陳慧芳，黃綺亮, 胡智超, 潘曉婷, 吳志勝, 白銀山

外泌體microRNA在豬成熟和閉鎖卵泡中的表達(dá)差異及功能分析

陳慧芳，黃綺亮, 胡智超, 潘曉婷, 吳志勝, 白銀山

佛山科學(xué)技術(shù)學(xué)院生命科學(xué)與工程學(xué)院，廣東佛山528231

【】通過(guò)分析成熟卵泡液外泌體（mature follicular fiuid Exosomes, mffEXs）和閉鎖卵泡液外泌體（atretic follicular fiuid Exosomes, affEXs）miRNA的表達(dá)差異，探索卵泡液外泌體（EXs）miRNA在卵泡發(fā)育和閉鎖過(guò)程中的調(diào)控作用。本研究通過(guò)抽提4—6 mm豬成熟發(fā)育和閉鎖卵泡的卵泡液分離外泌體，進(jìn)行粒徑分析及Western Blot檢測(cè)對(duì)EXs進(jìn)行鑒定，接下來(lái)對(duì)特征性EXs攜帶的miRNA測(cè)序和功能富集分析，篩選關(guān)鍵信號(hào)通路和差異基因。最后，將mffEXs和affEXs作為添加劑進(jìn)行顆粒細(xì)胞培養(yǎng)，利用Q-PCR檢測(cè)技術(shù)分析關(guān)鍵基因的表達(dá)，驗(yàn)證兩類卵泡液內(nèi)EXs miRNA在卵泡發(fā)育中的調(diào)控功能。成功分離了mffEXs和affEXs，對(duì)比mffEXs測(cè)序結(jié)果，affEXs中有90個(gè)miRNA上調(diào)表達(dá)，220個(gè)miRNA下調(diào)表達(dá)，表明了卵泡液中的miRNA表達(dá)水平與調(diào)控卵泡發(fā)育有關(guān)；KEGG富集分析結(jié)果顯示兩類卵泡的差異信號(hào)通路主要集中在Ras、cAMP、P53和MAPK等信號(hào)通路，涉及調(diào)控卵母細(xì)胞發(fā)育、減數(shù)分裂以及顆粒細(xì)胞細(xì)胞周期等生物學(xué)功能。在閉鎖卵泡中，上調(diào)表達(dá)的ssc-let-7a和ssc-miR-133a-3p分別潛在靶向調(diào)控細(xì)胞周期蛋白依賴性激酶（）和胰島素生長(zhǎng)因子（），抑制了G1和G2/M期的運(yùn)轉(zhuǎn)和類固醇激素代謝，促使顆粒細(xì)胞周期運(yùn)轉(zhuǎn)受阻和顆粒細(xì)胞凋亡，引起卵泡閉鎖的發(fā)生；下調(diào)的ssc-miR-21-5p潛在靶向腫瘤抑癌基因（），抑制細(xì)胞周期運(yùn)轉(zhuǎn)，促使顆粒細(xì)胞凋亡。在體外培養(yǎng)的顆粒細(xì)胞中分別添加mffEXs和affEXs，Q-PCR結(jié)果顯示在mffEXs中顯著上調(diào)表達(dá)，而顯著下調(diào)表達(dá)，表明了測(cè)序分析結(jié)果的可靠性。這些結(jié)果均顯示了affEXs中miRNA表達(dá)水平的變化促使顆粒細(xì)胞凋亡和細(xì)胞周期阻滯，引起卵泡閉鎖。豬affEXs攜帶miRNA增加了對(duì)、和的表達(dá)調(diào)控，抑制顆粒細(xì)胞細(xì)胞周期運(yùn)轉(zhuǎn)和類固醇激素代謝等信號(hào)通路，引起顆粒細(xì)胞凋亡，導(dǎo)致卵泡閉鎖。

豬；成熟卵泡液；閉鎖卵泡液；EXs；miRNA

0 引言

【研究意義】卵泡閉鎖是指一些卵泡發(fā)育到一定階段后停止發(fā)育并發(fā)生退化的一種普遍存在的現(xiàn)象。在很多雌性動(dòng)物卵泡發(fā)育過(guò)程中，只有極少數(shù)卵泡能夠發(fā)育成熟并且排卵，絕大多數(shù)卵泡都會(huì)發(fā)生閉鎖[1-2]，這可能與卵泡液所含營(yíng)養(yǎng)物質(zhì)的差異[3]、激素[4]、氧化應(yīng)激[5]和各種細(xì)胞因子[6]等相關(guān)，它們能夠通過(guò)不同途徑將刺激信號(hào)級(jí)聯(lián)放大，引起通路中相關(guān)作用基因或轉(zhuǎn)錄因子活性增加或減少進(jìn)而調(diào)控細(xì)胞凋亡[7-8]。以往研究顯示激素和營(yíng)養(yǎng)物質(zhì)均能參與調(diào)控卵泡閉鎖，而關(guān)于外泌體（exosomes, EXs）的調(diào)控作用，目前還知之甚少[9]。EXs是一類由細(xì)胞分泌的直徑大小約30—150 nm的細(xì)胞外囊泡，因攜帶多種生物活性物質(zhì)，可進(jìn)行細(xì)胞間信息傳遞[10]。已有報(bào)道在各種體液中均含有大量EXs[11-12]，且EXs可選擇性地包裹細(xì)胞內(nèi)的多種活性調(diào)節(jié)物質(zhì)（miRNA、LncRNA和CircRNA等），其中miRNA含量最豐富，廣泛進(jìn)行細(xì)胞間物質(zhì)轉(zhuǎn)運(yùn)和信號(hào)轉(zhuǎn)導(dǎo)，調(diào)節(jié)功能基因表達(dá)[13-14]。近年來(lái)，相關(guān)卵泡液EXs miRNA的研究越來(lái)越多，但關(guān)于EXs miRNA對(duì)卵泡閉鎖的調(diào)控機(jī)制仍未見(jiàn)報(bào)道?！厩叭搜芯窟M(jìn)展】適宜的卵泡液微環(huán)境是促使卵泡發(fā)育的主要場(chǎng)所，研究顯示卵泡液EXs中的miRNA廣泛參與調(diào)控顆粒細(xì)胞類固醇激素代謝的相關(guān)通路[15]，涉及調(diào)節(jié)顆粒細(xì)胞增殖和雌二醇的合成與分泌[16]。卵泡液EXs miRNA的相關(guān)研究已在多種女性生殖系統(tǒng)疾病中作為檢測(cè)和治療的關(guān)鍵靶標(biāo)[17-20]。如高水平表達(dá)的EXs-miR-199可以抑制芳香化酶的合成與代謝，促使雄激素升高，引起了多囊卵巢綜合癥的發(fā)生發(fā)展[21]。卵泡閉鎖也可能直接受到卵泡液EXs miRNA的調(diào)控，但目前miRNA在卵泡閉鎖過(guò)程中的調(diào)控機(jī)制還并不清楚?！颈狙芯壳腥朦c(diǎn)】卵泡液攜帶的EXs miRNA在調(diào)控卵泡閉鎖方面的研究有待解析。本研究對(duì)豬EXs miRNA進(jìn)行測(cè)序分析，通過(guò)GO和KEGG功能富集分析，探究EXs miRNA對(duì)卵泡閉鎖的調(diào)控機(jī)制。【擬解決的關(guān)鍵問(wèn)題】通過(guò)對(duì)比成熟及閉鎖卵泡液中的EXs miRNA的表達(dá)情況，擬探究豬卵泡液EXs攜帶的miRNA在調(diào)控成熟卵泡發(fā)育和閉鎖卵泡發(fā)生的潛在分子機(jī)制。

1 材料與方法

試驗(yàn)于2018年10月至2019年12月于廣東省佛山科學(xué)技術(shù)學(xué)院生命科學(xué)與工程學(xué)院和華南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)學(xué)院進(jìn)行。

1.1 卵泡液的收集

從廣州市嘉禾屠宰場(chǎng)采集豬卵巢，裝入38.5℃含有青霉素和鏈霉素生理鹽水中的保溫瓶?jī)?nèi)，2 h內(nèi)運(yùn)回實(shí)驗(yàn)室。用37℃添加青鏈霉素的生理鹽水清洗3遍，隨后使用10 mL注射器吸取直徑為4—6 mm卵泡中的液體，4℃，12 000 r/min離心3 min去除細(xì)胞碎片；然后使用0.22mm過(guò)濾器過(guò)濾，進(jìn)行EXs提取。

1.2 外泌體的分離與鑒定

使用ExoQuick Exosome Isolation Kit（System Bioscience, SBI）分離mffEXs和affEXs，詳細(xì)步驟均參照試劑盒說(shuō)明書。分離完成后，一部分用于檢測(cè)鑒定，另一部分置于-80℃冰箱中保存?zhèn)溆谩?/p>

1.3 外泌體粒徑分析（NTA）

取一部分mffEXs和affEXs，用PBS重懸，隨后將液體添加到Nanosight（NS300）納米顆粒跟蹤分析儀（NTA）中，利用NTA對(duì)分離出的EXs的大小和形態(tài)進(jìn)行分析。

1.4 Western Blot技術(shù)檢測(cè)

將RIPA裂解液混合加入mffEXs和affEXs中，進(jìn)行裂解和變性。根據(jù)Western-Blot步驟檢測(cè)外泌體表面標(biāo)志蛋白CD9、CD63和CD81（Rabbit anti-CD9, 1﹕2000 Dilution, ab92726 Abcam; Rabbit anti-CD63, 1﹕2000 Dilution, ab231975, Abcam和Rabbit anti-CD81, 1﹕2000 Dilution, ab109201, Abcam）的表達(dá)，4℃過(guò)夜孵育，1×TBST洗脫3次后，添加二抗（sc-2030, Santa Cruz, 1﹕2000 Dilution），室溫孵育2 h，1×TBST洗脫3次，添加顯色劑顯色后使用凝膠成像系統(tǒng)拍照[22-23]。

1.5 外泌體總RNA提取、cDNA文庫(kù)構(gòu)建和RNA測(cè)序

采用Trizol（Life teachnologies）傳統(tǒng)法提取mffEXs和affEXs總RNA，通過(guò)NanoDrop（Nanodrop 2000，Thermo，USA）以及Agilent 2100（Agilent Technologies, Palo Alto, CA, USA）對(duì)提取的總RNA的濃度和完整性進(jìn)行檢測(cè)，并構(gòu)建小RNA文庫(kù)，使用Illumina HiSeqTM2000（Illumina, San Diego, CA, USA）進(jìn)行測(cè)序，該部分由廣州基迪奧生物科技有限公司完成。

1.6 miRNA的差異分析與靶基因預(yù)測(cè)

評(píng)估m(xù)iRNA測(cè)序質(zhì)量，計(jì)算RNA的長(zhǎng)度分布，過(guò)濾掉原始數(shù)據(jù)中低質(zhì)量序列（質(zhì)量值＜20的堿基數(shù)超過(guò)1個(gè)或含N的序列），獲得高質(zhì)量的Reads。通過(guò)計(jì)算TPM（Tags per million）的表達(dá)量[24]，篩選出mffEXs和affEXs中差異表達(dá)的miRNA。使用RNAhybrid（v2.1.2）、Miranda（v3.3a）和TargetScan（Version：7.0）進(jìn)行miRNA靶基因預(yù)測(cè)和功能分析，并將獲得的靶基因用Cytoscape軟件繪制成可視化互作網(wǎng)絡(luò)圖。

1.7 GO和KEGG通路富集分析

篩選出卵泡液EXs中差異表達(dá)miRNA對(duì)應(yīng)的靶基因，進(jìn)行GO（http://www.geneontology.org/）和KEGG（kyoto encyclopedia of genesand genomes; http: //www.kegg.jp/kegg）顯著性富集分析[25]，分別描述GO的分子功能（molecular function）、細(xì)胞組分（cellular component）和生物進(jìn)程（biological process），并選擇與卵母細(xì)胞發(fā)育相關(guān)的主要調(diào)節(jié)基因和信號(hào)通路進(jìn)行分析。

1.8 顆粒細(xì)胞試驗(yàn)

將配制20 μg·mL-1的mffEXs和affEXs作為添加劑加到顆粒細(xì)胞培養(yǎng)基中進(jìn)行培養(yǎng)，48 h后提取顆粒細(xì)胞的RNA，反轉(zhuǎn)錄合成cDNA，通過(guò)Q-PCR分析和的表達(dá)情況，具體反應(yīng)條件為95℃預(yù)變性4 min；95℃變性30 s；59℃退火30 s；72℃延伸15 s，共33個(gè)循環(huán)。其中引物（上游AGAGTCGCTGGGGATCTACC；下游：TCATGGC TACCACTTGACCTG，擴(kuò)增產(chǎn)物134 bp；XM_ 005671013）、引物（上游AAGGGAATTTACGGGC CGAG；下游CACGCACCTCAAAGCTGTTC，擴(kuò)增產(chǎn)物228 bp；NM_213824）和內(nèi)參引物（上游TCGGAGTGAACGGATTTGGC；下游TGACAAG CTTCCCGTTCTCC，擴(kuò)增產(chǎn)物189 bp；NM_001206359）。

2 結(jié)果

2.1 成熟及閉鎖卵泡液EXs的分離鑒定與miRNA測(cè)序分析

將分離的成熟卵泡和閉鎖卵泡（圖1-A，B）EXs進(jìn)行粒徑分析和蛋白檢測(cè)，結(jié)果顯示粒徑峰值約100 nm（圖1-C，D）；Western-Blot結(jié)果顯示，CD9、CD63和CD81（圖1-E）這些標(biāo)志性蛋白均在分離的mffEXs和affEXs中表達(dá)。測(cè)序結(jié)果顯示，mffEXs和affEXs攜帶的miRNA序列長(zhǎng)度主要分布在20—22 nt范圍內(nèi)（圖1-F）；miRNA表達(dá)譜比較分析顯示，90個(gè)miRNA在affEXs中顯著上調(diào)表達(dá)，220個(gè)miRNA顯著下調(diào)表達(dá)（圖1-G）。

2.2 mffEXs和affEXs中主要差異的miRNA

根據(jù)mffEXs和affEXs攜帶的miRNA測(cè)序結(jié)果，以差異倍數(shù)|log2（F/C）|≥1.2，且＜0.05為閾值進(jìn)行篩選，發(fā)現(xiàn)這些miRNA在mffEXs和affEXs中存在顯著差異，且可能直接參與調(diào)控卵泡的成熟發(fā)育及閉鎖（表1）。

2.3 GO功能注釋分析

根據(jù)差異表達(dá)miRNA的靶基因進(jìn)行GO功能注釋分析，結(jié)果發(fā)現(xiàn)它們主要富集于11個(gè)生物過(guò)程條目（細(xì)胞周期進(jìn)程、蛋白絲氨酸/蘇氨酸激酶活性、MAPK級(jí)聯(lián)和有絲分裂細(xì)胞周期的G2/M轉(zhuǎn)變等）、11個(gè)細(xì)胞組分條目（紡錘、周期蛋白依賴的蛋白激酶全酶復(fù)合物、蛋白激酶復(fù)合物和泛素連接酶復(fù)合物等）和13個(gè)分子功能條目（磷酸酶結(jié)合、鈣調(diào)素結(jié)合、蛋白磷酸酶結(jié)合和腺苷酸環(huán)化酶活性等）。進(jìn)一步研究發(fā)現(xiàn)，這些差異基因被劃分為多種生物功能類別，包括細(xì)胞增殖、代謝和凋亡等。差異表達(dá)基因的分布及生物學(xué)過(guò)程類別如圖2所示。

A：閉鎖卵泡的形態(tài)觀察結(jié)果；B：成熟卵泡的形態(tài)觀察結(jié)果；C：mffEXs粒徑分析結(jié)果；D：affEXs粒徑分析結(jié)果；E：Westem blot檢測(cè)結(jié)果；F：miRNA的序列長(zhǎng)度分布；G：mffEXs和affEXs中miRNA的表達(dá)差異結(jié)果

2.4 KEGG富集分析

KEGG通路分析顯示差異表達(dá)的miRNA涉及調(diào)控169個(gè)靶基因，主要富集于Ras信號(hào)通路（ssc04014）、cAMP信號(hào)通路（ssc04024）、P53信號(hào)通路（ssc04115）、MAPK信號(hào)通路（ssc04010）、卵母細(xì)胞減數(shù)分裂（ssc04114）、泛素介導(dǎo)的蛋白水解作用（ssc04120）和細(xì)胞周期（ssc04110）等通路（圖3），可能參與調(diào)控卵泡的成熟與閉鎖。

2.5 富集關(guān)鍵信號(hào)通路中miRNA潛在的靶基因

差異表達(dá)的miRNA靶標(biāo)富集的主要信號(hào)通路顯示了靶基因的調(diào)控作用（表2）。通過(guò)富集通路篩選出一些參與顆粒細(xì)胞凋亡和卵母細(xì)胞發(fā)育的關(guān)鍵基因，結(jié)合信號(hào)傳導(dǎo)通路深入研究豬卵泡閉鎖調(diào)控機(jī)制，發(fā)現(xiàn)介導(dǎo)卵泡閉鎖的途徑顯著富集于7條信號(hào)通路，且這些通路可能直接參與調(diào)控卵泡閉鎖。

表1 卵泡液EXs中主要差異表達(dá)的miRNA

2.6 構(gòu)建miRNA-靶基因互作網(wǎng)絡(luò)

將篩選獲得的與卵泡成熟發(fā)育和閉鎖相關(guān)且差異表達(dá)顯著的miRNA及靶基因進(jìn)行了共表達(dá)網(wǎng)絡(luò)分析。在互作網(wǎng)絡(luò)中，我們檢測(cè)了3個(gè)特異性miRNA（ssc-miR-21-5p、ssc-miR-133a-3p和ssc-let-7a），發(fā)現(xiàn)它們分別作用于和等基因（圖4）。其中，是一種調(diào)節(jié)細(xì)胞周期運(yùn)轉(zhuǎn)的重要激酶，它能在特定時(shí)間被激活，通過(guò)底物磷酸化結(jié)合cyclin B驅(qū)動(dòng)細(xì)胞周期運(yùn)轉(zhuǎn)，而ssc-let-7a潛在靶向，抑制了細(xì)胞G2/M期的運(yùn)轉(zhuǎn)，抑制其增殖，誘導(dǎo)顆粒細(xì)胞凋亡；是調(diào)控細(xì)胞凋亡的關(guān)鍵因子，進(jìn)一步分析發(fā)現(xiàn)affEXs中ssc-miR-21-5p下調(diào)潛在靶向調(diào)控表達(dá)，調(diào)控顆粒細(xì)胞凋亡；胰島素生長(zhǎng)因子（）作為ssc-miR-133a-3p的潛在靶基因，在IGFBP-1作用下參與調(diào)控，增強(qiáng)了卵巢顆粒細(xì)胞芳香化酶的活性，抑制了類固醇激素代謝，誘導(dǎo)顆粒細(xì)胞凋亡，揭示了這些miRNA的靶標(biāo)可能潛在調(diào)控卵泡的成熟發(fā)育和閉鎖。

2.7 卵泡液EXs添加顆粒細(xì)胞的驗(yàn)證

根據(jù)以上mffEXs和affEXs的差異表達(dá)及功能富集分析結(jié)果，我們將mffEXs和affEXs分別添加到顆粒細(xì)胞中培養(yǎng)48 h。鏡檢結(jié)果顯示，mffEXs添加的顆粒細(xì)胞顯示出增殖狀態(tài)，長(zhǎng)勢(shì)較好；affEXs添加顆粒細(xì)胞，形態(tài)發(fā)生拉長(zhǎng)，部分出現(xiàn)凋亡現(xiàn)象（圖5-A、B）。Q-PCR結(jié)果顯示在mffEXs中顯著上調(diào)表達(dá)，而在affEXs中顯著上調(diào)（圖5-C、D），表明affEXs可能潛在促進(jìn)了顆粒細(xì)胞凋亡的發(fā)生。

圖2 卵泡液EXs中差異miRNA靶基因的GO富集分析

圖3 KEGG功能富集分析

表2 主要信號(hào)通路中miRNA調(diào)控靶基因信息表

圖4 差異性表達(dá)miRNA的靶基因互作網(wǎng)絡(luò)

A：添加mffEXs培養(yǎng)的顆粒細(xì)胞結(jié)果；B：添加affEXs培養(yǎng)顆粒細(xì)胞的結(jié)果；C和D：Q-PCR檢測(cè)CDK1和P53表達(dá)結(jié)果

3 討論

卵泡閉鎖是一種通過(guò)凋亡機(jī)制產(chǎn)生的生理性程序性細(xì)胞死亡，最終引起卵母細(xì)胞發(fā)育停滯和顆粒細(xì)胞凋亡，其中顆粒細(xì)胞凋亡是引起閉鎖卵泡發(fā)生的主要原因[26]。大量研究報(bào)道EXs介導(dǎo)細(xì)胞間信號(hào)轉(zhuǎn)導(dǎo)，涉及多種生理功能調(diào)控。已有研究顯示，卵泡液中的EXs攜帶大量miRNA[27-28]，參與卵泡發(fā)育調(diào)控。但EXs miRNA參與調(diào)控卵泡閉鎖的調(diào)控機(jī)制還不清楚。本研究通過(guò)抽提卵泡液，發(fā)現(xiàn)豬成熟及閉鎖卵泡中均含有粒徑峰值約100 nm，且表面表達(dá)特異性標(biāo)志蛋白CD9、CD63和CD81的EXs，這與先前的研究結(jié)果一致[29]。EXs是細(xì)胞向胞外分泌的一種囊泡小體[30]，在卵泡液[31]、輸卵管[32]和子宮液[33]中均存在。卵泡液中EXs攜帶大量調(diào)控分子可以直接調(diào)控卵母細(xì)胞和顆粒細(xì)胞，構(gòu)成卵母細(xì)胞和顆粒細(xì)胞生存的微環(huán)境，促使卵泡發(fā)育[34]。miRNA是一類EXs中含量最豐富的RNA小分子物質(zhì)，廣泛參與基因表達(dá)調(diào)控[35-36]，促使完成動(dòng)物機(jī)體的各項(xiàng)生命活動(dòng)[37]。

在卵泡發(fā)育過(guò)程中，EXs介導(dǎo)的信號(hào)傳遞發(fā)揮重要的調(diào)控作用，不僅影響卵母細(xì)胞發(fā)育，而且還會(huì)影響卵母細(xì)胞受精和胚胎質(zhì)量等[20,38-40]。本文通過(guò)對(duì)豬成熟和閉鎖卵泡液中的EXs miRNA進(jìn)行測(cè)序，發(fā)現(xiàn)許多miRNA呈現(xiàn)顯著差異表達(dá)，且參與介導(dǎo)關(guān)鍵基因和信號(hào)轉(zhuǎn)導(dǎo)調(diào)控，顯示了在卵泡發(fā)育過(guò)程中的重要調(diào)控作用。兩類差異表達(dá)的EXs miRNA功能富集分析發(fā)現(xiàn)這些miRNA對(duì)顆粒細(xì)胞凋亡、細(xì)胞周期運(yùn)轉(zhuǎn)和類固醇代謝等方面具有潛在的調(diào)控作用，表明了EXs中的miRNA可能直接參與調(diào)控卵泡的成熟發(fā)育和閉鎖[38,41]。

卵泡閉鎖是從顆粒細(xì)胞凋亡開(kāi)始的[42]，屬于一種促細(xì)胞凋亡基因，其功能主要涉及調(diào)控G1和G2/M期的運(yùn)轉(zhuǎn)，促進(jìn)細(xì)胞凋亡[40,43]。研究通過(guò)對(duì)豬[44]和牛[45-46]進(jìn)行miRNA表達(dá)譜分析，發(fā)現(xiàn)miR-21-5p在卵泡發(fā)育過(guò)程中呈現(xiàn)不同表達(dá)[45]；在培養(yǎng)小鼠顆粒細(xì)胞時(shí)發(fā)現(xiàn)miR-21-5p屬于一種抗凋亡因子，可以直接靶向同源基因，促進(jìn)細(xì)胞增殖[47]，證明了在顆粒細(xì)胞凋亡發(fā)生過(guò)程中起到重要調(diào)控作用[48]；本研究顯示affEXs中下調(diào)表達(dá)的ssc-miR-21-5p，潛在靶向，促使表達(dá)增加，引起顆粒細(xì)胞凋亡；在體外培養(yǎng)顆粒細(xì)胞時(shí)添加affEXs可增加的表達(dá)，引起卵巢顆粒細(xì)胞的凋亡，這說(shuō)明了affEXs中ssc-miR-21-5p下調(diào)可以引起卵母細(xì)胞凋亡；也有研究顯示miR-21高表達(dá)受到抑制時(shí)，體外胚胎發(fā)育停滯[49-50]。

let-7家族（let-7a、let-7b和let-7c等）在豬早期卵泡閉鎖和逐步閉鎖過(guò)程中特異性表達(dá)，且卵巢卵泡的數(shù)量也會(huì)隨卵泡閉鎖的發(fā)生逐漸減少[51]。隨著研究的深入，let-7a在動(dòng)物生長(zhǎng)發(fā)育、細(xì)胞增殖、分化和凋亡等方面發(fā)揮著重要調(diào)控作用[52]。相關(guān)報(bào)道顯示let-7a通過(guò)下調(diào)抑制哮喘氣道平滑肌細(xì)胞的增殖，促進(jìn)其凋亡[53]；還可以在細(xì)胞周期蛋白-D1的調(diào)控作用下，抑制肺腺癌細(xì)胞增殖[54]；在骨肉瘤細(xì)胞中l(wèi)et-7a潛在靶向，促使細(xì)胞生長(zhǎng)和肺轉(zhuǎn)移受到抑制[55-56]；let-7a還可以通過(guò)抑制和表達(dá)誘導(dǎo)滋養(yǎng)層細(xì)胞凋亡[57]；還有研究顯示let-7a靶向凋亡基因、和發(fā)揮調(diào)控作用[58-59]。miR-133a-3p在多種細(xì)胞中都起到了抑制增殖、侵襲，阻滯細(xì)胞周期等作用，促進(jìn)細(xì)胞凋亡[60-61]，如在口腔鱗狀細(xì)胞癌研究中發(fā)現(xiàn)miR-133a-3p下調(diào)表達(dá)，抑制癌細(xì)胞的增殖、侵襲和轉(zhuǎn)移[62-63]；它還可以通過(guò)阻斷自噬介導(dǎo)的谷氨酰胺分解，抑制胃癌細(xì)胞的生長(zhǎng)和轉(zhuǎn)移等[64]；在視網(wǎng)膜母細(xì)胞瘤中通過(guò)靶向，促進(jìn)細(xì)胞凋亡，阻滯細(xì)胞周期[65]；還有研究顯示胰島素生長(zhǎng)因子（）被認(rèn)為是卵泡發(fā)育所必須的生長(zhǎng)因子[66]，顯然缺失會(huì)對(duì)卵泡閉鎖的發(fā)生產(chǎn)生一定的影響。我們發(fā)現(xiàn)與mffEXs相比，affEXs中上調(diào)表達(dá)的ssc-let-7a和ssc-miR-133a-3p潛在靶向和，催化底物磷酸化，抑制了G2/M期的運(yùn)轉(zhuǎn)，降低了細(xì)胞的增殖能力，并使卵泡細(xì)胞與LH作用，誘導(dǎo)雄激素釋放，促使卵母細(xì)胞降解和顆粒細(xì)胞凋亡或其受IGFBP-1調(diào)控，增強(qiáng)卵巢顆粒細(xì)胞芳香化酶的活性，抑制類固醇激素代謝，解釋了EXs攜帶的miRNA上調(diào)表達(dá)可以促使顆粒細(xì)胞凋亡，進(jìn)而誘導(dǎo)卵泡閉鎖的發(fā)生，并且有望成為解析卵泡發(fā)育規(guī)律的分子機(jī)制[67]。

4 結(jié)論

近年來(lái)EXs已成為動(dòng)物生殖發(fā)育領(lǐng)域的研究熱點(diǎn)，許多潛在的功能機(jī)制逐漸被人們揭曉，然而卵泡液EXs攜帶的miRNA對(duì)豬卵泡發(fā)育調(diào)控作用仍然不清楚。本文研究顯示卵泡液EXs攜帶的miRNA（ssc-miR-133-3p、ssc-miR-21-5p和ssc-let-7a）在顆粒細(xì)胞凋亡、細(xì)胞周期調(diào)控和類固醇激素代謝等過(guò)程中具有重要的調(diào)控作用，這將為豬卵泡發(fā)育和閉鎖相關(guān)的主要調(diào)控機(jī)制提供重要數(shù)據(jù)。

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Expression Differences and Functional Analysis of Exosomes microRNA in Porcine Mature and Atretic Follicles

CHEN HuiFang, HUANG QiLiang, HU ZhiChao, PAN XiaoTing, WU ZhiSheng, BAI YinShan

School of Life Science and Engineering, Foshan University, Foshan 528231, Guangdong

【】To explore the regulatory role of follicular fluid Exosomes (EXs) miRNA in follicular development and atresia, the difference of miRNA expression between mature follicular fluid Exosomes (mffEXs) and atretic follicular fluid Exosomes (affEXs) were analyzed. 【】In this study, the follicular fluid of 4-6 mm porcine mature development and atresia follicles was extracted. Then EXs were identified by particle size analysis and Western Blot detection, respectively. the sequencing analysis of the characteristic EXs carried miRNA and functional enrichment analysis were carried out, and then the key signal pathways and differential genes were screened. Finally, mffEXs and affEXs were used as additives for granular cell culture, and Q-PCR detection technology was used to analyze the expression of key genes to verify and analyze the regulatory functions of EXs miRNA in the two types of follicular fluid in follicular development. 【】This study successfully separated mffEXs and affEXs. The sequencing results showed that compared with mffEXs, 90 miRNAs in affEXs were up-regulated and 220 miRNAs were down-regulated, indicating that the level of miRNA expression in follicular fluid could directly regulate follicular development. KEGG enrichment analysis showed that the differential signaling pathways of the two types of follicles were mainly concentrated in the signal pathways, such as Ras, cAMP, P53 and MAPK, which involved in the regulation of biological functions, such as oocyte development, meiosis, and granulosa cell cycle. In atretic follicles, the up-regulated expression of ssc-let-7a and ssc-miR-133a-3p potentially targeted and regulated cyclin-dependent kinase () and insulin growth factor (), which inhibited G1 and G2/M Phase operation, and steroid hormone metabolism promoted the obstruction of granular cell cycle and the apoptosis of granular cells, causing follicular atresia; down-regulated ssc-miR-21-5p potentially targeted tumor suppressor gene () and inhibited cell cycle operation to promote the apoptosis of granular cells. mffEXs and affEXs were added to granular cells cultured in vitro, and Q-PCR results showed thatwas significantly up-regulated in mffEXs, whilewas significantly down-regulated, indicating the reliability of the sequencing analysis results. These results all showed that changes in miRNA expression levels in affEXs promoted granular cell apoptosis and cell cycle arrest, causing follicular atresia. 【】 Porcine affEXs carry miRNAs increased the regulation of,andgene expression, and inhibited the cell cycle of granulosa cells and steroid hormone metabolism and other signal pathways, causing granulosa cell apoptosis and follicular atresia.

porcine; mature follicular fluid; atretic follicular fluid; EXs; miRNA

10.3864/j.issn.0578-1752.2021.21.015

2020-09-27；

2021-04-25

廣東省畜禽疫病防治研究重點(diǎn)實(shí)驗(yàn)室基金項(xiàng)目（YDWS1902）、佛山科學(xué)技術(shù)學(xué)院高層次人才科研啟動(dòng)項(xiàng)目（gg040969）

陳慧芳，E-mail：chenhuifang07@163.com。通信作者白銀山，E-mail：xuefei200403@163.com

（責(zé)任編輯 林鑒非）