去勢和非去勢公豬背最長肌circRNA差異表達(dá)分析

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

研究報告

去勢和非去勢公豬背最長肌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)動物科學(xué)學(xué)院，廣東省動物營養(yǎng)調(diào)控重點(diǎn)實(shí)驗(yàn)室/國家生豬種業(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

公豬去勢可減少異味和打斗，但去勢后產(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。為探究去勢后circRNAs對背最長肌發(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，當(dāng)體重達(dá)130 kg左右屠宰，采集背最長肌樣品，利用高通量測序篩選差異表達(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，去勢和非去勢組背最長肌樣品共獲得5866個circRNAs，兩組之間共有370個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circRNAs和8個miRNAs。選擇circRNA_2241和circRNA_4237進(jìn)行驗(yàn)證，結(jié)果發(fā)現(xiàn)這兩個circRNAs真實(shí)存在且表達(dá)趨勢與測序結(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)控豬去勢后背最長肌發(fā)育，從而為解析去勢對肌肉發(fā)育調(diào)控的分子機(jī)制提供參考。

環(huán)狀RNA；去勢；公豬；背最長肌

在豬生產(chǎn)中，去勢不僅減少公豬異味，還可減少打斗造成的經(jīng)濟(jì)損失。但去勢后，公豬產(chǎn)肉量和肌內(nèi)脂肪(intramuscular fat, IMF)含量與未去勢公豬差異很大，去勢公豬產(chǎn)肉量降低，脂肪沉積增加[1]。類似的，公牛去勢后肉質(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，有研究比較了去勢后背最長肌(longissimus dorsi, LD)和皮下脂肪mRNA、miRNA和lncRNA的表達(dá)變化。王璟等[5]比較了去勢和非去勢淮南公豬背最長肌轉(zhuǎn)錄組，共篩選到935個差異表達(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]對比23周齡去勢和非去勢豬皮下脂肪組織miRNA表達(dá)譜，發(fā)現(xiàn)177個差異表達(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]通過測序比較去勢和非去勢公豬皮下脂肪和背最長肌miRNAs表達(dá)差異，分別篩選到18個和7個差異表達(dá)miRNAs，其靶基因主要參與脂肪代謝和骨骼肌收縮。Wang等[9]在去勢和非去勢豬皮下脂肪組織篩選到18個差異表達(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，去勢和非去勢豬背最長肌有385個差異表達(dá)lncRNAs，主要與雌激素受體的信號傳導(dǎo)以及骨骼肌發(fā)育相關(guān)。雖然上述研究篩選了去勢后背最長肌和皮下脂肪組織全轉(zhuǎn)錄組差異表達(dá)譜，但去勢調(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通過競爭性吸附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在去勢后豬肌肉發(fā)育中的調(diào)控機(jī)制，本研究利用高通量測序比較了去勢公豬和未去勢公豬背最長肌circRNA的表達(dá)差異，為進(jìn)一步解析去勢對肌肉發(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，另1頭相同部位進(jìn)行偽手術(shù)處理，保證去勢組和非去勢組豬只所受手術(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)采集背最長肌樣品(體右側(cè)，第6～7肋骨)，液氮保存。

1.2 RNA提取和測序建庫

使用TRIzol (美國Invitrogen公司)分別提取6個背最長肌樣品總RNA。利用瓊脂糖凝膠電泳、Agilent 2100生物分析儀(美國安捷倫科技公司)和NanoDrop分光光度計(jì)(美國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Epicentre公司)去除核糖體RNA。去勢豬RNA樣品和非去勢豬RNA樣品分別混合后測序。使用Illumina TruSeq?RNA樣品制備試劑盒生成測序文庫，在Illumina Hiseq 2500平臺進(jìn)行測序。

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，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，每千個堿基的轉(zhuǎn)錄每百萬映射讀取的轉(zhuǎn)錄本數(shù)）對circRNAs進(jìn)行歸一化處理，計(jì)算每個circRNA在每個樣品的表達(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肌去勢相關(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)域沒有錯配，且能量< –18 kcal /摩爾的miRNAs。使用Cytoscape軟件對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。用RNase R (3 U/μg，美國Epicenter Biotechno-logies公司)處理背最長肌提取的總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circRNA_2241和circRNA_ 4237在去勢和非去勢豬背最長肌的表達(dá)水平，所用RNA與測序所用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)證去勢對肌肉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，分別模擬非去勢組和去勢組。當(dāng)細(xì)胞達(dá)到70%～80%融合時，在培養(yǎng)基中添加不同濃度睪酮，對照組不添加睪酮，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，檢測睪酮對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

去勢和非去勢公豬背最長肌共獲得5866個circRNAs，兩組共有circRNAs為5205個(圖1A)。這些circRNAs長度范圍150 bp～99,406 bp，平均長度5494 bp (圖1B)。這些circRNAs在全部染色體均有分布，6號染色體分布的circRNAs最多，占10.54%。X染色體和Y染色體分別有143個和9個，線粒體上僅有4個(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 去勢和非去勢組背最長肌鑒定circRNAs的特征

A：去勢組和非去勢組circRNAs差異；B：circRNAs分類；C：circRNAs長度；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

去勢組和非去勢組相比，共篩選到370個DECs，其中有217個上調(diào)，153個下調(diào)(| log2Foldchange | > 1,p< 0.8) (圖2)。對這些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)源競爭RNA (competing endogenous RNAs,ceRNA)機(jī)制，結(jié)合前期獲得的去勢和非去勢豬背最長肌差異表達(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circRNAs，8個miRNA，共234個edges，每個circRNAs最少有2個以上miRNA結(jié)合位點(diǎn)。其中miR-1靶circRNA最多，共38個，miR-133a-3p靶circRNA最少，共20個。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。

圖2 去勢和非去勢組差異表達(dá)circRNAs火山圖

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

圖3 去勢和非去勢組DECs來源基因KEGG富集分析

表2 去勢和非去勢組DECs來源基因GO富集分析

互作網(wǎng)絡(luò)圖中富集到的69個circRNAs中，有9個位于基因間區(qū)，剩下60個circRNAs來源于42個編碼基因。為了解這些circRNAs的功能，本研究對其來源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號染色體和2號染色體。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)行測序，證實(shí)這兩個circRNA反向剪切位點(diǎn)真實(shí)存在(圖7)。定量PCR分析表明，與非去勢組相比，circRNA_2241在去勢組表達(dá)量下調(diào)，circRNA_4237在去勢組表達(dá)量上調(diào)，表達(dá)變化趨勢和測序結(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在去勢組和非去勢組背最長肌的表達(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序比較去勢和非去勢淮南豬背最長肌circRNAs表達(dá)譜。測序共鑒定5866個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。

肌肉生長受細(xì)胞數(shù)量和蛋白合成降解兩個方面的調(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態(tài)平衡的過程[27]。之前研究表明，家畜去勢后產(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等信號通路參與去勢后肌肉發(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通路，低水平甲狀腺激素促進(jìn)骨骼肌生長，高水平抑制骨骼肌生長[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，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、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DECs (占差異circRNAs的18.65%)和8個miRNAs，平均每個miRNAs靶向29個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信號通路，促進(jìn)成肌細(xì)胞分化和骨骼肌生長[50]。Wnt/β-catenin信號通路通過誘導(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DECs可能通過競爭性吸附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DECs，對其來源基因進(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參與去勢后肌肉能量代謝的調(diào)控。

為驗(yàn)證高通量測序結(jié)果的準(zhǔn)確性，根據(jù)circRNAs的長度和表達(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。同時使用反向引物擴(kuò)增測序證實(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)變化趨勢和測序一致。同時在豬骨骼肌衛(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，非去勢組circRNA_2241表達(dá)量高于去勢組相一致。至于circRNA_2241是否能競爭性吸附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circRNA_2241通過吸附miR-1間接影響哪個靶基因參與肉質(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序篩選了豬去勢后背最長肌的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，為解析去勢后肌肉發(fā)育的分子調(diào)控機(jī)制提供了新思路。

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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

國家自然科學(xué)基金青年基金項(xiàng)目(編號：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：2020CX18)和河南省重點(diǎn)研發(fā)與推廣專項(xiàng)(編號：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)