circ-13267通過let-7-19/ERBB4通路調(diào)控蛋鴨卵泡顆粒細(xì)胞凋亡

吳艷，張昊，梁振華，潘愛鑾，申杰，蒲躍進，黃濤，皮勁松*，杜金平

circ-13267通過let-7-19/ERBB4通路調(diào)控蛋鴨卵泡顆粒細(xì)胞凋亡

吳艷1,2，張昊1，梁振華1，潘愛鑾1，申杰1，蒲躍進1，黃濤1，皮勁松1*，杜金平1

1湖北省農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)研究所/湖北省農(nóng)業(yè)科技創(chuàng)新中心，武漢 430064；2動物胚胎工程及分子育種湖北省重點實驗室，武漢 430064

【背景】蛋鴨卵泡發(fā)育是決定其產(chǎn)蛋性能的關(guān)鍵因素。已有研究表明，禽類卵泡發(fā)育是極為復(fù)雜的生物學(xué)過程，目前人們已對家禽的卵泡發(fā)育模式有了一定的了解，但作為決定產(chǎn)蛋量的重要因素，卵泡發(fā)育的具體調(diào)控機理仍需進一步深入研究。顆粒細(xì)胞是卵泡中主要的功能細(xì)胞，可調(diào)控卵泡膜細(xì)胞和卵母細(xì)胞的生長、分化和成熟，同時也精確的調(diào)控卵泡的生長發(fā)育，維持卵巢的正常功能（誘導(dǎo)排卵、維持成熟分裂的阻斷、為卵母細(xì)胞提供底物等）。circRNA是一類新型的內(nèi)源性非編碼RNA，在卵泡發(fā)育中發(fā)揮重要的調(diào)控作用。【目的】通過構(gòu)建circRNA過表達(dá)載體上調(diào)circ-13267的表達(dá)，研究circ-13267在蛋鴨卵泡顆粒細(xì)胞中的作用及其調(diào)控機制，為蛋鴨卵泡發(fā)育的調(diào)控機制解析提供依據(jù)?！痉椒ā渴紫龋肣-PCR檢測蛋鴨卵泡顆粒細(xì)胞的細(xì)胞質(zhì)與細(xì)胞核中circ-13267的表達(dá)水平；構(gòu)建circ-13267的過表達(dá)載體circ-13267-pLCDH，在蛋鴨顆粒細(xì)胞中過表達(dá)circ-13267后，利用Q-PCR法檢測circ-13267、let-7-19、ERBB4、FAS和BCL2的表達(dá)水平；分別轉(zhuǎn)染circ-13267-pLCDH和pLCDH-ciR于蛋鴨卵泡顆粒細(xì)胞，24 h后利用EdU法檢測蛋鴨卵泡顆粒細(xì)胞的增殖能力；將circ-13267的線性序列或ERBB4的3′UTR克隆到pmirGLo載體中，同時對上述野生型序列中的let-7-19結(jié)合位點進行突變，得到表達(dá)突變型序列的載體，利用雙熒光素酶報告基因驗證circ-13267與let-7-19及l(fā)et-7-19與靶基因ERBB4的結(jié)合關(guān)系；在蛋鴨卵泡顆粒細(xì)胞中轉(zhuǎn)染circ-13267-pLCDH和pLCDH-ciR，利用流式細(xì)胞術(shù)和Annexin V-FITC檢測蛋鴨卵泡顆粒細(xì)胞凋亡情況?！窘Y(jié)果】環(huán)狀RNA circ-13267在細(xì)胞質(zhì)和細(xì)胞核中均有表達(dá)；雙熒光素酶報告基因試驗結(jié)果顯示，let-7-19能與ERBB4結(jié)合，進而下調(diào)熒光素酶的活性，當(dāng)ERBB4序列中l(wèi)et-7-19的結(jié)合位點突變后，let-7-19則無法抑制熒光素酶的表達(dá)，說明ERBB4是let-7-19的一個靶基因；熒光定量檢測結(jié)果顯示，過表達(dá)環(huán)狀RNAcirc-13267后BCL2基因的表達(dá)顯著降低（＜0.05），而FAS和ERBB4基因的表達(dá)量顯著升高（＜0.05），當(dāng)過表達(dá)let-7-19后，ERBB4基因的表達(dá)量顯著升高（＜0.05），而抑制let-7-19后ERBB4基因的表達(dá)量顯著降低（＜0.05）；EdU試驗結(jié)果顯示，過表達(dá)circ-13267后蛋鴨卵泡顆粒細(xì)胞數(shù)量顯著減少，說明其促進了蛋鴨卵泡顆粒細(xì)胞的凋亡；然而，在蛋鴨卵泡顆粒細(xì)胞中共轉(zhuǎn)染circ-13267和let-7-19后，與對照組相比共轉(zhuǎn)染組中BCL2和FAS的表達(dá)量均無顯著變化（＞0.05），而與過表達(dá)circ-13267組相比，共轉(zhuǎn)組中BCL2基因的表達(dá)量極顯著降低（＜0.01）、FAS的表達(dá)量極顯著升高（＜0.01），說明circ-13267促進蛋鴨卵泡顆粒細(xì)胞凋亡的作用被抑制；利用流式細(xì)胞儀檢測轉(zhuǎn)染后的蛋鴨卵泡顆粒細(xì)胞發(fā)現(xiàn)，與共轉(zhuǎn)染circ-13267和let-7-19組相比，僅過表達(dá)circ-13267后，晚期凋亡細(xì)胞數(shù)和總凋亡細(xì)胞數(shù)均顯著增加（＜0.05），而活細(xì)胞數(shù)顯著降低（＜0.05）?！窘Y(jié)論】蛋鴨circ-13267在蛋鴨卵泡顆粒細(xì)胞的細(xì)胞質(zhì)和細(xì)胞核中均有表達(dá)，circ-13267可以吸附let-7-19并靶向ERBB4基因，從而促進了蛋鴨卵泡顆粒細(xì)胞的凋亡，為解析蛋鴨卵泡發(fā)育的調(diào)控機制提供理論依據(jù)。

circRNA；miRNA；蛋鴨；卵泡發(fā)育；顆粒細(xì)胞

0 引言

【研究意義】蛋鴨卵泡發(fā)育是決定其產(chǎn)蛋性能的關(guān)鍵因素。已有研究表明，禽類卵泡發(fā)育是極為復(fù)雜的生物學(xué)過程，目前人們已對家禽的卵泡發(fā)育模式有了一定的了解，但作為決定產(chǎn)蛋量的重要因素，卵泡發(fā)育的具體調(diào)控機理仍需進一步深入研究?！厩叭搜芯窟M展】Circular RNA（circRNA）是一類新型的內(nèi)源性非編碼RNA，它是在mRNA前體剪切過程中通過首尾相連形成的共價閉合環(huán)狀結(jié)構(gòu)，其形成主要依賴于經(jīng)典的剪接性位點和剪接性機制[1-3]，受特定順式作用元件和反式作用因子調(diào)節(jié)[4-5]，穩(wěn)定性顯著高于線性RNA，可通過海綿吸附微小RNA (microRNA，miRNA) 或其他分子，在轉(zhuǎn)錄和轉(zhuǎn)錄后水平發(fā)生調(diào)控作用[6-7]。已有研究表明，circRNA在卵泡發(fā)育過程和子宮內(nèi)膜細(xì)胞中發(fā)揮重要的調(diào)控作用，如ZHANG等[8]研究發(fā)現(xiàn)circRNA在卵巢激素生成等過程中發(fā)揮作用；JIA等[9]研究發(fā)現(xiàn)CircEGFR可能通過與miR-125a-3p競爭性結(jié)合調(diào)節(jié)Fyn，從而在小鼠卵巢GCs中發(fā)揮重要作用；TAO等[10]研究發(fā)現(xiàn)circNRA在山羊排卵前卵泡顆粒細(xì)胞中發(fā)揮重要作用；ZHANG等[11-12]在奶山羊中發(fā)現(xiàn)circRNA-9119通過吸附miR-26a調(diào)控奶山羊子宮內(nèi)膜上皮細(xì)胞PTGS2基因的表達(dá)，circRNA-miR182通過調(diào)控睪丸素抑制了山羊受孕前期子宮內(nèi)膜上皮細(xì)胞凋亡。XU等[13]鑒定獲得了卵巢中與豬產(chǎn)仔數(shù)相關(guān)的circRNA。有關(guān)circRNA在家禽卵泡發(fā)育中的研究相對較少，僅見SHEN等[14]分析了雞卵泡顆粒細(xì)胞circRNA 的動態(tài)表達(dá)及功能；ZHANG等[15]研究發(fā)現(xiàn)circRNA通過PPAR和脂肪酸代謝相關(guān)途徑調(diào)節(jié)miRNAs，從而潛在地影響脂肪的形成?！颈狙芯壳腥朦c】關(guān)于蛋鴨circRNA在卵泡發(fā)育中的研究僅見本團隊前期研究報道，其中circ-13267在白卵泡和黃卵泡組織中表達(dá)量存在顯著差異[16]。顆粒細(xì)胞是卵泡中主要的功能細(xì)胞，其可調(diào)控卵泡膜細(xì)胞和卵母細(xì)胞的生長、分化和成熟，同時也精確的調(diào)控卵泡的生長發(fā)育[17]，維持卵巢的正常功能（誘導(dǎo)排卵、維持成熟分裂的阻斷、為卵母細(xì)胞提供底物等）[18]?！緮M解決的關(guān)鍵問題】本研究擬通過構(gòu)建circRNA過表達(dá)載體上調(diào)circ-13267的表達(dá)，研究circ-13267調(diào)控蛋鴨卵泡顆粒細(xì)胞的機制，為蛋鴨卵泡發(fā)育的調(diào)控提供依據(jù)。

1 材料與方法

1.1 試驗材料

蛋鴨卵泡顆粒細(xì)胞：采集湖北農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)研究所家禽試驗場選育的蛋鴨卵泡組織，迅速置于含有2%雙抗（青霉素和鏈霉素）的PBS中，盡快返回實驗室，并分離卵泡顆粒細(xì)胞。

主要試劑：DMEM 基礎(chǔ)培養(yǎng)基、含EDTA 的胰酶稀釋液、PBS 緩沖液、胎牛血清、青霉素和鏈霉素購自Hyclone（美國）；Lipofectamine? 3000 轉(zhuǎn)染試劑盒、Opti-MEM?試劑購自Invitrogen（美國）；雙熒光素酶檢測試劑盒購自Promega公司；SYBR Green qPCR Mix 購自Thermo（美國）；HⅠ、RⅠ限制性內(nèi)切酶和T4 DNA連接酶購自Thermo公司；KOD FX酶購自TOYOBO，公司；凝膠DNA小量回收試劑盒和無內(nèi)毒素質(zhì)粒抽提試劑盒購自O(shè)MEGA公司（美國）；PrimeScriptTMRT reagent Kit反轉(zhuǎn)錄試劑盒購自寶生物工程（大連）有限公司；熒光定量PCR酶購自BIO-RAD公司；環(huán)狀RNA過表達(dá)載體PLCDH-ciR，購自吉賽生物科技有限公司；其余有機試劑均為國產(chǎn)分析純。

1.2 試驗方法

1.2.1 蛋鴨卵泡顆粒細(xì)胞分離培養(yǎng)與轉(zhuǎn)染 選擇產(chǎn)蛋期蛋鴨頸靜脈放血處死后取整個卵巢組織，置于裝有預(yù)冷PBS的無菌培養(yǎng)皿中迅速帶回實驗室，用加有雙抗的PBS 清洗數(shù)次。將漂洗干凈的卵泡移入裝有預(yù)冷PBS緩沖液的平皿中，剝凈卵泡外膜、結(jié)締組織及血管網(wǎng)，劃破卵泡后釋放卵黃，將漂洗干凈的卵泡膜盡量剪碎，置于15 mL離心管中，加4 mL培養(yǎng)基反復(fù)吹打1 min，4 ℃ 1 000 r/min離心后棄上清；加入4 mL 0.2%Ⅱ型膠原酶，重懸沉淀，置于37℃恒溫?fù)u床80 r/min消化30 min，加入4 mL M199完全培養(yǎng)基（含10%血清）終止消化；用200目不銹鋼篩過濾，收集濾液，4℃ 1 000 r/min離心10 min后收集細(xì)胞，加入M199完全培養(yǎng)基（含10% FBS及1 %雙抗）重懸后，測定細(xì)胞密度后接種于6孔培養(yǎng)皿，置于37℃、5 % CO2培養(yǎng)箱內(nèi)靜置培養(yǎng)，培養(yǎng)24 h后可用后續(xù)相關(guān)試驗。使用轉(zhuǎn)染試劑lip 3000按照轉(zhuǎn)染說明書對細(xì)胞進行轉(zhuǎn)染，轉(zhuǎn)染24 h后收獲細(xì)胞。

1.2.2 蛋鴨卵泡顆粒細(xì)胞核質(zhì)分離與RNA提取 根據(jù)PARIS? Kit（Invitrogen，ThermoFisher Scientific）試劑盒說明書進行蛋鴨卵泡顆粒細(xì)胞并提取細(xì)胞中的總RNA。

1.2.3 載體構(gòu)建 環(huán)狀RNA過表達(dá)載體pLCDH-ciR購自廣州吉賽生物技術(shù)有限公司。根據(jù)環(huán)狀RNA測序分析獲得的circ-13267基因序列，設(shè)計并合成circ-13267的全長序列引物（表1）。擴增circ-13267序列全長，切膠回收后，用RI和HI于37 ℃雙酶切PCR產(chǎn)物和pLCDH-ciR空載體。酶切后回收目的片段和空載體，利用T4連接酶將目的片段與pLCDH-ciR載體連接，將其命名為circ-13267-pLCDH。

1.2.4 RNA反轉(zhuǎn)錄與實時熒光定量PCR 使用TRIzol提取細(xì)胞和組織總RNA，利用RNase R處理RNA后按照TaKaRa反轉(zhuǎn)錄試劑盒說明書進行cDNA的合成，qPCR使用Bio-Rad公司的實時熒光定量PCR儀完成。circ-13267、β-actin、let-7-19、U6及ERBB4基因的定量引物見表1。反應(yīng)條件為: 95 ℃預(yù)變性10 min；95℃10 s，60℃ 30 s，72℃30s，重復(fù)40個循環(huán)，用2–ΔΔCt法計算基因表達(dá)。

表1 試驗所需引物序列

1.2.5 circRNA與miRNA及靶基因結(jié)合位點的預(yù)測 利用在線預(yù)測軟件BiBiServ中的rnahybrid 進行circ-13267與miRNA let-7-19及l(fā)et-7-19與靶基因ERBB4之間的結(jié)合位點，詳細(xì)預(yù)測網(wǎng)址如下：https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid。

1.2.6 雙熒光素酶報告實驗檢測熒光素酶活性 將circ-13267的線性序列或ERBB4的3′UTR克隆到pmirGLo載體中，同時對上述野生型序列中的let-7-19結(jié)合位點進行突變，得到表達(dá)突變型序列的載體，分別命名為circ-13267-pmirGLO-W、circ-13267-pmirGLO- M、pmirGLo- ERBB4-W和pmirGLo- ERBB4-M。將上述載體分別與let-7-19模擬物共轉(zhuǎn)染蛋鴨卵泡顆粒細(xì)胞，按照熒光素酶活性檢測試劑盒說明書進行，測定螢火蟲熒光素酶（firefly luciferase，F(xiàn)L）和海腎熒光素酶（ranilla luciferase，RL）強度，根據(jù)FL/RL比值來判斷相對熒光素酶活性。

1.2.7 EdU試驗檢測細(xì)胞增殖能力 將分離的蛋鴨卵泡顆粒細(xì)胞接種到24孔板, 使細(xì)胞匯合度達(dá)到80%，分別轉(zhuǎn)染circ-13267-pLCDH和pLCDH-ciR于蛋鴨卵泡顆粒細(xì)胞，24 h后進行EdU處理。每孔加300 μL稀釋EdU溶液孵育2 h。然后按照EdU試劑盒說明書（廣東銳博）進行細(xì)胞固定、Apollo染色和DNA染色。最后在熒光顯微鏡下（Olympus SZX16）拍照。

1.2.8 顆粒細(xì)胞凋亡檢測 參照杭州聯(lián)科生物技術(shù)股份有限公司的Annexin V-FITC/PI 雙染細(xì)胞凋亡檢測試劑盒說明書進行蛋鴨顆粒細(xì)胞凋亡檢測。取細(xì)胞接種于6 孔板，當(dāng)細(xì)胞匯合度達(dá)到80%左右，轉(zhuǎn)染circ-13267-pLCDH和pLCDH-ciR于顆粒細(xì)胞，培養(yǎng)24 h后消化并收集細(xì)胞。然后按照Annexin V-FITC/PI試劑盒說明書進行細(xì)胞處理，最后用流式細(xì)胞儀檢測分析（每個試驗組設(shè)3個重復(fù)）。

1.2.9 數(shù)據(jù)統(tǒng)計分析 每個試驗設(shè)3個重復(fù)，采用SPSS 19.0進行結(jié)果統(tǒng)計分析，兩組樣本均數(shù)比較采用t檢驗，利用GraphPad Prism 6進行作圖。

2 結(jié)果

2.1 circ-13267對蛋鴨卵泡顆粒細(xì)胞增殖、凋亡的影響

轉(zhuǎn)染circ-13267- pLCDH和pLCDH-ciR后的蛋鴨卵泡顆粒細(xì)胞后，利用熒光定量PCR法檢測細(xì)胞增殖凋亡關(guān)鍵基因BCL2、FAS的表達(dá)情況，結(jié)果見圖1-a，由圖1-a可以看出，過表達(dá)circ-13267后細(xì)胞增殖標(biāo)志基因BCL2的表達(dá)量極顯著降低（＜0.01），細(xì)胞凋亡標(biāo)志基因FAS的表達(dá)量顯著升高（＜0.01），說明circ-13267促進了蛋鴨卵泡顆粒細(xì)胞的凋亡；EdU法檢測細(xì)胞凋亡情況，結(jié)果表明過表達(dá)circ-13267后，蛋鴨卵泡顆粒細(xì)胞數(shù)量顯著減少（圖1-b）。

A：過表達(dá)circ-13267后熒光定量檢測；B：過表達(dá)circ-13267后EdU檢測

2.2 circ-13267抑制let-7-19在蛋鴨顆粒細(xì)胞中的表達(dá)

為探討circ-13267在蛋鴨卵泡顆粒細(xì)胞中的功能，分別提取細(xì)胞質(zhì)和細(xì)胞核中的RNA進行逆轉(zhuǎn)錄，熒光定量PCR檢測circ-13267的表達(dá)情況，結(jié)果表明circ-13267在細(xì)胞質(zhì)和細(xì)胞核中均有表達(dá)（圖2），說明circ-13267可能具備競爭性結(jié)合miRNA的作用。分析circ-13267中miRNA的結(jié)合位點，結(jié)果發(fā)現(xiàn)circ-13267中存在let-7-19的結(jié)合位點（圖3），并在前期研究中得到證明[16]。熒光定量檢測結(jié)果發(fā)現(xiàn)let-7-19在白卵泡中的表達(dá)顯著高于黃卵泡（＜0.05）（圖4）。

圖2 circ-13267在細(xì)胞質(zhì)與細(xì)胞核中的表達(dá)量

圖3 circ-13267與let-7-19的結(jié)合位點

2.3 circ-13267通過let-7-19上調(diào)ERBB4基因表達(dá)

分析let-7-19的下游靶基因，發(fā)現(xiàn)let-7-19在ERBB4基因3′UTR區(qū)域存在結(jié)合位點（圖5-a）。利用雙熒光素酶報告基因s試驗表明，let-7-19能與ERBB4結(jié)合，進而下調(diào)熒光素酶的活性，當(dāng)ERBB4序列中l(wèi)et-7-19的結(jié)合位點突變后，let-7-19則無法抑制熒光素酶的表達(dá)（圖5-b）。熒光定量結(jié)果顯示，ERBB4基因在白卵泡中的表達(dá)顯著低于黃卵泡（＜0.05）（圖5-c）；當(dāng)過表達(dá)let-7-19后，ERBB4基因的表達(dá)量顯著升高（＜0.05），而抑制let-7-19后ERBB4基因的表達(dá)量顯著降低（＜0.05）（圖5-d）。綜上可知，circ-13267可通過let-7-19上調(diào)ERBB4基因的表達(dá)。

** P＜0.01

A：let-7-19在ERBB4基因3′非編碼區(qū)（3′ untranslated region，3′UTR）中的結(jié)合位點；B：雙熒光素酶報告基因?qū)嶒灆z測let-7-19與ERBB4的結(jié)合能力；C：ERBB4在黃卵泡和白卵泡中的表達(dá)情況；D：過表達(dá)（抑制表達(dá)）let-7-19后ERBB4的表達(dá)變化情況。* P＜0.05; ** P＜0.01

2.4 circ-13267通過let-7-19/ERBB4途徑促進蛋鴨卵泡顆粒細(xì)胞的凋亡

進一步探討circ-13267是否通過let-7-19/ERBB4通路在蛋鴨卵泡顆粒細(xì)胞中發(fā)揮作用。在蛋鴨卵泡顆粒細(xì)胞中共轉(zhuǎn)染circ-13267和let-7-19后，與對照組相比共轉(zhuǎn)染組中BCL2和FAS的表達(dá)量均無顯著變化（＞0.05），而與過表達(dá)circ-13267組相比，共轉(zhuǎn)組中BCL2基因的表達(dá)量極顯著降低（＜0.01）、FAS的表達(dá)量極顯著升高（＜0.01）（圖6-a），circ-13267促進蛋鴨卵泡顆粒細(xì)胞凋亡的作用被抑制；利用流式細(xì)胞儀檢測轉(zhuǎn)染后的蛋鴨卵泡顆粒細(xì)胞發(fā)現(xiàn)，與共轉(zhuǎn)染circ-13267和let-7-19組相比，僅過表達(dá)circ-13267后，晚期凋亡細(xì)胞數(shù)和總凋亡細(xì)胞數(shù)均顯著增加（＜0.05），而活細(xì)胞數(shù)顯著降低（＜0.05）（圖6-b）。此外，熒光定量結(jié)果表明，過表達(dá)circ-13267后，ERBB4基因的表達(dá)量顯著升高（＜0.05），而同時過表達(dá)circ-13267和let-7-19后，ERBB4基因的表達(dá)量無顯著變化（＞0.05）（圖6-C）。綜上，說明circ-13267通過let-7-19/ERBB4途徑促進了蛋鴨卵泡顆粒細(xì)胞的凋亡。

A：過表達(dá)circ-13267及l(fā)et-7-19對顆粒細(xì)胞增殖凋亡的Q-PCR檢測；B：過表達(dá)circ-13267及l(fā)et-7-19對顆粒細(xì)胞增殖凋亡的流式細(xì)胞檢測；C：過表達(dá)circ-13267和let-7-19后ERBB4基因表達(dá)變化情況

3 討論

卵巢中卵泡的發(fā)育對蛋鴨的產(chǎn)蛋量具有直接影響，但作為決定產(chǎn)蛋量的重要因素，蛋鴨卵泡發(fā)育受多種因素的影響、調(diào)控機制復(fù)雜。因此，探討明確蛋鴨卵泡發(fā)育的具體調(diào)控機制具有重要的實踐意義。

家禽的卵泡發(fā)育按照其直徑的大小分為等級前卵泡（包括小白卵泡、大白卵泡和小黃卵泡）和等級卵泡（又稱為排卵前卵泡）[19-20]。雞卵泡發(fā)育的現(xiàn)有理論認(rèn)為，每當(dāng)一次排卵活動發(fā)生后，就會有一個等級前卵泡進入卵泡選擇，即有一個小黃卵泡被選擇進入等級發(fā)育階段[21-22]。由此可見，小黃卵泡的發(fā)育是決定等級卵泡的關(guān)鍵。前期研究發(fā)現(xiàn)circ-13267在白卵泡組織中的表達(dá)量顯著高于小黃卵泡組織[16]，提示circ-13267可能在蛋鴨的卵泡發(fā)育過程中發(fā)揮重要的作用。顆粒細(xì)胞的發(fā)育早于卵母細(xì)胞的發(fā)育[23]，由此可見顆粒細(xì)胞的生長分化是原始卵泡生長的關(guān)鍵因素，而顆粒細(xì)胞的凋亡是卵泡閉鎖的主要標(biāo)志[24]，因此本研究以蛋鴨卵泡顆粒細(xì)胞作為研究卵泡發(fā)育的細(xì)胞模型。本研究發(fā)現(xiàn)，過表達(dá)circ-13267后促進了蛋鴨卵泡顆粒細(xì)胞凋亡，而已有研究表明顆粒細(xì)胞的凋亡會導(dǎo)致生長卵泡和排卵前卵泡的閉鎖[25]，因此推測circ-13267對蛋鴨卵泡發(fā)育具有一定的抑制作用。

已有研究表明，circRNAs通常通過吸附miRNA從而調(diào)控下游靶基因的表達(dá)，且在雞的卵泡發(fā)育中發(fā)揮重要的作用[14]。已有研究表明ERBB4基因是表皮生長因子受體（EGFR）家族的成員之一，與EGFR基因具有高度的同源性[26]；調(diào)節(jié)細(xì)胞的增殖、遷移和存活等過程[27]。WU等[28]研究表明EGFR對鵪鶉卵泡顆粒細(xì)胞的增值具有促進作用，因此推測ERBB4基因與卵泡發(fā)育相關(guān)。且VEIKKOLAINEN等[29]最新研究發(fā)現(xiàn)ERBB4的確在卵泡發(fā)生過程中起著重要作用。此外，有多項研究表明ERBB4基因表達(dá)受到多個miRNA的調(diào)控，包括miR146a[30]、miR-551b[31]、miR-302b[32]和miR-193a-3p[33]。已有研究表明miRNA let-7家族let-7家族的miRNAs在多種癌癥中都有很好的抑癌作用[34-36]；過表達(dá)let-7 miRNAs后在某些白血病環(huán)境中抑制增殖和促進分化[37]；let-7家族在視網(wǎng)膜和玻璃體發(fā)育過程中具有重要作用，可能會調(diào)節(jié)透明質(zhì)酸的含量[38]；SPINT1-AS1通過調(diào)控miR-let-7a/b/ i-5p促進乳腺癌細(xì)胞增殖和遷移[39]；let-7b抑制t(8;21) AML細(xì)胞系的增殖[40]。綜上所述，miRNA let-7家族在細(xì)胞凋亡中具有重要的作用。本研究結(jié)果表明，circ-13267和靶基因ERBB4的3′UTR中均存在miRNA let-7-19的結(jié)合位點，且circ-13267可通過吸附let-7-19上調(diào)ERBB4基因的表達(dá)；且circ-13267通過let-7-19/ERBB4途徑促進了蛋鴨卵泡顆粒細(xì)胞的凋亡，但circ-13267調(diào)控蛋鴨卵泡發(fā)育的是否還通過其他途徑發(fā)揮作用尚需進一步研究證實。

4 結(jié)論

對前期篩選獲得的環(huán)狀RNA circ-13267的功能及其調(diào)控蛋鴨卵泡發(fā)育的機制進行研究，構(gòu)建了circ- 13267-miRNA調(diào)控網(wǎng)絡(luò)，初步證實circ-13267可通過let-7-19/ERBB4途徑促進蛋鴨卵泡顆粒細(xì)胞的凋亡，對蛋鴨卵泡發(fā)育的調(diào)控機制研究具有借鑒作用。

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circ-13267 Regulates Egg Duck Granulosa Cells Apoptosis Through Let-7-19/ERBB4 Pathway

WU Yan1,2, ZHANG Hao1, LIANG ZhenHua1, PAN AiLuan1, SHEN Jie1, PU YueJin1, Huang Tao1, PI JinSong1*, DU JinPing1

1Institute of Animal Husbandry and Vetervinary, Hubei Academy of Agricultural Science; Hubei Innovation Center of Agricultural Science and Technology, Wuhan 430064;2Hubei Key Lab of Animal Embryo Technology and Molecular Breeding, Wuhan 430064

【Background】 Follicle development is a key factor for laying performance of egg ducks.Previous studies have shown that follicular development is a very complex biological process in poultry.At present, the pattern of follicular development in poultry has been understood.However, as an important factor determining egg production, the specific regulation mechanism of follicular development still needs further study.Granulosa cells are the main functional cells in follicles.They can regulate the growth, differentiation and maturation of theca cells and oocytes.They also regulate the growth and development of follicles, maintain normal ovarian function, such as induce ovulation, maintain maturation division, and provide substrates for oocytes.Circular RNAs (circRNAs) are a new type of endogenous specific non-coding RNA, which plays an important role in follicular development.【Objective】The objective of this study was to explore the effects and regulatory mechanism of circ-13267 on apoptosis in egg duck granulosa cells, through regulating the expression of circ-13267 by constructing the overexpression vector, so as to provide the evidence for analysis the regulatory mechanism of egg duck follicular development.【Method】Firstly, the expression levels of circ-13267 in cytoplasm and nucleus of granulosa cells was detected by Q-PCR.The overexpression vector circ-13267-pLCDH was constructed.After transfection of circ-13267 in egg duck granulosa cells, the expression levels of circ-13267, let-7-19, ERBB4, FAS and BCL2 were detected by Q-PCR.The proliferation of egg duck granulosa cells was detected by EdU method after transfection circ-13267-pLCDH and pLCDH-ciR.The linear sequence of circ-13267 or the 3'UTR of ErbB4 was cloned into pmirGLo vector.At the same time, let-7-19 binding site in the wild-type sequence was mutated to obtain the vector expressing the mutant sequence.The targeting relationships between circ-13267 and let-7-19, let-7-19 and ERBB4 were verified by dual luciferase reporter assay, respectively.Then, after transfection of circ-13267-pLCDH and pLCDH-ciR into egg duck follicular granulosa cells, the flow cytometry and Annexin V-FITC were utilized to explore the effects of circ-13267 on duck granulosa cells.【Result】 In duck granulosa cells, circ-13267 was expressed in both cytoplasm and nucleus.The dual luciferase reporter gene assay confirmed that let-7-19 could bind to ERBB4 and down regulate the activity of luciferase; when the binding site of let-7-19 in ErbB4 sequence was mutated, let-7-19 could not inhibit the expression of luciferase, indicating that ERBB4 was a target gene of let-7-19.The results of Q-PCR showed that, after overexpression of circ-13267, the expression of BCL2 gene decreased significantly (＜0.05), while the expression of FAS and ERBB4 gene increased significantly (＜0.05); after overexpression of let-7-19, the expression of ERBB4 gene increased significantly (＜0.05), while after inhibition of let-7-19, the expression of ERBB4 gene decreased significantly (＜0.05).EdU test results showed that the number of follicular granulosa cells in egg ducks decreased significantly after overexpression of circ-13267, it was shown that circ-13267 promoted the apoptosis of follicular granulosa cells in egg ducks.However, after co-transfection of circ-13267 and let-7-19 into egg duck follicular granulosa cells, compared with the control group, there was no significant change in the expression of BCL2 and FAS (＞0.05); however, compared with overexpression of circ-13267, the expression of BCL2 gene decreased significantly (＜0.01) and FAS increased significantly (＜0.01).It was shown that circ-13267 could inhibit the apoptosis of egg duck follicular granulosa cells.In addition, flow cytometry was used to detect the transfected egg duck follicular granulosa cells.Compared with the co-transfection groups of circ-13267 and let-7-19, the number of late apoptotic cells and total apoptotic cells increased significantly (＜0.05), while the number of living cells decreased significantly (＜0.05).【Conclusion】 circ-13267 was expressed in the cytoplasm and nucleus of egg duck follicular granulosa cells.circ-13267 could sponge let-7-19 and target ERBB4 gene, which promoted the apoptosis of egg duck follicular granulosa cells.This results provided a theoretical basis for analysis of the regulatory mechanism of egg duck follicular development.

circRNA; miRNA; egg duck; follicular development; granulosa cells

2021-02-08；

2021-11-17

國家自然科學(xué)基金（32072709）、湖北省自然科學(xué)基金（2020CFB655）、湖北省技術(shù)創(chuàng)新專項（2019ABA084）、國家水禽現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系（CARS-42-47）、湖北省農(nóng)業(yè)科學(xué)院領(lǐng)軍人才項目（L2018017）、湖北省農(nóng)業(yè)科技創(chuàng)新中心項目（2016-620-000-001-023）、湖北省重點研發(fā)計劃（2020BBA034）

吳艷，E-mail：wuyanwh@163.com。通信作者皮勁松，E-mail：pijinsong@sina.com

（責(zé)任編輯 林鑒非）