唐春梅， 張 銘， 胡志琴， 朱杰寧， 符永恒， 張夢(mèng)珍， 林秋雄， 鄧春玉， 譚虹虹， 吳書(shū)林， 單志新, △

(1南方醫(yī)科大學(xué)，廣東 廣州 510515； 2廣東省人民醫(yī)院/廣東省醫(yī)學(xué)科學(xué)院，廣東 廣州 510080)

·論 著·

MicroRNA-34a靶向SIRT1參與阿霉素誘導(dǎo)的心肌細(xì)胞凋亡*

唐春梅1， 張 銘2， 胡志琴1， 朱杰寧2， 符永恒2， 張夢(mèng)珍2， 林秋雄2， 鄧春玉2， 譚虹虹2， 吳書(shū)林2， 單志新1, 2 △

(1南方醫(yī)科大學(xué)，廣東 廣州 510515；2廣東省人民醫(yī)院/廣東省醫(yī)學(xué)科學(xué)院，廣東 廣州 510080)

目的： 研究微小RNA-34a(microRNA-34a，miR-34a)在阿霉素誘導(dǎo)的心肌細(xì)胞凋亡中的作用及其作用靶基因。方法: 建立阿霉素(doxorubicin, Dox)誘導(dǎo)的大鼠H9c2心肌細(xì)胞凋亡模型；TUNEL染色觀察H9c2細(xì)胞凋亡;雙螢光素酶報(bào)告實(shí)驗(yàn)檢測(cè)miR-34a與潛在靶基因沉默信息調(diào)節(jié)因子1 (silent information regulator 1,SIRT1) 3’端非翻譯區(qū)(3’-untranslated region, 3’UTR)的結(jié)合作用；實(shí)時(shí)熒光定量PCR檢測(cè)miR-34a和SIRT1 mRNA表達(dá)水平，Western blot檢測(cè)SIRT1和凋亡相關(guān)蛋白表達(dá)水平。結(jié)果: 阿霉素處理H9c2細(xì)胞之后，細(xì)胞發(fā)生凋亡，miR-34a的表達(dá)顯著增強(qiáng)；雙螢光素酶報(bào)告實(shí)驗(yàn)提示miR-34a與SIRT1 3’UTR可相互作用，并證實(shí)miR-34a可在轉(zhuǎn)錄后水平抑制SIRT1的表達(dá)，SIRT1蛋白水平在阿霉素處理的心肌細(xì)胞中顯著下調(diào)；過(guò)表達(dá)miR-34a及沉默SIRT1均能一致性抑制Bcl-2表達(dá)，促進(jìn)Bax和p66shc的表達(dá)，而過(guò)表達(dá)SIRT1能有效抑制阿霉素誘導(dǎo)的H9c2細(xì)胞凋亡。結(jié)論:SIRT1是miR-34a的靶基因，并介導(dǎo)了miR-34a在阿霉素誘導(dǎo)的心肌細(xì)胞凋亡中的作用。

阿霉素； 心臟毒性； 微小RNA-34a； 沉默信息調(diào)節(jié)因子1； H9c2細(xì)胞； 細(xì)胞凋亡

阿霉素(doxorubicin，Dox)是臨床廣泛應(yīng)用的抗生素類廣譜抗腫瘤藥，它通過(guò)嵌入基因組DNA而抑制核酸的合成[1]。阿霉素具有強(qiáng)烈的細(xì)胞毒性作用，包括能引起嚴(yán)重的心臟毒性，因而對(duì)其臨床應(yīng)用產(chǎn)生了劑量限制[2]，而阿霉素誘導(dǎo)心肌細(xì)胞凋亡是其心臟毒性的重要機(jī)制[3-4]。

微小RNA(microRNA，miRNA，miR)是一類小的非編碼RNA，它能特異地與靶基因mRNA的3’端非翻譯區(qū)(3’-untranslated region, 3’-UTR)序列相互識(shí)別，在轉(zhuǎn)錄或轉(zhuǎn)錄后水平負(fù)調(diào)控基因表達(dá)[5]。研究表明，microRNA 參與調(diào)控包括細(xì)胞增殖、分化、凋亡等各種生物進(jìn)程[6]，由于其在心血管功能、疾病中的重要調(diào)控作用，逐漸成為心血管疾病的潛在生物標(biāo)志和治療靶點(diǎn)[7-8]。心肌細(xì)胞凋亡是導(dǎo)致心臟功能損傷的重要因素，已有報(bào)道顯示多種microRNA參與心肌細(xì)胞凋亡的過(guò)程。過(guò)表達(dá)miR-125b能有效抑制缺血再灌注以及膿毒癥導(dǎo)致的小鼠心肌細(xì)胞凋亡，從而減輕小鼠心功能損傷[9]。miR-24能抑制心肌細(xì)胞缺血缺氧條件下的凋亡水平，進(jìn)而改善心梗后心功能[10]。而過(guò)表達(dá)miR-200a能有效抑制低氧誘導(dǎo)的氧化應(yīng)激和心肌細(xì)胞凋亡，從而發(fā)揮心臟保護(hù)作用[11]。

miR-34a參與細(xì)胞凋亡、細(xì)胞周期阻滯和細(xì)胞衰老等過(guò)程[12-13]。miR-34a能促進(jìn)心梗大鼠的心肌細(xì)胞凋亡，過(guò)表達(dá)miR-34a可明顯增加乳大鼠心肌細(xì)胞的凋亡[14]。本文將主要研究miR-34a在阿霉素誘導(dǎo)的大鼠H9c2細(xì)胞凋亡中的表達(dá)及其參與阿霉素誘導(dǎo)心肌細(xì)胞凋亡過(guò)程的分子機(jī)制。

材 料 和 方 法

1 主要試劑

限制性內(nèi)切酶XhoI和EcoR I、轉(zhuǎn)染試劑Lipofectamine 2000、TRIzol、逆轉(zhuǎn)錄試劑盒、4×SDS loa-ding buffer(Invitrogen)；2×SYBR Green Mix、RNAase free water(TaKaRa)；miR-34a mimic、沉默信息調(diào)節(jié)因子1 (silent information regulator 1，SIRT1)siRNA(廣州銳博)；BCA蛋白定量試劑盒(Thermo)；SDS-PAGE凝膠配置試劑盒、TUNEL試劑盒(碧云天)；抗GAPDH、p66shc和Bax抗體(Protein Technology)；抗Bcl-2抗體(Abcam)；抗SIRT1 抗體(Sigma)；蛋白marker(Fermentas)； PVDF膜(Whatman)； ECL發(fā)光液(Bioword)；DMEM/F12細(xì)胞培養(yǎng)基(HyClone)；特級(jí)澳洲胎牛血清(Gibco)。其它生化試劑均為進(jìn)口分裝或國(guó)產(chǎn)分析純。

2 主要方法

2.1 大鼠心肌細(xì)胞(H9c2細(xì)胞)處理 將大鼠H9c2細(xì)胞(ATCC)穩(wěn)定培養(yǎng)，分別給予不同濃度(0、1、2、4、5 μmol/L)阿霉素處理24 h，誘導(dǎo)心肌細(xì)胞凋亡；分別將100 nmol/L scramble對(duì)照、miR-34a mimic和SIRT1 siRNA轉(zhuǎn)染H9c2細(xì)胞，24 h后結(jié)束實(shí)驗(yàn)；分別將空載體質(zhì)粒(pcDNA3)和SIRT1質(zhì)粒(pcDNA3-SIRT1)轉(zhuǎn)染H9c2細(xì)胞，之后給予或不給予阿霉素處理24 h。

2.2 TUNEL 染色 將H9c2細(xì)胞鋪在預(yù)先放置蓋玻片的6孔板中穩(wěn)定生長(zhǎng)，給予不同處理之后，吸掉培養(yǎng)基，用PBS漂洗2次，加入1 mL 4 %的多聚甲醛溶液固定，用PBS漂洗3次，加入新配置的100 μL TUNEL檢測(cè)液，37 ℃避光孵育60 min。用PBS漂洗3次，加入含DAPI的抗熒光猝滅封片液封片后熒光顯微鏡下觀察。

2.3 實(shí)時(shí)熒光定量PCR檢測(cè)SIRT1以及miR-34a的表達(dá) 用TRIzol試劑提取心肌細(xì)胞總RNA。取2.0 μg總RNA，加入5×的逆轉(zhuǎn)錄試劑4 μL(逆轉(zhuǎn)錄試劑盒)，用oligo(dT)15和random primers逆轉(zhuǎn)錄出cDNA用于檢測(cè)SIRT1的mRNA水平。取1.0 μg總RNA，用miR-34a特異的RT引物(5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGAT-ACGACACAACCAG-3’)逆轉(zhuǎn)錄出cDNA用于檢測(cè)miR-34a水平。分別用GAPDH和U6作為檢測(cè)SIRT1和miR-34a表達(dá)水平的內(nèi)參照。在ViiATM7 Real-Time PCR System(Applied Biosystems)上進(jìn)行PCR反應(yīng)和結(jié)果分析。以2-ΔΔCt法計(jì)算SIRT1和miR-34a的相對(duì)表達(dá)水平。所用PCR引物序列見(jiàn)表1。

表1 RT-qPCR的引物序列

F: forward; R: reverse.

2.4 Western blot法檢測(cè)蛋白水平 收集處理后的心肌細(xì)胞，加入RIPA蛋白裂解液，冰上裂解，于4 ℃ 12 000 r/min離心10 min，取上清進(jìn)行蛋白定量后分裝，加入4×上樣緩沖液，100 ℃加熱10 min使蛋白變性，然后進(jìn)行聚丙烯酰胺凝膠電泳。用聚偏二氟乙烯(PVDF)膜轉(zhuǎn)膜，5%脫脂奶粉封閉2 h，分別用相應(yīng)的SIRT1 (1∶1 000)、p66shc(1∶1 000)、Bcl-2(1∶200)和Bax(1∶1 000)Ⅰ抗4 ℃孵育過(guò)夜。TBST洗膜后，加Ⅱ抗(1∶5 000或1∶1 000)4 ℃孵育2 h。ECL發(fā)光試劑盒顯影，以GAPDH(1∶5 000)為內(nèi)參照，掃描灰度值并分析蛋白表達(dá)相對(duì)含量。

2.5 雙螢光素酶報(bào)告實(shí)驗(yàn)驗(yàn)證miR-34a與SIRT1 3’UTR的結(jié)合作用 參照我們已報(bào)道方法[15]分別構(gòu)建包含miR-34a潛在結(jié)合序列的SIRT1 3’UTR重組螢光素酶報(bào)告質(zhì)粒pGL3-SIRT1-746-752和pGL3-SIRT1-1236-1242，以及包含結(jié)合序列突變的重組質(zhì)粒pGL3-SIRT1-746-752-MUT和pGL3-SIRT1-1236-1242-MUT。將HEK293細(xì)胞(細(xì)胞密度約為每孔1×105)轉(zhuǎn)染200 ng重組螢光素酶報(bào)告質(zhì)粒、100 nmol/L miR-34a mimic以及10 ng pRL-TK(表達(dá)海腎螢光素酶的內(nèi)參照質(zhì)粒)。轉(zhuǎn)染后24 h，測(cè)定螢火蟲(chóng)螢光素酶(firefly luciferase，F(xiàn)L)及海腎螢光素酶(Renillaluciferase，RL)活性，兩者比值(FL/RL)變化可反映miR-34a與SIRT1 3’UTR結(jié)合的能力。

3 統(tǒng)計(jì)學(xué)處理

用SPSS 21.0統(tǒng)計(jì)軟件進(jìn)行分析。數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)誤(mean±SEM)表示，兩組間比較采用t檢驗(yàn)，多組間比較采用單因素方差分析(one-way ANOVA)，并用SNK-q檢驗(yàn)進(jìn)行各組間的兩兩比較，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 miR-34a在阿霉素處理的H9c2細(xì)胞中表達(dá)增強(qiáng)

TUNEL染色結(jié)果顯示，分別用1、2、4、5 μmol/L阿霉素處理H9c2細(xì)胞24 h后，隨著阿霉素濃度增加，心肌細(xì)胞凋亡率也明顯增加，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。RT-qPCR結(jié)果顯示，與空白組相比，阿霉素處理的H9c2細(xì)胞中miR-34a的表達(dá)顯著增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。以上實(shí)驗(yàn)結(jié)果顯示，阿霉素能有效誘導(dǎo)心肌細(xì)胞凋亡，濃度4～5 μmol/L時(shí)效果明顯，為后續(xù)實(shí)驗(yàn)選用4 μmol/L濃度提供了實(shí)驗(yàn)依據(jù)；且阿霉素能顯著上調(diào)miR-34a的表達(dá)，見(jiàn)圖1。

Figure 1.miR-34a was up-regulated in Dox-treated H9c2 cells. A: the apoptotic H9c2 cells was detected by TUNEL assay. The scale bar=100 μm. B: determination of the miR-34a expression in the H9c2 cells by RT-qPCR. Mean±SEM. n=3. *P<0.05, **P<0.01 vs 0 μmol/L group.

2 miR-34a作用靶基因SIRT1的鑒定

基于miRDB數(shù)據(jù)庫(kù)(www.mirdb.org)以及TargetScan(www.targetscan.org)的序列分析提示，SIRT1 3’UTR的746～752和1 236～1 242堿基可能是miR-34a的結(jié)合位點(diǎn)。雙螢光素酶報(bào)告基因檢測(cè)結(jié)果顯示，轉(zhuǎn)染pGL3-SIRT1-746-752和pGL3-SIRT1-1236-1242質(zhì)粒的HEK293細(xì)胞，其螢光素酶活性分別較對(duì)照組下降約50%(P<0.01)和 35%(P<0.05)；而轉(zhuǎn)染含突變結(jié)合序列的質(zhì)粒則對(duì)螢光素酶活性無(wú)顯著影響，說(shuō)明miR-34a能特異結(jié)合到SIRT1 3’UTR的746～752和1 236～1 242位點(diǎn)，并抑制結(jié)合位點(diǎn)上游的螢火蟲(chóng)螢光素酶的表達(dá)。RT-qPCR 和Western blot法檢測(cè)結(jié)果顯示，SIRT1在阿霉素誘導(dǎo)凋亡的H9c2細(xì)胞中表達(dá)顯著降低；同時(shí)證實(shí)，凋亡相關(guān)的Bax在阿霉素誘導(dǎo)凋亡的H9c2細(xì)胞中表達(dá)顯著增強(qiáng)，而B(niǎo)cl-2表達(dá)顯著降低。轉(zhuǎn)染miR-34a mimic不影響H9c2細(xì)胞中SIRT1的 mRNA表達(dá)，但可顯著抑制H9c2細(xì)胞中SIRT1的蛋白表達(dá)，提示miR-34a可在轉(zhuǎn)錄后水平調(diào)控SIRT1的表達(dá)，見(jiàn)圖2。

Figure 2.Identification of SIRT1 as a target of miR-34a in the H9c2 cells exposed to Dox. A: dual luciferase assay. Mean±SEM. n=3. *P<0.05, **P<0.01 vs pGL3-promoter group. B: the mRNA expression of SIRT1 in the H9c2 cells exposed to Dox. Mean±SEM. n=3. *P<0.05, **P<0.01 vs 0 μmol/L group. C: the protein expression of Bax, Bcl-2 and SIRT1 in the H9c2 cells exposed to Dox. Mean±SEM. n=3. *P<0.05, **P<0.01 vs 0 μmol/L group. D: the expression of SIRT1 in miR-34a-modified H9c2 cells. Mean±SEM. n=3. *P<0.05, **P<0.01 vs scramble control group.

3 SIRT1介導(dǎo)miR-34a的促H9c2細(xì)胞凋亡作用

為了鑒定過(guò)表達(dá)miR-34a與抑制SIRT1對(duì)心肌細(xì)胞凋亡的影響，分別將100 nmol/L miR-34a mimic和SIRT1 siRNA轉(zhuǎn)染入H9c2細(xì)胞中；另外將空載體質(zhì)粒(pcDNA3)和SIRT1質(zhì)粒(pcDNA3-SIRT1)轉(zhuǎn)染入阿霉素誘導(dǎo)的H9c2細(xì)胞中，驗(yàn)證過(guò)表達(dá)SIRT1對(duì)阿霉素的誘導(dǎo)H9c2細(xì)胞凋亡的影響。Western blot實(shí)驗(yàn)結(jié)果顯示，miR-34a mimic和SIRT1 siRNA能一致性地抑制H9c2細(xì)胞中SIRT1表達(dá)，SIRT1底物p66shc蛋白水平升高；同時(shí)Bax蛋白表達(dá)增強(qiáng)而B(niǎo)cl-2蛋白表達(dá)降低。TUNEL染色結(jié)果顯示，過(guò)表達(dá)SIRT1能有效抑制阿霉素誘導(dǎo)的H9c2細(xì)胞凋亡。Western blot實(shí)驗(yàn)結(jié)果顯示，阿霉素誘導(dǎo)的H9c2細(xì)胞中Bax和p66shc表達(dá)增強(qiáng)，Bcl-2表達(dá)減弱，而過(guò)表達(dá)SIRT1能顯著降低阿霉素處理的H9c2細(xì)胞中Bax和p66shc表達(dá)，增加Bcl-2表達(dá)，見(jiàn)圖3。

Figure 3.SIRT1 mediated the pro-apoptotic effect of miR-34a in Dox-treated H9c2 cells. A: the protein expression of Bax, Bcl-2, p66shc and SIRT1 in H9c2 cells treated with miR-34a or SIRT1 siRNA was determined by Western blot. Mean±SEM. n=3. *P<0.05, **P<0.01 vs scramble group. B: the apoptosis of H9c2 cells was detected by TUNEL assay. The scale bar=100 μm. C: the protein expression of Bax, Bcl-2, p66shc and SIRT1 in H9c2 cells treated with pcDNA3, pcDNA3+Dox or pcDNA3-SIRT1 (pcSIRT1)+Dox was determined by Western blot. Mean±SEM. n=3. *P<0.05, **P<0.01 vs pcDNA3 group; #P<0.05, ##P<0.01 vs pcDNA3+Dox group.

討 論

近期研究發(fā)現(xiàn)，阿霉素主要是通過(guò)激活心肌氧化應(yīng)激，促進(jìn)活性氧蓄積等途徑引起心肌細(xì)胞凋亡[16-17]。H9c2心肌細(xì)胞來(lái)源于胚胎BDIX大鼠心臟組織，這類細(xì)胞具有骨骼肌的功能，包括表達(dá)煙堿受體和產(chǎn)生特定肌肉的肌酸激酶同工酶[18]。已有研究證實(shí)，阿霉素可使H9c2細(xì)胞中活性氧累積并激活A(yù)MPK，進(jìn)而導(dǎo)致p53發(fā)生磷酸化，最后導(dǎo)致細(xì)胞凋亡[19]；而在普通細(xì)胞和腫瘤細(xì)胞中，阿霉素還可以通過(guò)激活ERK/p53通路或者激活caspase-2，-3來(lái)誘導(dǎo)細(xì)胞凋亡[20-21]。

miR-34家族包括miR-34a、miR-34b和miR-34c，它們具有組織表達(dá)特異性，其中miR-34a主要在心、腦、腎、肺組織中高表達(dá)[22]。Ito等[23]首次報(bào)道了miR-34家族在心血管系統(tǒng)中的功能。研究發(fā)現(xiàn)，miR-34a通過(guò)負(fù)調(diào)控SIRT1來(lái)促進(jìn)內(nèi)皮細(xì)胞衰老[22]；在衰老小鼠主動(dòng)脈中，高表達(dá)的miR-34a抑制SIRT1，從而促進(jìn)主動(dòng)脈平滑肌細(xì)胞的衰老及炎癥因子的分泌[24]；小鼠心梗后，miR-34a可直接調(diào)控SIRT1、Bcl-2和cyclin D1的表達(dá)來(lái)抑制心臟修復(fù)[25]；Boon等[26]發(fā)現(xiàn)miR-34a在心梗小鼠心臟中高表達(dá)，而沉默miR-34a可減少細(xì)胞死亡，絲氨酸/蘇氨酸蛋白磷酸酶1調(diào)節(jié)亞基10介導(dǎo)了miR-34a的促DNA損傷和心肌細(xì)胞凋亡及心肌收縮功能損傷作用。

本文中，我們證實(shí)miR-34a在阿霉素誘導(dǎo)凋亡的H9c2細(xì)胞中表達(dá)升高，這與既往關(guān)于小鼠靜脈注射Dox可使心臟miR-34a表達(dá)增加的報(bào)道是相符的[27]。研究顯示，抑制JNK和NF-κB信號(hào)通路可上調(diào)單核細(xì)胞RAW 264.7中miR-34a表達(dá)[28]，但目前Dox上調(diào)心肌細(xì)胞中miR-34a上調(diào)表達(dá)的機(jī)制還不清楚。

雙螢光素酶報(bào)告基因?qū)嶒?yàn)提示miR-34a可與SIRT1 3’UTR特異性結(jié)合。通過(guò)檢測(cè)miR-34a修飾的H9c2中SIRT1的表達(dá)證實(shí)miR-34a可在轉(zhuǎn)錄后水平抑制SIRT1表達(dá)。進(jìn)一步的功能性研究表明，過(guò)表達(dá)miR-34a及沉默SIRT1均能一致性地促進(jìn)H9c2細(xì)胞凋亡，而過(guò)表達(dá)SIRT1能有效抑制阿霉素誘導(dǎo)的H9c2細(xì)胞凋亡。因此，上述結(jié)果證實(shí)SIRT1是miR-34a的靶基因，并介導(dǎo)了miR-34a參與阿霉素誘導(dǎo)的H9c2細(xì)胞凋亡。而已有研究顯示，白藜蘆醇(SIRT1激動(dòng)劑)正是通過(guò)激活SIRT1來(lái)減輕Dox誘導(dǎo)的心肌毒性[29]。我們的結(jié)果也證實(shí)Bcl-2也是miR-34a的靶基因，miR-34a能特異地抑制H9c2細(xì)胞中Bcl-2表達(dá)，這與以往報(bào)道一致[25]。

沉默信息調(diào)節(jié)因子2同源蛋白家族是一類保守的去乙?；傅鞍准易? 包括SIRT1～7，具有明顯的分布特異性，其中SIRT1主要分布在細(xì)胞核中，細(xì)胞質(zhì)和細(xì)胞膜上少量分布[30]。SIRT1在心血管疾病中的作用已被廣泛關(guān)注和研究。壓力負(fù)荷、饑餓、運(yùn)動(dòng)、急性局部缺血等均會(huì)導(dǎo)致心臟中SIRT1上調(diào)表達(dá)，而缺血再灌注損傷使其下調(diào)表達(dá)[31]。全身敲除SIRT1的小鼠，其心臟存在嚴(yán)重的發(fā)育缺陷[32]，特異性降低心臟中SIRT1水平會(huì)加重缺血再灌注導(dǎo)致的心肌損傷[33]。上述結(jié)果提示SIRT1具有非常重要的心肌保護(hù)作用。本文結(jié)果也表明，SIRT1在阿霉素誘導(dǎo)的凋亡H9c2細(xì)胞中表達(dá)降低，升高的miR-34a在轉(zhuǎn)錄后水平進(jìn)一步抑制SIRT1表達(dá)，而過(guò)表達(dá)SIRT1可有效抑制阿霉素誘導(dǎo)的H9c2細(xì)胞凋亡。

p66shc是 shcA適配器分子的一種亞型，是一種氧化還原酶，能增強(qiáng)脂質(zhì)過(guò)氧化介導(dǎo)線粒體死亡途徑誘導(dǎo)的細(xì)胞凋亡[34]。既往研究顯示，p66shc是SIRT1的作用靶點(diǎn)，SIRT1通過(guò)抑制p66shc減輕肝細(xì)胞的氧化應(yīng)激和凋亡[35-36]。而在本文中，我們發(fā)現(xiàn)SIRT1過(guò)表達(dá)能顯著降低H9c2細(xì)胞中p66shc表達(dá)，進(jìn)而降低Bax，升高Bcl-2表達(dá)；miR-34a抑制H9c2細(xì)胞中SIRT1表達(dá)后，Bax表達(dá)升高，Bcl-2表達(dá)降低，促進(jìn)細(xì)胞凋亡。

綜上所述，本文證實(shí)miR-34a在阿霉素誘導(dǎo)凋亡的H9c2細(xì)胞中表達(dá)上調(diào)，通過(guò)雙螢光素酶報(bào)告基因?qū)嶒?yàn)、靶基因表達(dá)和相應(yīng)功能性實(shí)驗(yàn)證實(shí)SIRT1是miR-34a的作用靶基因，miR-34a/SIRT1/p66shc介導(dǎo)了阿霉素誘發(fā)的心肌細(xì)胞凋亡。在后續(xù)研究中，我們將在整體動(dòng)物水平進(jìn)一步明確miR-34a對(duì)SIRT1表達(dá)和阿霉素誘發(fā)心臟毒性的作用。

[1] Tacar O, Dass CR. Doxorubicin-induced death in tumour cells and cardiomyocytes: is autophagy the key to improving future clinical outcomes?[J]. J Pharm Pharmacol, 2013, 65(11):1577-1589.

[2] Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems[J]. J Pharm Pharmacol, 2013, 65(2):157-170.

[3] Abd El-Gawad HM, El-Sawalhi MM. Nitric oxide and oxidative stress in brain and heart of normal rats treated with doxorubicin: role of aminoguanidine[J]. J Biochem Mol Toxicol, 2004, 18(2):69-77.

[4] Deng S, Kruger A, Kleschyov AL, et al. Gp91phox-containing NAD(P)H oxidase increases superoxide formation by doxorubicin and NADPH[J]. Free Radic Biol Med, 2007, 42(4): 466-473.

[5] Chen K, Rajewsky N. The evolution of gene regulation by transcription factors and microRNAs[J]. Nat Rev Genet, 2007, 8(2):93-103.

[6] Maire G, Martin JW, Yoshimoto M,et al. Analysis of miRNA-gene expression-genomic profles reveals complex mechanisms of microRNA deregulation in osteosarcoma [J]. Cancer Genet, 2011, 204(3):138-146.

[7] Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology[J]. Nature, 2011, 469(7330):336-342.

[8] Wang F, Chen C, Wang D. Circulating microRNAs in cardiovascular diseases: from biomarkers to therapeutic targets [J]. Front Med, 2014, 8(4):404-418.

[9] Wang X, Ha T, Zou J, et al. MicroRNA-125b protects against myocardial ischaemia/reperfusion injury via targeting p53-mediated apoptotic signalling and TRAF6[J]. Cardiovasc Res, 2014, 102(3):385-395.

[10]王 玨， 黃偉聰， 鄭亮承， 等. MicroRNA-24對(duì)心肌梗死后心肌細(xì)胞凋亡的調(diào)控作用[J]. 中國(guó)病理生理雜志, 2013, 29(4):590- 596.

[11]Sun X, Zuo H, Liu C, et al. Overexpression of miR-200a protects cardiomyocytes against hypoxia-induced apoptosis by modulating the kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 signaling axis[J]. Int J Mol Med, 2016,38(4):1303-1311.

[12]Hermeking H. p53 enters the microRNA world [J]. Cancer Cell, 2007, 12(5): 414-418.

[13]Chen H, Sun Y, Dong R, et al. Mir-34a is upregulated during liver regeneration in rats and is associated with the suppression of hepatocyte proliferation [J]. PLoS One, 2011, 6(5): e20238.

[14] Fan F, Sun A, Zhao H, et al. MicroRNA-34a promotes cardiomyocyte apoptosis post myocardial infarction through down-regulating aldehyde dehydrogenase 2[J]. Curr Pharm Des, 2013, 19(27):4865-4873.

[15] Shan ZX, Lin QX, Deng CY, et al. miR-1/miR-206 re-gulate Hsp60 expression contributing to glucose-mediated apoptosis in cardiomyocytes[J]. FEBS Lett, 2010, 584(16):3592-3600.

[16] Berthiaume JM, Wallace KB. Adriamycin-induced oxidative mitochondrial cardiotoxicity [J]. Cell Biol Toxicol, 2007, 23(1):15-25.

[17] Ichikawa Y, Ghanefar M, Bayeva M, et al. Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation [J]. J Clin Invest, 2014, 124(2): 617-630.

[18]Kimes BW, Brandt BL. Properties of a clonal muscle cell line from rat heart [J]. Exp Cell Res, 1976, 98(2):367-381.

[19]Chen MB, Wu XY, Gu JH, et al. Activation of AMP-activated protein kinase contributes to doxorubicin-induced cell death and apoptosis in cultured myocardial H9c2 cells[J]. Cell Biochem Biophys, 2011, 60(3):311-322.

[20]Wang S, Konorev EA, Kotamraju S, et al. Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms. Intermediacy of H2O2- and p53-dependent pathways[J]. J Biol Chem, 2004, 279(24): 25535-25543.

[21]Yeh PY, Chuang SE, Yeh KH, et al. Phosphorylation of p53 on Thr55 by ERK2 is necessary for doxorubicin-induced p53 activation and cell death[J]. Oncogene, 2004, 23(20):3580-3588.

[22]Li N, Wang K, Li PF. MicroRNA-34 family and its role in cardiovascular disease[J]. Crit Rev Eukaryot Gene Expr, 2015, 25(4):293-297.

[23]Ito T, Yagi S, Yamakuchi M. MicroRNA-34a regulation of endothelial senescence[J]. Biochem Biophys Res Commun, 2010, 398(4):735-740.

[24]Badi I, Burba I, Ruggeri C, et al. MicroRNA-34a induces vascular smooth muscle cells senescence by SIRT1 downregulation and promotes the expression of age-asso-ciated pro-infammatory secretory factors[J]. J Gerontol A Biol Sci Med Sci, 2015, 70(11):1304-1311.

[25]Yang Y, Cheng HW, Qiu Y, et al. MicroRNA-34a plays a key role in cardiac repair and regeneration following myocardial infarction[J]. Circ Res, 2015, 117(5): 450-459.

[26]Boon RA, Iekushi K, Lechner S, et al. MicroRNA-34a regulates cardiac ageing and function [J]. Nature, 2013, 495(7439):107-110.

[27]Desai VG, Kwekel JC, Vijay V, et al. Early biomarkers of doxorubicin-induced heart injury in a mouse model [J]. Toxicol Appl Pharmacol, 2014, 281(2):221-229.

[28]Saba E, Jeon BR, Jeong DH, et al. A novel korean red ginseng compound gintonin inhibited inflammation by MAPK and NF-κB pathways and recovered the levels of mir-34a and mir-93 in RAW 264.7 cells[J]. Evid Based Complement Alternat Med, 2015, 2015:624132.

[29]Liu MH, Shan J, Li J, et al. Resveratrol inhibits doxorubicin-induced cardiotoxicity via sirtuin 1 activation in H9c2 cardiomyocytes[J]. Exp Ther Med, 2016, 12(2):1113-1118.

[30]Dali-Youcef N, Lagouge M, Froelich S, et al. Sirtuins: the ‘magnificent seven’, function, metabolism and longevity[J]. Ann Med, 2007, 39(5):335-345.

[31]Matsushima S, Sadoshima J. The role of sirtuins in cardiac disease [J]. Am J Physiol Heart Circ Physiol, 2015, 309(9): H1375-H1389.

[32]Sundaresan NR, Pillai VB, Wolfgeher D, et al. The deacetylase SIRT1 promotes membrane localization and activation of Akt and PDK1 during tumorigenesis and cardiac hypertrophy [J]. Sci Signal, 2011, 19:4(182):ra46.

[33] Hsu CP, Zhai P,Yamamoto T, et al. Silent information regulator 1 protects the heart from ischemia/reperfusion[J]. Circulation, 2010, 122(21): 2170-2182.

[34]Migliaccio E, Giorgio M, Mele S, et al. The p66shcadaptor protein controls oxidative stress response and life span in mammals[J]. Nature, 1999, 402 (6759): 309-313.

[35]Hu Y, Yao J, Zhang F, et al. Sirtuin 1-mediated inhibition of p66shc expression alleviates liver ischemia/reperfusion injury[J]. Crit Care Med, 2014, 42(5): e373-e381.

[36]Xu X, Hu Y, Zhai X, et al. Salvianolic acid A preconditioning confers protection against concanavalin-A-induced liver injury through SIRT1-mediated repression of p66shc in mice [J]. Toxicol Appl Pharmacol, 2013, 273(1): 68-76.

(責(zé)任編輯： 陳妙玲， 羅 森)

MicroRNA-34a participates in doxorubicin-induced cardiomyocyte apoptosis via targetingSIRT1

TANG Chun-mei1, ZHANG Ming2, HU Zhi-qin1, ZHU Jie-ning2, FU Yong-heng2, ZHANG Meng-zhen2, LIN Qiu-xiong2, DENG Chun-yu2, TAN Hong-hong2, WU Shu-lin2, SHAN Zhi-xin1, 2

(1SouthernMedicalUniversity,Guangzhou510515,China;2GuangdongGeneralHospital,GuangdongAcademyofMedicalSciences,Guangzhou510080,China.E-mail:zhixinshan@aliyun.com)

AIM: To investigate the role of microRNA-34a (miR-34a) in doxorubicin (Dox)-induced cardiomyocyte apoptosis and the potential target gene. METHODS: The apoptotic model of H9c2 cells was established by Dox induction. The apoptotic H9c2 cells was detected by TUNEL assay. Dual luciferase reporter assay was performed to confirm the interaction between miR-34a and the 3’UTR of silent information regulator 1 (SIRT1). The expression of miR-34a and SIRT1 mRNA was determined by RT-qPCR, and the protein expression of SIRT1 and apoptosis-related proteins was detected by Western blot. RESULTS: Cell apoptosis and miR-34a was markedly increased in Dox-induced H9c2 cells. Dual luciferase reporter assay revealed that miR-34a interacted with the 3’UTR of SIRT1, and miR-34a was observed to inhibit SIRT1 expression at the post-transcriptional level. The protein expression of SIRT1 was markedly decreased in the apoptotic H9c2 cells. Moreover, miR-34a mimic, in parallel toSIRT1 siRNA, inhibited Bcl-2 expression but increased the expression of Bax and p66shc. Overexpression of SIRT1 significantly inhibited Dox-induced apoptosis in the H9c2 cells. CONCLUSION:SIRT1 is a target gene of miR-34a, and mediates the effect of miR-34a on Dox-induced cardiomyocyte apoptosis.

Doxorubicin; Cardiotoxicity; MicroRNA-34a; Silent information regulator 1; H9c2 cells; Apoptosis

1000- 4718(2017)03- 0385- 07

2016- 10- 11

2016- 12- 09

國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 81270222； No. 81470439; No. 91649109)；廣東省醫(yī)學(xué)科學(xué)研究項(xiàng)目(No. 2014A030313635； No. 2013B022000083； No. A2015187)

△通訊作者 Tel: 020-83827812-51158； E-mail: zhixinshan@aliyun.com

R329.21

A

10.3969/j.issn.1000- 4718.2017.03.001