邢寶松，王璟，陳俊峰，馬強(qiáng)，任巧玲，張家慶，張華，滑留帥，孫加節(jié)，曹海

研究報(bào)告

去勢(shì)和非去勢(shì)公豬背最長(zhǎng)肌circRNA差異表達(dá)分析

邢寶松1，王璟1，陳俊峰1，馬強(qiáng)1，任巧玲1，張家慶1，張華1，滑留帥1，孫加節(jié)2，曹海3

1. 河南省農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)研究所，河南省畜禽繁育與營(yíng)養(yǎng)調(diào)控重點(diǎn)實(shí)驗(yàn)室，鄭州 450002 2. 華南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)學(xué)院，廣東省動(dòng)物營(yíng)養(yǎng)調(diào)控重點(diǎn)實(shí)驗(yàn)室/國(guó)家生豬種業(yè)工程技術(shù)中心，廣州 510642 3. 河南興銳農(nóng)牧科技有限公司，信陽 465550

公豬去勢(shì)可減少異味和打斗，但去勢(shì)后產(chǎn)肉量和肌內(nèi)脂肪沉積發(fā)生變化，其分子機(jī)制的解析對(duì)生產(chǎn)具有重要意義。近年來研究表明，環(huán)狀RNA (circRNA)在肌肉發(fā)育中具有重要調(diào)控作用。為探究去勢(shì)后circRNAs對(duì)背最長(zhǎng)肌發(fā)育的調(diào)控，本研究選擇6頭淮南公豬，隨機(jī)選擇3頭去勢(shì)，當(dāng)體重達(dá)130 kg左右屠宰，采集背最長(zhǎng)肌樣品，利用高通量測(cè)序篩選差異表達(dá)circRNAs (differentially expressed circRNAs, DECs)并進(jìn)行KEGG功能富集分析。結(jié)合前期篩選的公豬去勢(shì)相關(guān)miRNAs，構(gòu)建DECs-miRNAs調(diào)控網(wǎng)絡(luò)，最后使用豬骨骼肌衛(wèi)星細(xì)胞驗(yàn)證候選circRNA表達(dá)譜及其與miRNA互作關(guān)系。結(jié)果表明，去勢(shì)和非去勢(shì)組背最長(zhǎng)肌樣品共獲得5866個(gè)circRNAs，兩組之間共有370個(gè)DECs (| log2Foldchange | > 1,p<0.8)，KEGG富集分析表明，DECs來源母基因主要富集于肌肉發(fā)育、肌纖維類型轉(zhuǎn)化、能量代謝等相關(guān)通路。構(gòu)建的DECs-miRNA調(diào)控網(wǎng)絡(luò)共包括69個(gè)circRNAs和8個(gè)miRNAs。選擇circRNA_2241和circRNA_4237進(jìn)行驗(yàn)證，結(jié)果發(fā)現(xiàn)這兩個(gè)circRNAs真實(shí)存在且表達(dá)趨勢(shì)與測(cè)序結(jié)果一致。進(jìn)一步在豬骨骼肌衛(wèi)星細(xì)胞初步驗(yàn)證circRNA_2241與miR-1互作關(guān)系，結(jié)果表明睪酮顯著促進(jìn)circRNA_2241表達(dá)，同時(shí)抑制miR-1表達(dá)。本研究結(jié)果提示circRNAs可能通過與miRNAs互作調(diào)控豬去勢(shì)后背最長(zhǎng)肌發(fā)育，從而為解析去勢(shì)對(duì)肌肉發(fā)育調(diào)控的分子機(jī)制提供參考。

環(huán)狀RNA；去勢(shì)；公豬；背最長(zhǎng)肌

在豬生產(chǎn)中，去勢(shì)不僅減少公豬異味，還可減少打斗造成的經(jīng)濟(jì)損失。但去勢(shì)后，公豬產(chǎn)肉量和肌內(nèi)脂肪(intramuscular fat, IMF)含量與未去勢(shì)公豬差異很大，去勢(shì)公豬產(chǎn)肉量降低，脂肪沉積增加[1]。類似的，公牛去勢(shì)后肉質(zhì)性狀例如IMF含量、大理石花紋、脂肪酸組成等都顯著提高，但產(chǎn)肉量降低[2～4]。近年來，有研究比較了去勢(shì)后背最長(zhǎng)肌(longissimus dorsi, LD)和皮下脂肪mRNA、miRNA和lncRNA的表達(dá)變化。王璟等[5]比較了去勢(shì)和非去勢(shì)淮南公豬背最長(zhǎng)肌轉(zhuǎn)錄組，共篩選到935個(gè)差異表達(dá)基因，KEGG富集到肌肉發(fā)育和脂質(zhì)代謝相關(guān)通路。其中硬脂酰輔酶A去飽和酶1 (stearoyl-CoA desaturase-1, SCD-1)、激素敏感酯酶(hormone-sensitive lipase, HSL)、葡萄糖轉(zhuǎn)運(yùn)體4 (glucose transporter 4, GLUT4)等基因能同時(shí)或部分參與激素分泌、脂肪沉積和肌肉發(fā)育調(diào)控。Bai等[6]對(duì)比23周齡去勢(shì)和非去勢(shì)豬皮下脂肪組織miRNA表達(dá)譜，發(fā)現(xiàn)177個(gè)差異表達(dá)miRNAs，KEGG富集分析發(fā)現(xiàn)這些miRNAs參與肌細(xì)胞增殖、分化和凋亡和脂肪組織發(fā)育。Cai等[7,8]通過測(cè)序比較去勢(shì)和非去勢(shì)公豬皮下脂肪和背最長(zhǎng)肌miRNAs表達(dá)差異，分別篩選到18個(gè)和7個(gè)差異表達(dá)miRNAs，其靶基因主要參與脂肪代謝和骨骼肌收縮。Wang等[9]在去勢(shì)和非去勢(shì)豬皮下脂肪組織篩選到18個(gè)差異表達(dá)lncRNAs，其靶基因與脂肪酸、胰島素和脂肪細(xì)胞因子有關(guān)。Xing等[10]研究表明，去勢(shì)和非去勢(shì)豬背最長(zhǎng)肌有385個(gè)差異表達(dá)lncRNAs，主要與雌激素受體的信號(hào)傳導(dǎo)以及骨骼肌發(fā)育相關(guān)。雖然上述研究篩選了去勢(shì)后背最長(zhǎng)肌和皮下脂肪組織全轉(zhuǎn)錄組差異表達(dá)譜，但去勢(shì)調(diào)控肌肉發(fā)育和脂質(zhì)代謝的分子機(jī)制尚不清楚。

近期研究表明，環(huán)狀RNA (circular RNA, circRNAs)參與肌肉發(fā)育和脂肪沉積調(diào)控，例如成肌細(xì)胞分化過程中circ-ZNF609表達(dá)量上調(diào)，可特異性抑制成肌細(xì)胞增殖[11]。線粒體分裂和凋亡相關(guān)circRNA (mitochondrial fission and apoptosis-related circRNA, MFACR)可通過抑制MTP18翻譯減少心肌細(xì)胞死亡[12]。來源于雞Supervillin基因的circSVIL通過競(jìng)爭(zhēng)性吸附miR-203促進(jìn)成肌細(xì)胞增殖和分化[13]。牛circFGF3可吸附miR-107，釋放其對(duì)Wnt3a的抑制作用，進(jìn)而促進(jìn)成肌細(xì)胞分化[14]。circFUT10- miR-133a通路抑制成肌細(xì)胞增殖，并促進(jìn)分化[15]。牛circHUWE1通過miR-29b-AKT3-AKT信號(hào)通路，促進(jìn)成肌細(xì)胞增殖，抑制凋亡和分化[16]。circINSR通過海綿吸附miR-34a，減輕miR-34a對(duì)Bcl-2和CyclinE2的抑制，促進(jìn)成肌細(xì)胞增殖減少細(xì)胞凋亡[17]。環(huán)狀RNA SAMD4A通過miR-138-5p-EZH2促進(jìn)前脂肪細(xì)胞分化[18]。CDR1as促進(jìn)源自人臍帶的間充質(zhì)干細(xì)胞增殖和分化[19]。circFUT10通過let-7c-PPARGC1B促進(jìn)牛脂肪細(xì)胞增殖抑制分化[20]?；谶@些結(jié)果，為探究circRNAs在去勢(shì)后豬肌肉發(fā)育中的調(diào)控機(jī)制，本研究利用高通量測(cè)序比較了去勢(shì)公豬和未去勢(shì)公豬背最長(zhǎng)肌circRNA的表達(dá)差異，為進(jìn)一步解析去勢(shì)對(duì)肌肉發(fā)育調(diào)控機(jī)制提供參考。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

在河南興銳農(nóng)牧科技有限公司選擇6頭出生體重相近的半同胞淮南豬公豬，每2頭來源于同一窩。于7日齡每窩隨機(jī)選擇1頭去勢(shì)，另1頭相同部位進(jìn)行偽手術(shù)處理，保證去勢(shì)組和非去勢(shì)組豬只所受手術(shù)應(yīng)激一致。按照標(biāo)準(zhǔn)飼養(yǎng)流程飼喂，豬只體重達(dá)到130 kg左右(大約300～315日齡)屠宰，屠宰30 min內(nèi)采集背最長(zhǎng)肌樣品(體右側(cè)，第6～7肋骨)，液氮保存。

1.2 RNA提取和測(cè)序建庫

使用TRIzol (美國(guó)Invitrogen公司)分別提取6個(gè)背最長(zhǎng)肌樣品總RNA。利用瓊脂糖凝膠電泳、Agilent 2100生物分析儀(美國(guó)安捷倫科技公司)和NanoDrop分光光度計(jì)(美國(guó)Nano-Drop科技公司)分析所提RNA的純度、質(zhì)量和完整性。RNA完整性數(shù)(RIN)大于8的樣品用于構(gòu)建文庫。使用DNase I (美國(guó)QIAGEN公司)消化所提RNA，去除殘留基因組DNA。使用Ribo-Zero?rRNA試劑盒(美國(guó)Epicentre公司)去除核糖體RNA。去勢(shì)豬RNA樣品和非去勢(shì)豬RNA樣品分別混合后測(cè)序。使用Illumina TruSeq?RNA樣品制備試劑盒生成測(cè)序文庫，在Illumina Hiseq 2500平臺(tái)進(jìn)行測(cè)序。

1.3 circRNA鑒定

原始數(shù)據(jù)(Raw data)去除接頭和低質(zhì)量數(shù)據(jù)得到有效數(shù)據(jù)(clean data)，用TopHat2軟件將有效數(shù)據(jù)與豬參考基因組(11.1)比對(duì)分析。通過find_circ軟件鑒定circRNAs，其基本原理是：提取與基因組未比對(duì)上序列兩端20 nt的anchor序列，反向拼接anchor序列獲得短序列讀段，將短序列讀段再次與基因組進(jìn)行比對(duì)，選取序列吻合且有GT-AG剪接位點(diǎn)的作為候選circRNA。將read count小于2的circRNA留作鑒定的circRNAs。與circBase數(shù)據(jù)庫比對(duì)區(qū)分已知circRNAs和新發(fā)現(xiàn)circRNAs。進(jìn)一步根據(jù)circRNAs在染色體的位置，分為反義，有義重疊，外顯子，內(nèi)含子和基因間五類。

1.4 circRNA表達(dá)分析

使用TPM（transcripts per kilobase of exon model per million mapped reads，每千個(gè)堿基的轉(zhuǎn)錄每百萬映射讀取的轉(zhuǎn)錄本數(shù)）對(duì)circRNAs進(jìn)行歸一化處理，計(jì)算每個(gè)circRNA在每個(gè)樣品的表達(dá)量[21]。使用DESeq軟件分析circRNAs在不同樣品中的表達(dá)差異，差異表達(dá)circRNA (differently expressed circRNA,DEC)篩選條件為| log2Foldchange |≥1且p≤0.8[22]。利用Bowtie2軟件鑒定circRNA的母源基因，對(duì)DECs母源基因進(jìn)行GO和KEGG分析，<0.05視為有統(tǒng)計(jì)意義。

1.5 circRNA-miRNA網(wǎng)絡(luò)圖的構(gòu)建

為進(jìn)一步分析DECs的生物學(xué)功能，結(jié)合前期研究篩選的公豬背最長(zhǎng)肌去勢(shì)相關(guān)miRNAs[23]，使用miRanda軟件分析DECs與這些miRNAs之間的關(guān)系，保留種子區(qū)域沒有錯(cuò)配，且能量< –18 kcal /摩爾的miRNAs。使用Cytoscape軟件對(duì)DECs-miRNA互作網(wǎng)絡(luò)進(jìn)行繪圖。

1.6 circRNA驗(yàn)證和定量分析

根據(jù)長(zhǎng)度和表達(dá)量，選擇circRNA_2241和circRNA_4237鑒定所篩選circRNA真實(shí)性和表達(dá)趨勢(shì)。用RNase R (3 U/μg，美國(guó)Epicenter Biotechno-logies公司)處理背最長(zhǎng)肌提取的總RNA，1 μg RNA使用3 U的RNase R于37℃孵育15 min。根據(jù)circRNA_2241和circRNA_4237的剪切位點(diǎn)，設(shè)計(jì)特異性反向擴(kuò)增剪切位點(diǎn)的引物，引物信息見表1，引物由上海生工生物工程有限公司設(shè)計(jì)合成。擴(kuò)增后通過Sanger測(cè)序鑒定circRNA的真實(shí)性(上海生工生物工程有限公司)。

使用qRT-PCR檢測(cè)circRNA_2241和circRNA_ 4237在去勢(shì)和非去勢(shì)豬背最長(zhǎng)肌的表達(dá)水平，所用RNA與測(cè)序所用RNA相同。使用SYBR Green PCR試劑盒，擴(kuò)增體系包括20 ng cDNA、10 μL 2×SYBR Premix ExTM和10 μmol/L上下游引物。qPCR擴(kuò)增程序：95℃預(yù)變性5 min；95℃變性20 s，60℃復(fù)性20 s，72℃延伸20 s，40個(gè)循環(huán)。所有反應(yīng)重復(fù)3次，并通過2–ΔΔCt法計(jì)算circRNAs相對(duì)表達(dá)量。

1.7 豬骨骼肌衛(wèi)星細(xì)胞培養(yǎng)

circRNA_2241包含在DECs-miRNA互作網(wǎng)絡(luò)中，所以選擇circRNA_2241-miR-1做進(jìn)一步驗(yàn)證。按文獻(xiàn)[24]的方法分離豬骨骼肌衛(wèi)星細(xì)胞。為驗(yàn)證去勢(shì)對(duì)肌肉circRNA表達(dá)的影響，在豬骨骼肌衛(wèi)星細(xì)胞中，通過添加不同濃度睪酮和不添加睪酮，分別模擬非去勢(shì)組和去勢(shì)組。當(dāng)細(xì)胞達(dá)到70%～80%融合時(shí)，在培養(yǎng)基中添加不同濃度睪酮，對(duì)照組不添加睪酮，實(shí)驗(yàn)組睪酮添加量分別為10–9mol/L和10–10mol/L。添加睪酮48 h后收獲細(xì)胞，檢測(cè)睪酮對(duì)circRNA_2241和miR-1表達(dá)的影響。

1.8 統(tǒng)計(jì)分析

使用SPSS統(tǒng)計(jì)軟件進(jìn)行方差分析和顯著性檢驗(yàn)，所有數(shù)據(jù)均以mean±s.e.m.表示。<0.05表示差異顯著，<0.01表示差異極顯著。

2 結(jié)果與分析

2.1 circRNA特征分析

去勢(shì)和非去勢(shì)公豬背最長(zhǎng)肌共獲得5866個(gè)circRNAs，兩組共有circRNAs為5205個(gè)(圖1A)。這些circRNAs長(zhǎng)度范圍150 bp～99,406 bp，平均長(zhǎng)度5494 bp (圖1B)。這些circRNAs在全部染色體均有分布，6號(hào)染色體分布的circRNAs最多，占10.54%。X染色體和Y染色體分別有143個(gè)和9個(gè)，線粒體上僅有4個(gè)(圖1C)。根據(jù)在基因組的位置，這些circRNAs中有義重疊最多(77%)，其次是基因間區(qū)(14%)，反義circRNAs和位于外顯子區(qū)的circRNAs均為4%，內(nèi)含子區(qū)最少，僅有1% (圖1D)。

表1 circRNA引物序列信息

圖1 去勢(shì)和非去勢(shì)組背最長(zhǎng)肌鑒定circRNAs的特征

A：去勢(shì)組和非去勢(shì)組circRNAs差異；B：circRNAs分類；C：circRNAs長(zhǎng)度；D：染色體分布情況。MT：線粒體。

2.2 circRNAs差異表達(dá)和功能分析

去勢(shì)組和非去勢(shì)組相比，共篩選到370個(gè)DECs，其中有217個(gè)上調(diào)，153個(gè)下調(diào)(| log2Foldchange | > 1,p< 0.8) (圖2)。對(duì)這些DECs來源基因進(jìn)行功能分析，GO分析主要富集的細(xì)胞組分為細(xì)胞器，生物學(xué)過程主要是各種代謝過程，分子功能主要富集于酶、蛋白質(zhì)、核苷酸的結(jié)合(表2)。KEGG分析主要富集于肌肉發(fā)育、肌纖維類型轉(zhuǎn)化和能量代謝相關(guān)通路，例如Wnt、泛素介導(dǎo)的蛋白水解、甲狀腺激素、淀粉和蔗糖代謝、AMPK等信號(hào)通路(圖3)。

2.3 circRNAs-miRNAs互作網(wǎng)絡(luò)的構(gòu)建和分析

為進(jìn)一步解析這些DECs的功能，基于內(nèi)源競(jìng)爭(zhēng)RNA (competing endogenous RNAs,ceRNA)機(jī)制，結(jié)合前期獲得的去勢(shì)和非去勢(shì)豬背最長(zhǎng)肌差異表達(dá)的miRNAs，構(gòu)建DECs-miRNA互作網(wǎng)絡(luò)。如圖4所示，網(wǎng)絡(luò)圖共富集69個(gè)circRNAs，8個(gè)miRNA，共234個(gè)edges，每個(gè)circRNAs最少有2個(gè)以上miRNA結(jié)合位點(diǎn)。其中miR-1靶circRNA最多，共38個(gè)，miR-133a-3p靶circRNA最少，共20個(gè)。其中circ_1060、circ_5230、circ_6457、circ_7356和circ_7733的miRNA結(jié)合位點(diǎn)最多，都有8個(gè)。

圖2 去勢(shì)和非去勢(shì)組差異表達(dá)circRNAs火山圖

圖中一個(gè)點(diǎn)代表一個(gè)circRNA，每個(gè)點(diǎn)的橫坐標(biāo)值是該circRNA的log2Foldchange (Foldchange=去勢(shì)組TPM/非去勢(shì)組TPM)，每個(gè)點(diǎn)的縱坐標(biāo)為該circRNA在兩組的–log10value。

圖3 去勢(shì)和非去勢(shì)組DECs來源基因KEGG富集分析

表2 去勢(shì)和非去勢(shì)組DECs來源基因GO富集分析

互作網(wǎng)絡(luò)圖中富集到的69個(gè)circRNAs中，有9個(gè)位于基因間區(qū)，剩下60個(gè)circRNAs來源于42個(gè)編碼基因。為了解這些circRNAs的功能，本研究對(duì)其來源mRNA進(jìn)行了KEGG富集分析，結(jié)果發(fā)現(xiàn)這些mRNA顯著富集于蛋白、脂質(zhì)和糖類代謝通路(圖5)。

圖4 去勢(shì)相關(guān)miRNA與DECs互作分析

圖5 miRNA-DECs網(wǎng)絡(luò)富集circRNAs的KEGG分析

2.4 circRNAs的驗(yàn)證

為驗(yàn)證所篩選circRNAs真實(shí)性，綜合考慮轉(zhuǎn)錄本長(zhǎng)度和表達(dá)量水平，本研究選擇circRNA_2241和circRNA_4237進(jìn)行驗(yàn)證，二者分別位于14號(hào)染色體和2號(hào)染色體。結(jié)果如圖6所示，RNase R消化前后circRNA_2241和circRNA_4237擴(kuò)增量略有差異，而線性的GAPDH在RNase R消化后沒有擴(kuò)增產(chǎn)物。進(jìn)一步將擴(kuò)增產(chǎn)物進(jìn)行測(cè)序，證實(shí)這兩個(gè)circRNA反向剪切位點(diǎn)真實(shí)存在(圖7)。定量PCR分析表明，與非去勢(shì)組相比，circRNA_2241在去勢(shì)組表達(dá)量下調(diào)，circRNA_4237在去勢(shì)組表達(dá)量上調(diào)，表達(dá)變化趨勢(shì)和測(cè)序結(jié)果一致(圖8)。

圖6 RNase R消化法驗(yàn)證circRNA_2241和circRNA_ 4237

RNase R+：添加RNase R組，RNase R-：不添加RNase R組。

為驗(yàn)證預(yù)測(cè)的DECs-miRNAs互作關(guān)系，進(jìn)一步選取circRNA_2241和miR-1進(jìn)行驗(yàn)證。在豬骨骼肌衛(wèi)星細(xì)胞添加不同劑量睪酮，qRT-PCR發(fā)現(xiàn)睪酮抑制miR-1表達(dá)，促進(jìn)circRNA_2241表達(dá)(圖10)，睪酮對(duì)二者表達(dá)調(diào)控作用是相反的，提示circRNA_ 2241可能是miR-1靶基因。

圖7 circRNA_2241和circRNA_4237剪切位點(diǎn)測(cè)序結(jié)果

紅色箭頭表示反向剪切位點(diǎn)。

圖8 circRNA_2241和circRNA_4237在去勢(shì)組和非去勢(shì)組背最長(zhǎng)肌的表達(dá)

*<0.05表示差異顯著，**<0.01表示差異極顯著。

3 討論

以往的研究從mRNA、miRNA以及l(fā)ncRNA等角度探討了去勢(shì)后肌肉發(fā)育變化的分子機(jī)制。近年來研究表明circRNAs也參與肌肉發(fā)育調(diào)控，所以本研究首次通過高通量測(cè)序比較去勢(shì)和非去勢(shì)淮南豬背最長(zhǎng)肌circRNAs表達(dá)譜。測(cè)序共鑒定5866個(gè)circRNAs，主要為同義重疊類型，位于內(nèi)含子區(qū)的最少，這與之前研究結(jié)果類似[25,26]。這些circRNAs大于2000 bp的最多，小于2000 bp中，200～400 bp最多。這些circRNAs分布于所有染色體，其中Y染色體和線粒體最少。

圖9 睪酮對(duì)circRNA_2241和miR-1表達(dá)量的影響

**<0.01表示差異極顯著。

肌肉生長(zhǎng)受細(xì)胞數(shù)量和蛋白合成降解兩個(gè)方面的調(diào)控，其中肌細(xì)胞數(shù)量在胚胎期已固定，與成肌細(xì)胞增殖分化相關(guān)，需要肌源性蛋白適時(shí)合成和降解。出生后肌肉肥大主要是蛋白質(zhì)分解代謝和合成代謝動(dòng)態(tài)平衡的過程[27]。之前研究表明，家畜去勢(shì)后產(chǎn)肉量降低，即肌肉量減少。本研究中，KEGG富集分析結(jié)果提示，DECs可能通過泛素系統(tǒng)、甲狀腺激素、Wnt、AMPK等信號(hào)通路參與去勢(shì)后肌肉發(fā)育、肌纖維類型轉(zhuǎn)化以及能量代謝的調(diào)控。

DECs來源基因最主要富集到的是泛素介導(dǎo)的蛋白水解信號(hào)通路，其中泛素蛋白酶體系統(tǒng)(ubiquitin- proteasomesystem, UPS)參與調(diào)控肌肉蛋白降解，泛素蛋白連接酶肌肉降解因子(muscle atrophy F-box, MAFbx)泛素化并降解分化蛋白，抑制肌肉蛋白合成[28]。肌肉環(huán)指蛋白1 (muscle RING finger 1, MuRF-1)通過泛素化導(dǎo)致集鈣蛋白1 (calsequestrin 1, CASQ1)和肌球蛋白重鏈(myosin heavy chain, MYH)降解[29,30]?？炻∈怯绊懭馄焚|(zhì)的重要因素[31]，而MAFbx和MuRF-1降解快慢肌速度不同[32,33]。所以UPS系統(tǒng)一方面調(diào)控肌肉分化相關(guān)蛋白降解，調(diào)控肌肉分化，另一方面通過對(duì)快慢肌降解速度不同，間接調(diào)控肉品質(zhì)。KEGG分析還富集到甲狀腺激素信號(hào)通路，低水平甲狀腺激素促進(jìn)骨骼肌生長(zhǎng)，高水平抑制骨骼肌生長(zhǎng)[34]。甲狀腺激素可促進(jìn)糖和脂肪氧化，增加脂肪分解[35]。

Wnt信號(hào)通路中Wnt5a促進(jìn)生肌性定向分化[36]，Wnt10b抑制成肌細(xì)胞的成脂分化[37]，Wnt5a促進(jìn)慢肌纖維增多而Wnt11促進(jìn)快肌纖維增加[38]，myostatin通過Wnt/β-catenin信號(hào)通路調(diào)控慢肌纖維發(fā)育[39]。AMPK在肌肉能量代謝中起重要調(diào)控作用，AMPK活化促進(jìn)GLUT4表達(dá)，促進(jìn)其轉(zhuǎn)運(yùn)到細(xì)胞膜，提高酵解型肌纖維的葡萄糖吸收[40]。當(dāng)肌肉中葡萄糖過量時(shí)，AMPK可降低磷酸化糖原合成酶(glycogen synthase, GS)活性抑制糖原合成[41]。此外，AMPK也參與調(diào)控骨骼肌的生長(zhǎng)、肥大和再生[42]。

為進(jìn)一步分析這些circRNAs的調(diào)控機(jī)制，基于ceRNA機(jī)制，結(jié)合前期獲得的去勢(shì)相關(guān)miRNAs，繪制了DECs-miRNA互作網(wǎng)絡(luò)。該網(wǎng)絡(luò)共富集69個(gè)DECs (占差異circRNAs的18.65%)和8個(gè)miRNAs，平均每個(gè)miRNAs靶向29個(gè)circRNAs。富集到的miR-1和miR-133都是肌肉特異性miRNAs，也是重要的非肌性基因表達(dá)的抑制因子。轉(zhuǎn)錄因子如成肌分化抗原(myogenic differentiation, MyoD)、肌細(xì)胞生成素(myogenin, MyoG)、血清應(yīng)答因子(serum response factor, SRF)、肌肉增強(qiáng)因子2 (myocyte enhancer factor 2, AMEF2)都是miR-1和miR-133a的調(diào)節(jié)因子。miR-1靶基因有組蛋白脫乙?；? (histone deacetylase 4, HDAC4)，轉(zhuǎn)錄因子YY1 (ying-yang 1)和調(diào)寧蛋白3 (calponin 3, CNN3)，其中HDAC4是肌肉表達(dá)基因的轉(zhuǎn)錄抑制因子[43,44]，YY1在肌肉基因轉(zhuǎn)錄中起負(fù)調(diào)控作用[45]，CNN3調(diào)控肌動(dòng)蛋白和肌球蛋白的重組和分解[46]。Hong等[47]發(fā)現(xiàn)豬miR-1的2個(gè)SNPs位點(diǎn)與I型和II型肌纖維面積和組成相關(guān)。miR-133靶向SRF促進(jìn)成肌細(xì)胞增殖[43]，調(diào)控骨骼肌分化過程中的主腦樣蛋白1 (ma-stermind like transcriptional coactivator 1, MAML1)、胰島素樣生長(zhǎng)因子1 (insulin like growth factor 1, IGF-1)和神經(jīng)多嘧啶束結(jié)合蛋白(polypyrimidine tract binding protein 1, PTBP1)[48]。此外miR-133通過細(xì)胞外信號(hào)調(diào)節(jié)激酶(extracellular regulated protein kinases, ERK)促進(jìn)成肌細(xì)胞分化[49]。谷氨酰胺乙酸(alpha glucosidase, GAA)通過miR-133a-3p和miR- 1a-3p激活A(yù)KT/mTOR/S6K信號(hào)通路，促進(jìn)成肌細(xì)胞分化和骨骼肌生長(zhǎng)[50]。Wnt/β-catenin信號(hào)通路通過誘導(dǎo)miR-133b和miR-206抑制Pax7表達(dá)，誘導(dǎo)肌源性分化[51]。由此可見，這69個(gè)DECs可能通過競(jìng)爭(zhēng)性吸附miR-1、miR-133等參與肌細(xì)胞增殖、分化、肌纖維發(fā)育等過程，進(jìn)而參與肌肉發(fā)育和肉質(zhì)性狀的調(diào)控。為進(jìn)一步驗(yàn)證這69個(gè)DECs，對(duì)其來源基因進(jìn)行KEGG分析，主要富集于碳水化合物、脂類和蛋白代謝相關(guān)通路，提示這些DECs參與去勢(shì)后肌肉能量代謝的調(diào)控。

為驗(yàn)證高通量測(cè)序結(jié)果的準(zhǔn)確性，根據(jù)circRNAs的長(zhǎng)度和表達(dá)量，選擇circRNA_2241和circRNA_ 4237進(jìn)行驗(yàn)證。結(jié)果發(fā)現(xiàn)使用RNase R處理對(duì)circRNA_2241和circRNA_4237的表達(dá)量影響不顯著，但RNase R處理后，GAPDH無擴(kuò)增產(chǎn)物。同時(shí)使用反向引物擴(kuò)增測(cè)序證實(shí)circRNA_2241和circRNA_4237確實(shí)以環(huán)狀存在。RT-qPCR結(jié)果提示circRNA_2241和circRNA_4237在兩組表達(dá)變化趨勢(shì)和測(cè)序一致。同時(shí)在豬骨骼肌衛(wèi)星細(xì)胞中，添加睪酮促進(jìn)circRNA_2241表達(dá)，抑制miR-1表達(dá)。這與本研究中，非去勢(shì)組circRNA_2241表達(dá)量高于去勢(shì)組相一致。至于circRNA_2241是否能競(jìng)爭(zhēng)性吸附miR-1還需要進(jìn)一步構(gòu)建載體，通過雙熒光素酶系統(tǒng)進(jìn)行驗(yàn)證。同時(shí)circRNA_2241通過吸附miR-1間接影響哪個(gè)靶基因參與肉質(zhì)性狀的調(diào)控，也需進(jìn)一步的細(xì)胞試驗(yàn)驗(yàn)證。

綜上所述，本研究通過高通量測(cè)序篩選了豬去勢(shì)后背最長(zhǎng)肌的DECs，構(gòu)建了DECs-miRNAs互作網(wǎng)絡(luò)，使用反向引物和RT-PCR證實(shí)篩選circRNAs的真實(shí)性，并通過細(xì)胞試驗(yàn)證實(shí)睪酮對(duì)circRNA_ 2241和miR-1的表達(dá)調(diào)控。這些結(jié)果提示circRNAs可能通過與miRNAs互作，參與去勢(shì)后肌肉發(fā)育、肌纖維類型和能量代謝的調(diào)控，為解析去勢(shì)后肌肉發(fā)育的分子調(diào)控機(jī)制提供了新思路。

[1] Trefan L, Doeschl-Wilson A, Rooke JA, Terlouw C, Bünger L. Meta-analysis of effects of gender in combination with carcass weight and breed on pork quality., 2013, 91(3): 1480–1492.

[2] Li Y, Wang MM, Li QF, Gao YX, Li Q, Li JG, Cao YF. Transcriptome profiling of longissimus lumborum in Holstein bulls and steers with different beef qualities., 2020, 15(6): e0235218.

[3] Zhou ZK, Gao X, Li JY, Chen JB, Xu SZ. Effect of castration on carcass quality and differential gene expression of longissimus muscle between steer and bull., 2011, 38(8):5307–5312.

[4] Zhang YY, Wang HB, Wang YN, Wang HC, Zhang S, Hong JY, Guo HF, Chen D, Yang Y, Zan LS. Transcriptome analysis of mRNA and microRNAs in intramuscular fat tissues of castrated and intact male Chinese Qinchuan cattle., 2017, 12(10): e0185961.

[5] Wang J, Hua LS, Chen JF, Zhang JQ, Ren QL, Bai HJ, Guo HX, Xu ZX, Xing BS, Bai XX, Cao H. Effect of castration on gene expression in Longissimus dorsi muscle of Huainan male pig by transcriptome analysis., 2019, 50(9): 1746–1758.

王璟, 滑留帥, 陳俊峰, 張家慶, 任巧玲, 白紅杰, 郭紅霞, 徐照學(xué), 邢寶松, 白獻(xiàn)曉, 曹海. 去勢(shì)對(duì)淮南公豬背最長(zhǎng)肌轉(zhuǎn)錄組的影響. 畜牧獸醫(yī)學(xué)報(bào), 2019, 50(9): 1746–1758.

[6] Bai Y, Huang JM, Liu G, Zhang JB, Wang JY, Liu CK, Fang MY. A comprehensive microRNA expression profile of the backfat tissue from castrated and intact full-sib pair male pigs., 2014, 15: 47.

[7] Cai ZW, Zhang LF, Chen ML, Jiang XL, Xu NY. Castration-induced changes in microRNA expression profiles in subcutaneous adipose tissue of male pigs., 2014, 55(2): 259–266.

[8] Cai ZW, Zhang LF, Jiang XL, Sheng YF, Xu NY. Differential miRNA expression profiles in the longissimus dorsi muscle between intact and castrated male pigs., 2015, 99: 99–104.

[9] Wang J, Hua LS, Chen JF, Zhang JQ, Bai XX, Gao BW, Li CJ, Shi ZH, Sheng WD, Gao Y, Xing BS. Identification and characterization of long non-coding RNAs in subcu-taneous adipose tissue from castrated and intact full-sib pair Huainan male pigs., 2017, 18(1): 542.

[10] Xing BS, Bai XX, Guo HX, Chen JF, Hua LS, Zhang JQ, Ma Q, Ren QL, Wang HS, Wang J. Long non-coding RNA analysis of muscular responses to testosterone deficiency in Huainan male pigs., 2017, 88(9): 1451– 1456.

[11] Legnini I, Di Timoteo G, Rossi F, Morlando M, Briganti F, Sthandier O, Fatica A, Santini T, Andronache A, Wade M, Laneve P, Rajewsky N, Bozzoni I. Circ-ZNF609 is a circular rna that can be translated and functions in myogenesis., 2017, 66(1): 22–37.e29.

[12] Wang K, Gan TY, Li N, Liu CY, Zhou LY, Gao JN, Chen C, Yan KW, Ponnusamy M, Zhang YH, Li PF. Circular RNA mediates cardiomyocyte death via miRNA-dependent upregulation of MTP18 expression., 2017, 24(6): 1111–1120.

[13] Ouyang HJ, Chen XL, Li WM, Li ZH, Nie QH, Zhang XQ. Circular RNA circSVIL promotes myoblast proliferation and differentiation by sponging miR-203 in chicken., 2018, 9: 172.

[14] Li H, Wei XF, Yang JM, Dong D, Hao D, Huang YZ, Lan XY, Plath M, Lei CZ, Ma Y, Lin FP, Bai YY, Chen H. circFGFR4 promotes differentiation of myoblasts via binding miR-107 to relieve its inhibition of Wnt3a., 2018, 11: 272–283.

[15] Li H, Yang JM, Wei XF, Song CC, Dong D, Huang YZ, Lan XY, Plath M, Lei CZ, Ma Y, Qi XL, Bai YY, Chen H. CircFUT10 reduces proliferation and facilitates differen-tiation of myoblasts by sponging miR-133a., 2018, 233(6): 4643–4651.

[16] Yue BL, Wang J, Ru WX, Wu JY, Cao XK, Yang HY, Huang YZ, Lan XY, Lei CZ, Huang BZ, Chen H. The circular RNA circHUWE1 sponges the miR-29b-AKT3 axis to regulate myoblast development., 2020, 19:1086–1097.

[17] Shen XM, Zhang XY, Ru WX, Huang YZ, Lan XY, Lei CZ, Chen H. circINSR promotes proliferation and reduces apoptosis of embryonic myoblasts by sponging miR-34a., 2020, 19: 986–999.

[18] Liu YJ, Liu HT, Li Y, Mao R, Yang HW, Zhang YC, Zhang Y, Guo PS, Zhan DF, Zhang TT. circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/ EZH2 axis., 2020, 10(10): 4705–4719.

[19] Yang LY, Bin Z, Hui S, Rong L, You BS, Wu PP, Han XY, Qian H, Xu WR. The role of CDR1as in proliferation and differentiation of human umbilical cord-derived mesen-chymal stem cells., 2019, 2019: 2316834.

[20] Jiang R, Li H, Yang JM, Shen XM, Song CC, Yang ZX, Wang XG, Huang YZ, Lan XY, Lei CZ, Chen H. circRNA profiling reveals an abundant circFUT10 that promotes adipocyte proliferation and inhibits adipocyte differentia-tion via sponging let-7., 2020, 20: 491–501.

[21] Zhou L, Chen JH, Li ZZ, Li XX, Hu XD, Huang Y, Zhao XK, Liang CZ, Wang Y, Sun L, Shi M, Xu XH, Shen F, Chen MS, Han ZJ, Peng ZY, Zhai QN, Chen J, Zhang ZF, Yang RL, Ye JX, Guan ZC, Yang HM, Gui YT, Wang J, Cai ZM, Zhang XQ. Integrated profiling of microRNAs and mRNAs: microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma., 2010, 5(12): e15224.

[22] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2., 2014, 15(12): 550.

[23] Cai ZW, Zhang LF, Chen ML, Jiang XL, Xu NY. Castration-induced changes in microRNA expression profiles in subcutaneous adipose tissue of male pigs., 2014, 55(2): 259–266.

[24] Li JX, Su T, Zou C, Luo WZ, Shi GL, Chen L, Fang CC, Li CC. Long non-coding RNAregulates porcine satellite cell differentiation through/and., 2020, 8: 518724.

[25] Sun WX, Sun XC, Chu WW, Yu SG, Dong FL, Xu GF. circRNA expression profiles in human visceral preadi-pocytes and adipocytes., 2020, 21(2): 815–821.

[26] Xu TY, Wu J, Han P, Zhao ZM, Song XF. circular RNA expression profiles and features in human tissues: a study using RNA-seq data., 2017, 18(Suppl 6): 680.

[27] Mohammadabadi M, Bordbar F, Jensen J, Du M, Guo W. Key genes regulating skeletal muscle development and growth in farm animals., 2021, 11(3): 835.

[28] Chen K, Cheng HH, Zhou RJ. Molecular mechanisms and functions of autophagy and the ubiq-uitin-proteasome pathway., 2012, 34(1): 5–18.

陳科, 程漢華, 周榮家. 自噬與泛素化蛋白降解途徑的分子機(jī)制及其功能. 遺傳, 2012, 34(1):5–18.

[29] Gregorio CC, Perry CN, McElhinny AS. Functional properties of the titin/connectin-associated proteins, the muscle-specific RING finger proteins (MURFs), in striated muscle., 2005, 26(6–8): 389–400.

[30] Kedar V, McDonough H, Arya R, Li HH, Rockman HA, Patterson C. Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I., 2004, 101(52): 18135–18140.

[31] Shen LY, Zhang SH, Wu ZH, Zheng MY, Li XW, Zhu L. The influence of satellite cells on meat quality and its differential regulation., 2013, 35(9): 1081–1086.

沈林園, 張順華, 吳澤輝, 鄭夢(mèng)月, 李學(xué)偉, 朱礪. 骨骼肌衛(wèi)星細(xì)胞對(duì)肉品質(zhì)的影響及其分化調(diào)控. 遺傳, 2013, 35(9): 1081–1086.

[32] Phillips SM, Glover EI, Rennie MJ. Alterations of protein turnover underlying disuse atrophy in human skeletal muscle., 2009, 107(3): 645–654.

[33] Salanova M, Schiffl G, Püttmann B, Schoser BG, Blottner D. Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures., 2008, 212(3): 306–318.

[34] Millward DJ. Interactions between growth of muscle and stature: mechanisms involved and their nutritional sensitivity to dietary protein: the protein-stat revisited., 2021, 13(3): 729.

[35] Volke L, Krause K. Effect of thyroid hormones on adipose tissue flexibility., 2021, 10(1): 1–9.

[36] Reggio A, Rosina M, Palma A, Cerquone Perpetuini A, Petrilli LL, Gargioli C, Fuoco C, Micarelli E, Giuliani G, Cerretani M, Bresciani A, Sacco F, Castagnoli L, Cesareni G. Adipogenesis of skeletal muscle fibro/adipogenic progenitors is affected by the WNT5a/GSK3/β-catenin axis., 2020, 27(10): 2921–2941.

[37] Park YK, Park B, Lee S, Choi K, Moon Y, Park H. Hypoxia-inducible factor-2α-dependent hypoxic induction of Wnt10b expression in adipogenic cells., 2013, 288(36): 26311–26322.

[38] Anakwe K, Robson L, Hadley J, Buxton P, Church V, Allen S, Hartmann C, Harfe B, Nohno T, Brown AMC, Evans DJR, Francis-West P. Wnt signalling regulates myogenic differentiation in the developing avian wing., 2003, 130(15): 3503–3514.

[39] Jiang YL, Lian ZX, Li N, Wu CX. Myostatin: a negative regulator of skeletal muscle mass., 2000, 22(2): 119–121.

姜運(yùn)良, 連正興, 李寧, 吳常信. 肌肉生長(zhǎng)抑制素基因的研究進(jìn)展. 遺傳, 2000, 22(2): 119–121.

[40] Kido K, Egawa T, Fujiyoshi H, Suzuki H, Kawanaka K, Hayashi T. AMPK is indispensable for overload-induced muscle glucose uptake and glycogenesis but dispensable for inducing hypertrophy in mice., 2021, 35(4): e21459.

[41] Liu XH, Bauman WA, Cardozo CP. Myostatin inhibits glucose uptake via suppression of insulin-dependent and -independent signaling pathways in myoblasts., 2018, 6(17): e13837.

[42] Thomson DM. The role of AMPK in the regulation of skeletal muscle size, hypertrophy, and regeneration., 2018, 19(10): 3125.

[43] Chen JF, Mandel EM, Thomson JM, Wu QL, Callis TE, Hammond SM, Conlon FL, Wang DZ. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation., 2006, 38(2): 228–233.

[44] Backs J, Worst BC, Lehmann LH, Patrick DM, Jebessa Z, Kreusser MM, Sun Q, Chen L, Heft C, Katus HA, Olson EN. Selective repression of MEF2 activity by PKA- dependent proteolysis of HDAC4., 2011, 195(3): 403–415.

[45] Lu LN, Zhou L, Chen EZ, Sun K, Jiang PY, Wang LJ, Su XX, Sun H, Wang HT. A novel YY1-miR-1 regulatory circuit in skeletal myogenesis revealed by genome-wide prediction of YY1-miRNA network., 2012, 7(2): e27596.

[46] Tang ZL, Liang RY, Zhao SP, Wang RQ, Huang RH, Li K. CNN3 is regulated by microRNA-1 during muscle development in pigs., 2014, 10(4): 377–385.

[47] Hong JS, Noh SH, Lee JS, Kim JM, Hong KC, Lee YS. Effects of polymorphisms in the porcine microRNA miR-1 locus on muscle fiber type composition and miR-1 expression., 2012, 506(1): 211–216.

[48] Iqbal A, Ping J, Ali S, Zhen G, Juan L, Kang JZ, Ziyi P, Huixian L, Zhihui Z. Role of microRNAs in myogenesis and their effects on meat quality in pig - A review., 2020, 33(12): 1873–1884.

[49] Feng Y, Niu LL, Wei W, Zhang WY, Li XY, Cao JH, Zhao SH. A feedback circuit between miR-133 and the ERK1/2 pathway involving an exquisite mechanism for regulating myoblast proliferation and differentiation., 2013, 4(11): e934.

[50] Wang YJ, Ma JD, Qiu WL, Zhang JW, Feng SY, Zhou XK, Wang X, Jin L, Long K, Liu LY, Xiao WH, Tang QZ, Zhu L, Jiang YZ, Li XW, Li MZ. Guanidinoacetic acid regulates myogenic differentiation and muscle growth through miR-133a-3p and miR-1a-3p co-mediated Akt/ mTOR/S6K signaling pathway., 2018, 19(9): 2837.

[51] Cui S, Li L, Mubarokah SN, Meech R. Wnt/β-catenin signaling induces the myomiRs miR-133b and miR-206 to suppress Pax7 and induce the myogenic differentiation program., 2019, 120(8): 12740–12751.

Analysis of differentially expressed circRNAs in longissimus muscle between castrated and intact male pigs

Baosong Xing1, Jing Wang1, Junfeng Chen1, Qiang Ma1, Qiaoling Ren1, Jiaqing Zhang1, Hua Zhang1, Liushuai Hua1, Jiajie Sun2, Hai Cao3

Castration can reduce odor and fights in boars, but the carcass yield is reduced, and the intramuscular fat content is increased. Understanding its molecular mechanism is of great significance for production. Recent studies have shown that circular RNAs (circRNAs) play an important role(s) in the regulation of muscle development. To explore the effects of circRNAs on the development of longissimus dorsi (LD) muscle after castration, six Huainan male pigs were selected and three of which were randomly castrated. Six pigs were slaughtered when their body weight reached around 130 kg, and the LD muscle samples were collected. The differentially expressed circRNAs (DECs) were screened by high-throughput sequencing and functionally analyzed using the KEGG databases. DECs-miRNAs network was constructed, and the expression profiles of candidate circRNAs and their interactions with miRNAs were verified in porcine skeletal muscle satellite cells. The results showed that a total of 5866 circRNAs were obtained, and 370 DECs were identified in LD muscle between the castrated and intact groups (| log2Foldchange | > 1,p<0.8). KEGG enrichment indicated that the parental genes for the DECs were mainly enriched in the pathways associated with muscle development, muscle fiber type transformation, and energy metabolism. There were 8 miRNAs and 69 circRNAs enriched in the DECs-miRNA network. circRNA_2241 and circRNA_4237 were selected for verification, which showed that these two circRNAs really existed and their expression profiles were consistent with the sequencing results. Further, preliminary analysis showed that circRNA_2241 interacted with miR-1, and testosterone promoted circRNA_2241 but inhibited miR-1 expression. These results confirmed that circRNAs might participate in the regulation of LD muscle development after castration by interacting with miRNAs, thereby providing new materials and references for analyses on the molecular mechanisms of castration on the regulation of muscle development.

circRNAs; castration; male pigs; longissimus muscle

2021-04-27;

2021-07-28

國(guó)家自然科學(xué)基金青年基金項(xiàng)目(編號(hào)：31601927)，河南省農(nóng)業(yè)科學(xué)院科技創(chuàng)新創(chuàng)意項(xiàng)目(編號(hào)：2020CX18)和河南省重點(diǎn)研發(fā)與推廣專項(xiàng)(編號(hào)：212102110010)資助[Supported by the National Natural Science Foundation of China (No. 31601927), Technological Innovation and Creative Project from the Henan Academy of Agricultural Sciences (No. 2020CX18) and Financial Budget Project of Henan Province (No. 212102110010)]

邢寶松，博士，副研究員，研究方向：豬的育種與管理。E-mail: bsxing@126.com

王璟，博士，副研究員，研究方向：遺傳育種。E-mail: wangjing_0407@163.com

10.16288/j.yczz.21-162

2021/8/27 12:31:16

URI: https://kns.cnki.net/kcms/detail/11.1913.r.20210825.1518.002.html

(責(zé)任編委: 李明洲)