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        miR-124對Müller細(xì)胞轉(zhuǎn)分化機(jī)制的影響

        2016-07-07 13:41:59范文斌張介平呂立夏徐國彤
        關(guān)鍵詞:轉(zhuǎn)分化神經(jīng)細(xì)胞熒光素酶

        范文斌, 張介平, 王 娟, 呂立夏, 徐國彤

        (1. 同濟(jì)大學(xué)醫(yī)學(xué)院眼科研究所,上海 200092; 2. 同濟(jì)大學(xué)醫(yī)學(xué)院再生醫(yī)學(xué)系,上海 200092;3. 同濟(jì)大學(xué)醫(yī)學(xué)院干細(xì)胞研究中心,上海 200092)

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        ·基礎(chǔ)研究·

        miR-124對Müller細(xì)胞轉(zhuǎn)分化機(jī)制的影響

        范文斌1,2,3, 張介平1,2,3, 王 娟1,2,3, 呂立夏1,2,3, 徐國彤1,2,3

        (1. 同濟(jì)大學(xué)醫(yī)學(xué)院眼科研究所,上海 200092; 2. 同濟(jì)大學(xué)醫(yī)學(xué)院再生醫(yī)學(xué)系,上海 200092;3. 同濟(jì)大學(xué)醫(yī)學(xué)院干細(xì)胞研究中心,上海 200092)

        目的 探討過表達(dá)miR-124對Müller細(xì)胞轉(zhuǎn)分化機(jī)制的影響。方法 構(gòu)建pSuper-EGFP-miR-124質(zhì)粒并測序驗(yàn)證,獲得miR-124的過表達(dá)載體。并用其轉(zhuǎn)染Müller細(xì)胞系,用G418進(jìn)行篩選,獲得Müller細(xì)胞穩(wěn)轉(zhuǎn)株,將樣本送基因芯片檢測。利用TargetScan和miRBase Targets數(shù)據(jù)庫預(yù)測差異表達(dá)明顯的miR-124靶基因,通過Real-Time PCR和熒光素酶活性測定實(shí)驗(yàn)驗(yàn)證分析miR-124的靶基因,篩選變化比較顯著的基因進(jìn)一步研究。miR-124轉(zhuǎn)染Müller細(xì)胞,提取mRNA和蛋白,采用Real-Time PCR、Western印跡法檢測相關(guān)基因的變化。結(jié)果 成功構(gòu)建pSuper-EGFP-miR-124質(zhì)粒并獲得了穩(wěn)定轉(zhuǎn)染的Müller細(xì)胞系。熒光素酶報(bào)告分析表明,miR-124與STAT3 3′UTR相互作用。體外結(jié)果顯示,pSuper-miR124轉(zhuǎn)染Müller細(xì)胞后,STAT3表達(dá)下降并開始表達(dá)光感受器細(xì)胞標(biāo)志物CRX和RCVRN。結(jié)論 miR-124可能通過下調(diào)STAT3,上調(diào)CRX和RECVN,誘導(dǎo)Müller細(xì)胞向感光細(xì)胞轉(zhuǎn)分化。

        miR-124; STAT3; Müller細(xì)胞; 轉(zhuǎn)分化

        MicroRNA(miRNA)是一類廣泛存在于真核生物中,長度為21~23個(gè)堿基的內(nèi)源性非編碼單鏈小RNA。共有78種miRNA在成年小鼠的視網(wǎng)膜中表達(dá),其中21種是視網(wǎng)膜特有的,以維持視網(wǎng)膜功能[1-3]。

        miR-124在線蟲體內(nèi)的許多感覺神經(jīng)元中表達(dá)[4],可誘導(dǎo)胚胎干細(xì)胞分化為神經(jīng)細(xì)胞,與轉(zhuǎn)錄因子STAT[5-6]一起調(diào)控神經(jīng)元和膠質(zhì)細(xì)胞的比例[7-8]。因此,miR-124可能與特定的靶分子結(jié)合對哺乳動物視網(wǎng)膜起到多效性的保護(hù)作用[9]。

        Müller細(xì)胞是視網(wǎng)膜中重要膠質(zhì)細(xì)胞,對視網(wǎng)膜神經(jīng)元起著支持和營養(yǎng)的作用,同時(shí)還參與維持視網(wǎng)膜神經(jīng)細(xì)胞的新陳代謝內(nèi)環(huán)境的穩(wěn)態(tài)[10-11]。Müller細(xì)胞可能是一種晚期視網(wǎng)膜祖細(xì)胞,具有分化、增殖并轉(zhuǎn)分化為視網(wǎng)膜神經(jīng)細(xì)胞的潛能[12-13]。

        本研究采用過表達(dá)miR-124的Müller細(xì)胞作為細(xì)胞模型,在基因芯片結(jié)果的基礎(chǔ)上,經(jīng)TargetScan、miRBase Targets數(shù)據(jù)庫預(yù)測及熒光素酶報(bào)告基因分析,采用qPCR和Western B印跡法鑒定miR-124調(diào)控Müller細(xì)胞靶基因的關(guān)鍵分子及其在視網(wǎng)膜變性早期發(fā)揮的作用。

        1 材料與方法

        1.1 構(gòu)建pSuper-EGFP-miR-124質(zhì)粒

        miR-124前體擴(kuò)增引物的設(shè)計(jì): 檢索Sanger miRNA數(shù)據(jù)庫(http://microrna.sanger.ac.uk/sequences),獲得miR-124前體序列。利用primer3在線引物設(shè)計(jì)軟件(http://frodo.wi.mit.edu/primer3/),根據(jù)擬插入pSuper質(zhì)粒的酶切位點(diǎn)(BglⅡ/HindⅢ)設(shè)計(jì)合成擴(kuò)增miR-124前體片段的引物,連接轉(zhuǎn)化大腸桿菌并測序鑒定。

        1.2 轉(zhuǎn)染pSuper-EGFP-miR-124至Müller細(xì)胞系

        Müller細(xì)胞培養(yǎng)基為含10%胎牛血清、100U/ml青霉素、100U/ml鏈霉素的DMEM/F12培養(yǎng)基,置37℃、5%CO2培養(yǎng)箱培養(yǎng)。常規(guī)培養(yǎng)Müller細(xì)胞于24孔板中,至細(xì)胞密度達(dá)80%鋪滿時(shí),分5個(gè)濃度組加入含G418的培養(yǎng)液1ml/孔(G418用雙蒸水配制后過濾除菌,終濃度分別為0、250、500、750、1000μg/ml),換液時(shí)保持G418濃度穩(wěn)定不變,持續(xù)培養(yǎng)12d。選取在篩選12d能完全殺死所有Müller細(xì)胞的最小G418濃度作為最佳篩選濃度。

        將Müller細(xì)胞鋪于24孔板,待細(xì)胞長至孔板底面積80%左右時(shí),利用脂質(zhì)體LipofectamineTM2000將pSuper-miR-124重組質(zhì)粒轉(zhuǎn)入Müller細(xì)胞。細(xì)胞轉(zhuǎn)染48h后,熒光顯微鏡觀察轉(zhuǎn)染效率,加入按最佳篩選濃度配制好的G418篩選培養(yǎng)基篩選,最后獲得單克隆穩(wěn)定生長的Müller細(xì)胞株。收集活細(xì)胞樣本,做基因芯片。

        1.3 結(jié)合芯片結(jié)果預(yù)測差異表達(dá)明顯的miR-124的靶基因

        結(jié)合過表達(dá)miR-124的Müller細(xì)胞系的DNA芯片結(jié)果,利用TargetScan(http://genes.mit.edu/targetscan.test/ucsc.html)和miRBase Targets(http://cbio.mskcc.org/cgi-bin/mirnaviewer/mirna-viewer.pl)數(shù)據(jù)庫預(yù)測miR-124靶基因。

        1.4 Real-Time PCR驗(yàn)證miR-124的靶基因芯片

        收集G418篩選的miR-124穩(wěn)轉(zhuǎn)Müller細(xì)胞系,加入TRIzol抽提總RNA,采用反轉(zhuǎn)錄試劑盒(TaKaRa公司)完成反轉(zhuǎn)錄。將cDNA稀釋至10ng/μl,用于定量PCR。采用ΔΔCt=ΔCt處理組-ΔCt對照組的方法計(jì)算靶基因的表達(dá)量。

        1.5 psiCHECK-2-STAT3 3′UTR雙熒光素酶重組載體的構(gòu)建

        以Müller細(xì)胞系RNA為模板進(jìn)行反轉(zhuǎn)錄得到cDNA,設(shè)計(jì)rno-STAT3 3′UTR引物,以反轉(zhuǎn)錄后產(chǎn)物為模板進(jìn)行PCR擴(kuò)增,程序: 94℃ 10s,94℃ 30s、55℃ 30s、72℃ 30s,35個(gè)循環(huán)。然后電泳,目的片段90nt,割膠回收(天根DNA純化回收試劑盒),過柱純化。將純化產(chǎn)物與psiCHECK-2質(zhì)粒(C8021,Promega公司)同時(shí)用NotⅠ和XhoⅠ酶切,經(jīng)凝膠電泳分離、凝膠回收試劑盒回收目的片段并連接。將連接產(chǎn)物轉(zhuǎn)化感受態(tài)大腸桿菌,挑選轉(zhuǎn)化陽性克隆接種培養(yǎng),取菌液用小量法提取質(zhì)粒,用NotⅠ和XhoⅠ進(jìn)行雙酶切,電泳分析酶切片段并送測序鑒定,將測序結(jié)果正確的質(zhì)粒保種,濃度為300ng/μl。

        1.6 Western印跡法

        細(xì)胞轉(zhuǎn)染miR-124后刮取細(xì)胞,加入RIPA緩沖液(含10mg/ml PMSF)50l,冰上孵育30min,用BCA試劑盒測定細(xì)胞總蛋白濃度。30μg總蛋白樣品電泳,分離,轉(zhuǎn)膜,抗體孵育,化學(xué)發(fā)光顯色。采用β-actin作為內(nèi)參。

        1.7 免疫熒光組織化學(xué)染色

        細(xì)胞爬片用PBS漂洗2次,在24孔板內(nèi)加入4%多聚甲醛,室溫固定10~15min,PBS漂洗,0.3%Triton X-100的PBS透膜,5%BSA的PBS封閉。一抗孵育: 一抗按適當(dāng)?shù)味认♂屧诤?%BSA的PBS中,將一抗加入24孔板,4℃過夜,一抗分別為: GS(1∶200),Vimentin(1∶200),GFAP(1∶500),STAT3(1∶200),RCVRN(1∶200),CRX(1∶200);PBS漂洗3次。二抗孵育: 避光,加入相應(yīng)一抗來源的按比例稀釋的熒光素標(biāo)記二抗,室溫孵育60min,二抗分別為Cy3標(biāo)記山羊抗兔IgG(1∶500),Cy3標(biāo)記山羊抗小鼠IgG(1∶500);PBS漂洗3次,每次10min;0.5mg/ml DAPI(PBS配制)染細(xì)胞核3min,PBS漂洗3次,每次10min;取載玻片,滴少量DAKO,熒光顯微鏡或激光共聚焦顯微鏡觀察。

        1.8 細(xì)胞轉(zhuǎn)染及熒光素酶活性檢測

        將報(bào)告基因質(zhì)粒與pSuper-miR-124共轉(zhuǎn)染HEK293細(xì)胞,在轉(zhuǎn)染前1d,接種293細(xì)胞到24孔板,在正常條件下培養(yǎng)至80%匯合率時(shí)轉(zhuǎn)染,LipofectamineTM2000脂質(zhì)體共轉(zhuǎn)pSuper-miR-124和Psicheck-STAT3-3UTR后36h,進(jìn)行熒光素酶活性分析。

        1.9 統(tǒng)計(jì)學(xué)處理

        2 結(jié) 果

        2.1 pSuper-EGFP-miR-124重組質(zhì)粒的構(gòu)建

        檢索Sanger miRNA數(shù)據(jù)庫(http://microrna.sanger.ac.uk/sequences),獲得miR-124前體序列,并于NCBI數(shù)據(jù)庫檢索miR-124前體序列基因組定位及序列。利用primer3在線引物設(shè)計(jì)軟件(http://frodo.wi.mit.edu/primer3/),根據(jù)擬插入pSuper質(zhì)粒的酶切位點(diǎn)(BglⅢ/HindⅢ)設(shè)計(jì)合成擴(kuò)增miR-124。

        2.2 pSuper-EGFP-miR-124穩(wěn)定轉(zhuǎn)染的Müller細(xì)胞株的構(gòu)建

        選取在篩選12d充分殺死所有Müller細(xì)胞的最小G418濃度(500μg/ml)作為最佳篩選濃度。細(xì)胞轉(zhuǎn)染48h后,熒光顯微鏡下觀察轉(zhuǎn)染效率,綠色熒光的細(xì)胞數(shù)約占70%(圖1A、圖1B)。綠色熒光的細(xì)胞為表達(dá)綠色熒光蛋白(green fluorescent protein, GFP)的細(xì)胞,提示該轉(zhuǎn)染條件下能夠保證外源基因高效率地進(jìn)入Müller細(xì)胞。G418篩選2周后,Müller細(xì)胞均發(fā)出較為明亮的綠色熒光(圖1C、圖1D),通過提取細(xì)胞mRNA進(jìn)行檢測,進(jìn)一步驗(yàn)證了篩選得到的Müller細(xì)胞株穩(wěn)定表達(dá)了miR-124基因(圖2A),提示穩(wěn)定表達(dá)pSuper-miR-124的Müller細(xì)胞株成功獲得。

        2.3 miR-124靶基因的分析及驗(yàn)證

        miRBase Targets分析顯示,miR-124的靶基因有727個(gè);TargetScan 分析顯示,miR-124的靶基因共有1104個(gè)。miRBase Targets和TargetScan分析中miR-124共有的靶基因有233個(gè)。根據(jù)前期miR-124在Müller細(xì)胞中過表達(dá)的芯片結(jié)果,本研究選定STAT3作為進(jìn)一步重點(diǎn)研究的對象。經(jīng) RT-PCR 驗(yàn)證,與芯片結(jié)果一致(圖2A、圖2B)。同時(shí),Western印跡法也驗(yàn)證miR-124對STAT3有負(fù)調(diào)控作用(圖2C、圖2D)。

        2.4 熒光素酶活性分析

        與pSuper-miR-124共轉(zhuǎn)染時(shí),psiCHECK-2-STAT3 3′UTR共轉(zhuǎn)染組熒光比率較psiCHECKTM-2空載體對照組都有明顯下降,抑制率可以達(dá)到40%,兩組抑制作用差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。而當(dāng)psiCHECK-2-STAT3 3′UTR和psiCHECKTM-2與pSuper空載體共轉(zhuǎn)染時(shí),兩組間熒光比率差異無統(tǒng)計(jì)學(xué)意義,說明STAT3的3′-UTR能與miR-124相互作用,抑制海腎熒光素酶的活性(圖3)。因此,STAT3是miR-124的直接靶基因(P<0.05)。

        2.5 構(gòu)建psiCHECK-2-STAT3 3′UTR雙熒光素酶重組載體

        與未轉(zhuǎn)染miR124的Müller細(xì)胞相比,轉(zhuǎn)染miR-124后細(xì)胞的RECVN的mRNA水平顯著上調(diào),(P<0.05)。由于RECVN是感光細(xì)胞的高表達(dá)基因,故本結(jié)果提示轉(zhuǎn)染了miR-124 Müller細(xì)胞可能已發(fā)生向光感受器細(xì)胞的轉(zhuǎn)分化(圖4)。

        圖1 穩(wěn)定轉(zhuǎn)染pSuper-EGFP-miR-124的Müller細(xì)胞的篩選(標(biāo)尺: 20μm)Fig.1 Transfection of miR-124 into Müller cells and screening(scale bar: 20μm)A、B: Müller細(xì)胞轉(zhuǎn)染pSuper-EGFP-miR-124后48h;C、D: Müller細(xì)胞轉(zhuǎn)染pSuper-EGFP-miR-124后G418篩選2周;A、C為明場;綠色表示GFP

        圖2 Müller細(xì)胞中過表達(dá)miR-124對STAT3的影響Fig.2 Effect of miR-124 overexpression on STAT3 in Müller cellsA: miR-124表達(dá)水平;B: STAT3的mRNA水平;C: STAT3和p-STAT3的蛋白水平;D: 蛋白表達(dá)的灰度分析

        圖3 miR-124與STAT3 3′UTR相互作用Fig.3 The interaction between miR-124 and 3′UTR of STAT3

        圖4 RT-PCR檢測Müller細(xì)胞中Recoverin表達(dá)Fig.4 RT-PCR analysis of Recoverin expression in Müller cells

        3 討 論

        本研究通過miR-124轉(zhuǎn)染Müller細(xì)胞,鑒定miR-124能夠靶向STAT3基因,從而上調(diào)RECVN基因的表達(dá)。

        研究[14-15]發(fā)現(xiàn),在小鼠的腦組織和由P19多能干細(xì)胞分化而來的神經(jīng)樣細(xì)胞中miR-124被特異性地高表達(dá),提示miR-124可能與神經(jīng)細(xì)胞的分化相關(guān)[16]。miR-124的一個(gè)靶基因是多聚嘧啶序列結(jié)合蛋白(PTB)的編碼基因,PTB是一種選擇性剪切的調(diào)控蛋白,它能阻止選擇性外顯子的接入[17]。在神經(jīng)發(fā)育分化過程中,PTB和神經(jīng)元PTB(nPTB)的表達(dá)轉(zhuǎn)換,導(dǎo)致了神經(jīng)元重要功能基因的剪切模式的變化,PTB能改變nPTB的剪切方式,使nPTB蛋白的翻譯提前終止,從而不能得到成熟的nPTB蛋白[18]。而miR-124通過直接抑制PTB蛋白的表達(dá),而間接激活了nPTB的表達(dá),從而促進(jìn)細(xì)胞向神經(jīng)元方向分化。

        研究[19]表明,miR-124能直接抑制羧基端小結(jié)構(gòu)域磷酸酶1(SCP1),而SCP1是REST發(fā)揮抑制神經(jīng)元基因表達(dá)的必需成分,因此miR-124能間接地抑制REST的活性[20-21]。這表明存在一種負(fù)反饋機(jī)制: 在非神經(jīng)元細(xì)胞和神經(jīng)元前體細(xì)胞中,神經(jīng)元特異性基因(包括miR-124)的表達(dá)是被REST和SCP1抑制的,隨著細(xì)胞向神經(jīng)元方向分化以及REST受轉(zhuǎn)錄抑制,miR-124通過在轉(zhuǎn)錄后水平抑制REST的輔助因子SCP1的表達(dá)而快速終止REST的生物功能。

        以上的研究均提示,miR-124可能通過靶向PTB及SCP1等靶基因,而間接促進(jìn)神經(jīng)元基因表達(dá),使細(xì)胞向神經(jīng)元方向分化,這也是本研究在視網(wǎng)膜膠質(zhì)細(xì)胞(Müller細(xì)胞)中過表達(dá)miR-124并檢測視網(wǎng)膜神經(jīng)元特異基因改變的主要原因之一。

        Müller細(xì)胞作為視網(wǎng)膜中極為重要的膠質(zhì)細(xì)胞,具有分化、增殖并轉(zhuǎn)分化為視網(wǎng)膜神經(jīng)細(xì)胞的潛能。本研究利用miRBase Targets和TargetScan分析miR-124的靶基因并鑒定STAT3為miR-124靶基因。STAT3是一種潛在的細(xì)胞質(zhì)中固有的被JAK激酶磷酸化調(diào)節(jié)的轉(zhuǎn)錄因子,JAK/STAT是重要的細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)通路,調(diào)控細(xì)胞生長、分化和凋亡等重要過程。研究[22-25]報(bào)道,IL-6會導(dǎo)致視網(wǎng)膜STAT3活化[22],并進(jìn)一步影響視覺功能[23-25]: 如視紫紅質(zhì)蛋白水平的降低和光感受器外節(jié)的縮短。暗視視網(wǎng)膜電圖的a波的振幅也減小等。此外,還有文獻(xiàn)指出,STAT3的激活會導(dǎo)致視紫紅質(zhì)的泛素化和迅速降解,這一結(jié)論已經(jīng)在體外實(shí)驗(yàn)中得到了驗(yàn)證。應(yīng)用JKA的抑制劑抑制IL-6介導(dǎo)的STAT3的激活,可以阻止視紫紅質(zhì)的降解,進(jìn)而保護(hù)視功能[24,26]。

        在給予AAV2/8-EGFP-miR-124處理后,處理組STAT3的表達(dá)被抑制,而RCVN的表達(dá)量顯著上升。該結(jié)果進(jìn)一步提示了在視網(wǎng)膜Müller細(xì)胞中miR-124與STAT3的密切關(guān)系,RCVN的上升預(yù)示著Müller細(xì)胞很有可能已經(jīng)開始向視網(wǎng)膜神經(jīng)細(xì)胞轉(zhuǎn)分化。miR-124對Müller細(xì)胞具體的轉(zhuǎn)分化機(jī)制還有待于進(jìn)一步研究。從目前這些數(shù)據(jù)結(jié)果來看,抑制STAT3通路在Müller細(xì)胞轉(zhuǎn)分化成神經(jīng)細(xì)胞過程中起到了十分重要的作用。通過對miR-124功能的進(jìn)一步探索,發(fā)現(xiàn)其參與調(diào)節(jié)了細(xì)胞內(nèi)信號通路的轉(zhuǎn)導(dǎo),因此,通過過表達(dá)miR-124靶向抑制STAT3的表達(dá)從而干預(yù)Müller細(xì)胞轉(zhuǎn)分化過程,促進(jìn)神經(jīng)細(xì)胞再生可能成為視網(wǎng)膜退行性疾病治療的新方法。

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        [4] Clark AM, Goldstein LD, Tevlin M, et al. The micro-RNA miR-124 controls gene expression in the sensory nervous system of Caenorhabditis elegans[J]. Nucleic Acids Res, 2010,38(11): 3780-3793.

        [5] Bhattacharya S, Das AV, Mallya KB, et al. Ciliary neurotrophic factor-mediated signaling regulates neuronal versus glial differentiation of retinal stem cells/progenitors by concentration-dependent recruit-ment of mitogen-activated protein kinase and Janus kinase-signal transducer and activator of transcription pathways in conjunction with Notch signaling[J]. Stem Cells, 2008,26(10): 2611-2624.

        [6] Rhee KD, Goureau O, Chen S, et al. Cytokine-induced activation of signal transducer and activator of transcription in photoreceptor precursors regulates rod differentiation in the developing mouse retina[J]. J Neurosci, 2004,24(44): 9779-9788.

        [7] Yoo AS, Sun AX, Li L, et al. MicroRNA-mediated conversion of human fibroblasts to neurons[J]. Nature, 2011,476(7359): 228-331.

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        [9] Wu JH, Gao Y, Ren AJ, et al. Altered microRNA expression profiles in retinas with diabetic retinopa-thy[J]. Ophthalmic Res, 2012,47(4): 195-201.

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        [12] Fischer AJ,Reh TA. Muller glia are a potential source of neural regeneration in the postnatal chicken retina[J]. Nat Neurosci, 2001,4(3): 247-252.

        [13] Garcia M,Vecino E. Role of Muller glia in neuroprotection and regeneration in the retina[J]. Histol Histopathol, 2003,18(4): 1205-1218.

        [14] Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouse and human[J]. RNA, 2003,9(2): 175-179.

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        [16] Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs[J]. Nature, 2005,433(7027): 769-773.

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        [19] Baudet ML, Zivraj KH, Abreu-Goodger C, et al. miR-124 acts through CoREST to control onset of Sema3A sensitivity in navigating retinal growth cones[J]. Nat Neurosci, 2011,15(1): 29-38.

        [20] Coulson JM. Transcriptional regulation: Cancer, neurons and the REST[J]. Curr Biol, 2005,15(17): R665-668.

        [21] Visvanathan J, Lee S, Lee B, et al. The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development[J]. Genes Deve, 2007,21(7): 744-749.

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        Mechanisms of miR-124-induced transdifferentiation in Müller cells

        FANWen-bin1,2,3,ZHANGJie-ping1,2,3,WANGJuan1,2,3,LVLi-xia1,2,3,XUGuo-tong1,2,3

        (1. Tongji Eye Institute, Medical College, Tongji University, Shanghai 200092, China;2. Dept. of Regenerative Medicine, Medical College, Tongji University, Shanghai 200092, China;3. Stem Cell Research Center, Medical College, Tongji University, Shanghai 200092, China)

        Objective To investigate the mechanism of miR-124-induced transdifferentiation in Müller cells. Methods The pSuper-EGFP-miR-124 plasmid was constructed and sequenced, then transfected in Müller cells and verified by microarray analysis. miR-124 target genes were predicted with the combination of DNA microarray analysis, TargetScan and miRBase Targets databases. Real-time PCR and luciferase were employed to verify those target genes, and the differentially expressed genes were picked up for future study. The changes of related genes were detected by Real-time PCR and western blot in pSuper-miR-124 transfected Müller cells. Results pSuper-EGFP-miR-124 plasmid was cloned and sequenced, its sequence was confirmed. The stable transfected Müller cells were obtained under the selection of G418. Based on the results of microarray analysis, differentially expressed genes of interest were verified, especially STAT3 which showed significant trend. It showed that miR-124 regulated expression of STAT3 by directly targeting its 3′-UTR, and inhibition of STAT3 activation increased the expression of photoreceptor cell markers CRX and RCVRN in cultured Müller cells. Conclusion The miR-124 targets STAT3 to up-regulate CRX and RCVRN expression in Müller cells, which suggests that miR-124 may induce transdifferentiation of Müller cells to photoreceptor. It can be speculated that miR-124 could protect against retinal degeneration via inhibition of Müller cell gliosis and induction of Müller cell transdifferentiation.

        miR-124; STAT3; Müller cell; transdifferentiation

        10.16118/j.1008-0392.2016.01.001

        2015-06-09

        國家“九七三”重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(2013CB967501)

        范文斌(1989—),女,碩士研究生.E-mail: fanwenbing.1@163.com

        徐國彤.E-mail: gtxu@#edu.cn

        R 774.1

        A

        1008-0392(2016)01-0001-06

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