīng)的circRNA數(shù)據(jù)庫(kù)信息，再結(jié)合生物信息學(xué)分析手段，預(yù)測(cè)circRNA生物學(xué)功能，完成后續(xù)功能驗(yàn)證。

2.1 circRNA的發(fā)現(xiàn)與鑒定手段

近年來(lái)，研究非編碼RNA在特定細(xì)胞或組織中某一特定時(shí)刻的表達(dá)和功能，RNA-seq成為有力的研究工具，也是目前circRNA研究中最常見的測(cè)序手段[31]。針對(duì)circRNA研究，RNA-seq包括文庫(kù)構(gòu)建、上機(jī)測(cè)序、數(shù)據(jù)分析處理與功能預(yù)測(cè)等基本流程[32]。根據(jù)不同的研究目的與需求，研究者通常選擇全轉(zhuǎn)錄組測(cè)序或circRNA測(cè)序來(lái)實(shí)現(xiàn)對(duì)circRNA的篩選。前者可同時(shí)探究編碼和非編碼RNA的表達(dá)模式，適用于circRNA生物學(xué)功能研究；后者則適用于circRNA的富集，有助于挖掘未知circRNA。兩者主要差異在于測(cè)序文庫(kù)的構(gòu)建，circRNA測(cè)序文庫(kù)除了要求去除絕大多數(shù)rRNA和poly(A)外，需用核糖核酸外切酶RNase R處理去除線性RNA的干擾。大多數(shù)研究報(bào)道顯示去線性RNA后得到的circRNA豐度降低，因?yàn)槟承ヽircRNA對(duì)RNase R敏感而可能被消化[33,34]。目前已發(fā)現(xiàn)的circRNA數(shù)量差異很大，從幾百到幾千不等。這種可變性可能與RNA樣本預(yù)處理的方式、測(cè)序的深度以及用于數(shù)據(jù)篩選的過濾器等因素有關(guān)。值得注意的是，circRNA的可變剪接對(duì)區(qū)分測(cè)序結(jié)果中的正反義鏈來(lái)源有要求，因此circRNA測(cè)序最好構(gòu)建鏈特異性文庫(kù)，能夠更準(zhǔn)確地檢測(cè)轉(zhuǎn)錄表達(dá)水平[35]。

得到circRNA測(cè)序數(shù)據(jù)后，通常基于find_circ[36~38]、CIRCexplorer2[36,38]與CIRI36~38]等識(shí)別軟件，對(duì)circRNA進(jìn)行預(yù)測(cè)鑒定。Hansen等[39]建議結(jié)合使用MapSplice與circRNA finder這兩個(gè)軟件算法，更能提高預(yù)測(cè)的可靠性。由于軟件算法使用的要求各不相同，假陽(yáng)性率存在很大差異，故由算法檢測(cè)到的circRNA需要進(jìn)一步驗(yàn)證。為鑒定circRNA的存在，通常使用實(shí)時(shí)熒光定量PCR (quantitative real-time PCR, qRT-PCR)[40]、Northern印跡雜交(Northern blot)[27]、原位雜交(hybri-dization, ISH)[41]、RPAD(RNase R treatment, polya-denylation, and poly(A)+ RNA Depletion)[42]等技術(shù)檢測(cè)circRNA。

Microarray芯片也是分析檢測(cè)circRNA的有效工具，在許多疾病腫瘤的臨床診斷和監(jiān)測(cè)中廣泛應(yīng)用。Microarray的檢測(cè)成本及分析難度都低于RNA-seq，它們主要區(qū)別在于：(1) Microarray檢測(cè)circRNA必須有已知參考序列，而RNA-seq能檢測(cè)未知circRNA；(2) Microarray檢測(cè)的本質(zhì)是核酸雜交，可定量circRNA表達(dá)，而RNA-seq則不能準(zhǔn)確定量circRNA[43]；(3) Microarray可高效檢測(cè)反向剪接位點(diǎn)序列，相比RNA-seq可得到更大數(shù)量的circRNA[44]。Microarray為circRNA的發(fā)現(xiàn)提供了高靈敏度和特異性的平臺(tái)，也提供了高效的circRNA標(biāo)記系統(tǒng)。然而Microarray檢測(cè)的缺點(diǎn)之一是對(duì)樣品進(jìn)行預(yù)處理需要高的總RNA輸入，利用Microarray芯片也得不到全轉(zhuǎn)錄組測(cè)序所得對(duì)應(yīng)的線性RNA數(shù)據(jù)[45]。在參考序列未知的情況下，很多研究通常先用RNA-seq測(cè)得全轉(zhuǎn)錄組序列，再通過Microarray對(duì)circRNA進(jìn)行深入分析。

2.2 circRNA生物學(xué)功能的研究策略

如圖1所示，circRNA的功能調(diào)控機(jī)制多樣，包括競(jìng)爭(zhēng)性吸附miRNA、與RNA結(jié)合蛋白互作、在翻譯水平編碼蛋白質(zhì)等。研究人員通常利用實(shí)驗(yàn)技術(shù)調(diào)控circRNA表達(dá)水平，揭示circRNA與其他分子的相互作用以闡明circRNA的功能。

2.2.1 circRNA競(jìng)爭(zhēng)性吸附miRNA

驗(yàn)證circRNA競(jìng)爭(zhēng)性結(jié)合miRNA從而調(diào)控下游效應(yīng)基因是目前circRNA功能研究中最為常見、且相對(duì)較為成熟的研究方向。通常情況下，利用RNA-seq數(shù)據(jù)找到可能具有生物學(xué)功能的差異表達(dá)circRNA，分別將預(yù)測(cè)的靶miRNA、靶mRNA和差異表達(dá)miRNA、mRNA取交集，構(gòu)建circRNA- miRNA-mRNA網(wǎng)絡(luò)調(diào)控圖，再利用軟件進(jìn)行GO (gene ontology)功能注釋和KEGG (Kyoto encyclo-pedia of genes and genomes)通路分析[46,47]，預(yù)測(cè)相關(guān)circRNA的生物學(xué)功能和調(diào)控通路。結(jié)果表明，大量circRNA具有miRNA的結(jié)合位點(diǎn)，但它們的結(jié)合關(guān)系和相互作用還需要嚴(yán)謹(jǐn)?shù)膶?shí)驗(yàn)驗(yàn)證。通常利用雙熒光素酶報(bào)告系統(tǒng)、RNA pull down等實(shí)驗(yàn)技術(shù)進(jìn)行結(jié)合關(guān)系的驗(yàn)證<[48~51]，通過circRNA功能獲得缺失實(shí)驗(yàn)，驗(yàn)證其對(duì)靶miRNA和靶基因的功能調(diào)控作用[51,52]。

2.2.2 circRNA與RBPs互作

雖然與對(duì)應(yīng)線性RNA相比，circRNA與RBPs的結(jié)合位點(diǎn)很少被發(fā)現(xiàn)有富集，但也有不少研究證實(shí)了兩者之間的功能調(diào)控關(guān)系。目前circRNA與蛋白的相互作用主要通過RNA pull-down和RNA免疫沉淀法(RNA immunoprecipitation, RIP)分析[53,54]。根據(jù)circRNA反向剪接位點(diǎn)(back-splice junction region, BSJ)設(shè)計(jì)探針，獲取目標(biāo)circRNA，其相關(guān)蛋白可以利用Western Blot或質(zhì)譜法進(jìn)行分析鑒定[55,56]。另外，為了鑒定與感興趣的蛋白相關(guān)的circRNA，也可以使用針對(duì)該蛋白的抗體進(jìn)行RIP分析[57,58]，并使用交聯(lián)免疫沉淀法(crosslinking immunoprecipita-tion, CLIP)定位circRNA上確切的蛋白結(jié)合位點(diǎn)[54,57]。為避免線性RNA的干擾，在RNA pull-down和免疫沉淀之前，必須去除線性RNA。

2.2.3 circRNA編碼蛋白質(zhì)

目前已發(fā)現(xiàn)有的circRNAs能夠被翻譯成多肽，形成特殊的功能蛋白質(zhì)。研究發(fā)現(xiàn)，編碼蛋白的circRNA具有開放閱讀框(open reading frame, ORF)、內(nèi)部核糖體進(jìn)入位點(diǎn)(internal ribosome entry site, IRES)或N6-甲基腺苷(N6-methyladenosine, m6A)基序[59~62]。目前ORFfinder[63]、IRESfinder[64]、m6Apred[65]等多種生物學(xué)工具能夠輔助預(yù)測(cè)circRNA中潛在的ORF、IRES和m6A序列。預(yù)測(cè)circRNA編碼潛能后，還需從多個(gè)方面對(duì)circRNA的編碼功能進(jìn)行驗(yàn)證。通過深度測(cè)序結(jié)合核糖體印記技術(shù)，可以篩選出與核糖體結(jié)合的circRNA[66]；在circRNA預(yù)測(cè)的ORF終止密碼子上游插入特異蛋白標(biāo)簽，通過檢測(cè)標(biāo)簽蛋白免疫熒光探針來(lái)驗(yàn)證circRNA的翻譯功能[27,61,67]。通過標(biāo)簽研究circRNA編碼的肽段，既能識(shí)別可能與circRNA衍生肽段互作的RNA或蛋白，又能揭示circRNA肽段的亞細(xì)胞定位[27,28,68]。研究circRNA衍生肽的生物學(xué)功能，通過敲低或過表達(dá)目的circRNA或設(shè)計(jì)特定細(xì)胞刺激，檢測(cè)circRNA蛋白產(chǎn)物水平，分析其對(duì)基因表達(dá)譜及表型的影響[68]。

3 circRNA參與骨骼肌發(fā)育調(diào)控

隨著circRNA研究策略和方法的不斷成熟，circRNA在動(dòng)物體內(nèi)發(fā)揮的多種生物學(xué)功能被逐漸揭示。在動(dòng)物骨骼肌發(fā)育進(jìn)程中，成肌細(xì)胞的增殖和分化、肌衛(wèi)星細(xì)胞活化促進(jìn)肌肉再生等一系列過程，都離不開肌生成相關(guān)編碼基因家族的調(diào)控[8,9]。隨著研究技術(shù)與手段的更迭發(fā)展，越來(lái)越多circRNA被發(fā)現(xiàn)與肌生成、肌纖維類型轉(zhuǎn)化和骨骼肌疾病發(fā)生等過程密切相關(guān)。

3.1 circRNA參與骨骼肌正常生長(zhǎng)發(fā)育的調(diào)控

動(dòng)物骨骼肌從胚胎期到出生后期，經(jīng)歷了骨骼肌衛(wèi)星細(xì)胞活化以及成肌細(xì)胞增殖、分化、凋亡等過程[2]。研究表明，人、模式動(dòng)物和家養(yǎng)動(dòng)物的骨骼肌中都存在circRNA，并且不同生長(zhǎng)發(fā)育階段的骨骼肌中circRNA呈時(shí)序表達(dá)特異性[69~71]。

3.1.1 不同circRNA參與骨骼肌不同生長(zhǎng)發(fā)育進(jìn)程

Wei等[72]從幼年到老年的恒河猴()骨骼肌樣本中鑒定出12,007個(gè)circRNAs，發(fā)現(xiàn)并證實(shí)其中有5個(gè)circRNAs表達(dá)水平隨年齡增長(zhǎng)而下調(diào)。同樣，豬()不同胚胎時(shí)期肌肉中circRNA也呈現(xiàn)動(dòng)態(tài)表達(dá)，并隨著胚胎骨骼肌的生長(zhǎng)發(fā)育進(jìn)程，部分circRNA表達(dá)水平顯著下調(diào)[73]。這種動(dòng)態(tài)表達(dá)模式提示不同circRNA在動(dòng)物骨骼肌發(fā)育各階段起到不同的調(diào)控作用。

從骨骼肌發(fā)育相關(guān)測(cè)序結(jié)果中發(fā)現(xiàn)，一個(gè)基因可轉(zhuǎn)錄剪接成多個(gè)circRNA亞型，表達(dá)量較高的亞型在骨骼肌發(fā)育調(diào)控中發(fā)揮作用，并且在不同的骨骼肌發(fā)育階段，宿主基因會(huì)產(chǎn)生不同的circRNA亞型[69,70,73]。Ling等[69]首次揭示了山羊()骨骼肌中circRNA時(shí)序動(dòng)態(tài)表達(dá)模式，發(fā)現(xiàn)多個(gè)基因都產(chǎn)生了不同circRNA亞型，其中SLX4相互作用蛋白基因()的兩種circRNA亞型在山羊骨骼肌發(fā)育各階段均表現(xiàn)出完全相反的表達(dá)趨勢(shì)，表明不同circRNA亞型在骨骼肌發(fā)育過程中可能發(fā)揮相反作用，表明circRNA的多樣性與其調(diào)控機(jī)制的復(fù)雜性。

3.1.2 circRNA參與成肌細(xì)胞增殖、分化、凋亡等過程調(diào)控

近年來(lái)，研究者們利用測(cè)序分析技術(shù)挖掘出許多參與調(diào)控生物體發(fā)育過程的miRNA、lncRNA和circRNA，也鑒定出相當(dāng)數(shù)量的circRNA可能參與調(diào)控動(dòng)物骨骼肌生長(zhǎng)發(fā)育的生物學(xué)過程(圖2)。研究發(fā)現(xiàn)，經(jīng)功能驗(yàn)證的大部分circRNA作為miRNA的競(jìng)爭(zhēng)性內(nèi)源RNA (competing endogenous RNA, ceRNA)，參與調(diào)控成肌細(xì)胞增殖、分化等過程(表1)。Ouyang等[74]鑒定了不同發(fā)育階段肌肉中差異表達(dá)circRNAs的miRNA結(jié)合能力，發(fā)現(xiàn)1401個(gè)circRNA中有946個(gè)都具有一個(gè)或多個(gè)miRNA結(jié)合位點(diǎn)，涉及150個(gè)已知miRNA。

骨骼肌組織或肌細(xì)胞中circRNA通過競(jìng)爭(zhēng)性結(jié)合來(lái)抑制miRNA下游通路。Wang等[77]預(yù)測(cè)發(fā)現(xiàn)上具有4個(gè)結(jié)合位點(diǎn)，在C2C12細(xì)胞中競(jìng)爭(zhēng)性結(jié)合，從而解除后者對(duì)基因的抑制作用，抑制C2C12成肌分化。Ouyang等[76]發(fā)現(xiàn)在雞()晚期胚胎骨骼肌發(fā)育中高表達(dá)，并驗(yàn)證發(fā)現(xiàn)雞作為的分子海綿，使和基因表達(dá)上調(diào)，促進(jìn)雞成肌細(xì)胞增殖和分化，調(diào)控雞晚期胚胎骨骼肌發(fā)育過程。另外，Li[81]和聶露[86]等在山羊胚胎中期骨骼肌中高表達(dá)，并驗(yàn)證了MRFs家族中基因作為轉(zhuǎn)錄因子對(duì)發(fā)揮正向調(diào)控作用，同時(shí)通過靶向結(jié)合，阻遏其對(duì)基因表達(dá)的抑制，從而揭示了促進(jìn)山羊骨骼肌衛(wèi)星細(xì)胞成肌分化的調(diào)控通路。

圖2 circRNA在骨骼肌增殖分化中的作用

circRNA-miRNA-mRNA在骨骼肌發(fā)育過程中的調(diào)控網(wǎng)絡(luò)圖，circRNA參與調(diào)控骨骼肌衛(wèi)星細(xì)胞活化、成肌細(xì)胞增殖以及成肌細(xì)胞分化為肌管的生理過程。

表1 circRNA參與不同動(dòng)物骨骼肌發(fā)育的調(diào)控

除此之外，還有研究揭示了circRNA對(duì)骨骼肌生長(zhǎng)發(fā)育的其他調(diào)控機(jī)制。2017年，Legnini等[28]首次利用測(cè)序數(shù)據(jù)篩選出通過參與蛋白質(zhì)編碼發(fā)揮調(diào)控作用的circRNA，將其命名為。在人和小鼠中有較高同源性，反向剪接時(shí)形成了一個(gè)開放閱讀框，能夠在應(yīng)激條件下被特殊翻譯成蛋白質(zhì)發(fā)揮作用，可能促進(jìn)成肌細(xì)胞增殖。此外，Pandey等[85]發(fā)現(xiàn)與PUR蛋白相結(jié)合，阻遏PUR蛋白對(duì)(myosin heavy chain)基因轉(zhuǎn)錄的拮抗作用，從而促進(jìn)成肌細(xì)胞分化，加快骨骼肌生長(zhǎng)發(fā)育進(jìn)程。這是首次發(fā)現(xiàn)circRNA通過與RBPs協(xié)同作用發(fā)揮對(duì)骨骼肌生長(zhǎng)發(fā)育的調(diào)控作用。

3.1.3 circRNA參與骨骼肌纖維類型轉(zhuǎn)換過程調(diào)控

不同類型骨骼肌纖維具有不同的生理學(xué)特性，在動(dòng)物骨骼肌發(fā)育過程中，成熟分化的肌管將逐漸轉(zhuǎn)變?yōu)椴煌δ茴愋偷募±w維，包括氧化型和酵解型肌纖維，又稱慢肌和快肌纖維。目前有研究表明，circRNA可能參與骨骼肌纖維生理功能調(diào)控及肌纖維類型轉(zhuǎn)換調(diào)控。Shen等[87]選取處于生長(zhǎng)拐點(diǎn)青峪豬的腰大肌和背最長(zhǎng)肌，測(cè)序獲得這兩種類型肌肉的mRNA、lncRNA和circRNA差異表達(dá)譜，發(fā)現(xiàn)氧化型和酵解型肌肉不同的生理特性與其中差異表達(dá)的circRNA存在相關(guān)，這些circRNA的宿主基因被發(fā)現(xiàn)參與ATP代謝、快慢肌纖維轉(zhuǎn)換等生理過程。值得關(guān)注的是，其中3個(gè)circRNA是由肌纖維分型相關(guān)的、和基因轉(zhuǎn)錄形成。研究提示circRNA在不同類型骨骼肌發(fā)育過程中起到特異的調(diào)控功能，并且circRNA還可能參與骨骼肌纖維類型轉(zhuǎn)換過程的調(diào)控，其分子機(jī)制有待深入研究。

Li等[88]通過測(cè)序技術(shù)從豬快肌(肱二頭肌)和慢肌(比目魚肌)中分析circRNA差異表達(dá)譜，找到242個(gè)差異表達(dá)circRNAs，通過GO和KEGG分析發(fā)現(xiàn)這些差異表達(dá)circRNA宿主基因也與骨骼肌纖維類型形成的相關(guān)通路和生理過程有關(guān)，包括cGMP- PKG和AMPK信號(hào)通路，肌肉收縮、肌肉結(jié)構(gòu)發(fā)育等生理學(xué)過程。此外，該研究還發(fā)現(xiàn)其中許多差異表達(dá)circRNA可能通過競(jìng)爭(zhēng)性結(jié)合，參與調(diào)控肌纖維類型相關(guān)的骨骼肌疾病發(fā)生過程。circRNA在動(dòng)物骨骼肌纖維發(fā)育形成及發(fā)揮生理功能上具有潛在作用，有助于進(jìn)一步理解動(dòng)物骨骼肌生長(zhǎng)發(fā)育的表觀遺傳機(jī)制。

3.2 circRNA參與骨骼肌病理變化過程的調(diào)控

circRNA可變剪接在不同組織和發(fā)育階段受到嚴(yán)格的調(diào)控[89]，其降解調(diào)控可能影響增殖信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞凋亡、血管生成等生物學(xué)過程，從而導(dǎo)致廣泛的人類疾病[90]。在動(dòng)物正常的骨骼肌中通?？蓹z測(cè)到高表達(dá)的circRNA[70,91]，而在肌肉疾病病例中存在circRNA表達(dá)失調(diào)[92]。研究發(fā)現(xiàn)circRNA表達(dá)水平在肌營(yíng)養(yǎng)不良癥及心肌病等疾病中發(fā)生改變[93,94]，這暗示了circRNA的表達(dá)失調(diào)可能與肌肉病理狀態(tài)相關(guān)(表2)。

杜氏肌營(yíng)養(yǎng)不良(Duchenne muscular dystrophy, DMD)是一種由于基因發(fā)生移碼突變，主要是外顯子缺失引起肌營(yíng)養(yǎng)不良蛋白缺失，從而產(chǎn)生肌無(wú)力等癥狀的疾病[95]。由基因轉(zhuǎn)錄產(chǎn)物剪接生成的circRNA是最早在骨骼肌中被識(shí)別的環(huán)狀RNA之一[96]。有研究表明，由基因45~55號(hào)外顯子區(qū)域剪接產(chǎn)生的circRNA，可能使基因外顯子缺失的患者癥狀減輕[92,97]。也有研究分析了來(lái)自DMD患者和正常人成肌細(xì)胞的RNA測(cè)序結(jié)果，發(fā)現(xiàn)DMD患者來(lái)源的成肌細(xì)胞在circRNA表達(dá)水平方面確實(shí)具有獨(dú)特的差異特征[28]。有趣的是，在正常成肌細(xì)胞體外分化過程中上調(diào)的和在DMD患者來(lái)源的成肌細(xì)胞中表達(dá)下調(diào)[98]；相反，在正常成肌細(xì)胞肌生成過程中下調(diào)的編碼蛋白的，在DMD成肌細(xì)胞分化過程中表達(dá)水平上調(diào)[28]。另外，Song等[99]從DMD模型小鼠的肌肉中鑒定出197個(gè)與對(duì)照組差異表達(dá)的circRNAs；Weng等[100]在坐骨神經(jīng)損傷后的萎縮肌肉中鑒定出236個(gè)差異表達(dá)circRNAs，這些研究都表明circRNA與肌肉疾病病理過程有密切聯(lián)系。

另一種肌營(yíng)養(yǎng)不良癥為1型肌強(qiáng)直性營(yíng)養(yǎng)不良(myotonic dystrophy type 1, DM1)是一種由肌強(qiáng)直性營(yíng)養(yǎng)不良蛋白激酶(myotonic dystrophy protein kinase,)基因3′UTR的CTG重復(fù)擴(kuò)增導(dǎo)致mRNA剪接異常的多系統(tǒng)疾病[101]。與在DMD患者來(lái)源成肌細(xì)胞中的結(jié)果相似，Czubak等[102]發(fā)現(xiàn)DM1肌肉組織或細(xì)胞中，circRNA也呈現(xiàn)特異性表達(dá)，還初步發(fā)現(xiàn)DM1疾病程度與circRNA可變剪接變化之間的關(guān)聯(lián)，并且circRNA整體表達(dá)水平呈上調(diào)趨勢(shì)，在疾病個(gè)體中、、和的環(huán)狀/線性比均升高[28,103](表2)。目前需要對(duì)circRNA可變剪接相關(guān)疾病中大量表達(dá)的circRNA進(jìn)行識(shí)別與鑒定，以突出其在發(fā)病機(jī)制中的作用，并開發(fā)未來(lái)的疾病治療方法。

表2 circRNA在肌肉病理過程的表達(dá)變化

DMD：杜氏肌營(yíng)養(yǎng)不良；DM1：I型肌強(qiáng)直性營(yíng)養(yǎng)不良；Sarcopenia：老年性肌肉衰減癥；HCM：肥厚型心肌?。籇CM：擴(kuò)張型心肌病。

4 結(jié)語(yǔ)與展望

circRNA的動(dòng)態(tài)表達(dá)模式、復(fù)雜的調(diào)控機(jī)制和在不同細(xì)胞水平上扮演的新角色共同表明，它們并不是非正常剪接的“噪聲”，而是在生物體發(fā)揮重要作用的新型調(diào)控分子。盡管大部分circRNA尚未被解析驗(yàn)證，但已有數(shù)千個(gè)circRNAs在不同物種、不同組織中被識(shí)別。

目前針對(duì)動(dòng)物骨骼肌circRNA的大量研究，展現(xiàn)了circRNA在調(diào)控骨骼肌正常生長(zhǎng)發(fā)育和骨骼肌相關(guān)疾病發(fā)生過程中的重要性及其分子機(jī)制的復(fù)雜性，仍存在許多科學(xué)問題有待深入探究。已經(jīng)發(fā)現(xiàn)相當(dāng)數(shù)量的circRNAs作為ceRNA在骨骼肌細(xì)胞增殖、分化等過程發(fā)揮調(diào)控作用，也有愈來(lái)愈多的研究者開始關(guān)注circRNA與骨骼肌纖維分型以及骨骼肌異常代謝發(fā)育的關(guān)系，其在骨骼肌相關(guān)疾病診斷、監(jiān)測(cè)過程中有重要意義。目前circRNA形成機(jī)制與circRNA轉(zhuǎn)錄后調(diào)控機(jī)制均未被完全闡明，circRNA與蛋白質(zhì)互作、調(diào)控親本基因轉(zhuǎn)錄、參與編碼蛋白質(zhì)等一系列復(fù)雜的作用機(jī)制仍有待探索。

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circRNA on animal skeletal muscle development regulation

Ting Zheng1, Mailin Gan1, Linyuan Shen1, Lili Niu1, Zongyi Guo2, Jinyong Wang2, Shunhua Zhang1, Li Zhu1

Circular RNA (circRNA) is a type of closed circular RNA molecules formed by reverse splicing, which exists widely in organisms and has become a research hotspot in non-coding RNAs in recent years. Skeletal muscle plays the role of coordinating movement and maintaining normal metabolism and endocrine in organisms. With the development of sequencing and bioinformatics analysis technology, the functions and regulation mechanisms of circRNAs in skeletal muscle development have been gradually revealed. In this review, we summarize the types of molecular regulatory mechanisms, the classical research ideas and the functional research methods of circRNAs, and the research progress of circRNAs involved in normal development of skeletal muscle and regulation of skeletal muscle disease, in order to provide a reference to further study of the genetic mechanisms of circRNAs in the regulation of skeletal muscle development.

circRNA; skeletal muscle development; skeletal muscle diseases; RNA-seq

2020-07-06;

2020-10-13

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：31972524)，國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系四川生豬創(chuàng)新團(tuán)隊(duì)項(xiàng)目(編號(hào)：SCSZTD-3-008)和四川省科技支撐計(jì)劃項(xiàng)目(編號(hào)：2016NYZ0050)資助[Supported by the National Natural Science Foundation of China (No. 31972524), Sichuan Pig Innovation Team Project of National Modern Agricultural Industrial Technology System (No. SCSZTD-3-008), and the Science and Technology Program of Sichuan Province (No. 2016NYZ0050)]

鄭婷，在讀碩士研究生，專業(yè)方向：動(dòng)物遺傳育種。E-mail: 741377392@qq.com

張順華，博士，助理研究員，碩士生導(dǎo)師，研究方向：動(dòng)物遺傳育種。E-mail: 363445986@qq.com

朱礪，博士，教授，博士生導(dǎo)師，研究方向：動(dòng)物遺傳育種。E-mail: zhuli7508@163.com

10.16288/j.yczz.20-207

2020/12/2 15:43:23

URI: https://kns.cnki.net/kcms/detail/11.1913.R.20201202.1335.002.html

(責(zé)任編委: 蔣思文)