郭行雅,章瑞南,何崇信,孫 芳,范建高,潘 勤
上海交通大學(xué)醫(yī)學(xué)院附屬新華醫(yī)院消化內(nèi)科,上海 200092
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人環(huán)狀RNA-0046366與肝細(xì)胞脂肪變性的相關(guān)性及其機(jī)制研究
郭行雅,章瑞南,何崇信,孫 芳,范建高,潘 勤
上海交通大學(xué)醫(yī)學(xué)院附屬新華醫(yī)院消化內(nèi)科,上海 200092
目的 探討人環(huán)狀RNA-0046366(circRNA-0046366)表達(dá)變化對肝細(xì)胞脂肪變性的影響及其機(jī)制。方法 將肝HepG2細(xì)胞株隨機(jī)分為正常對照組(n=3)和脂肪變性模型組(n=3),分別給予高糖DMEM、高脂培養(yǎng)基(油酸∶棕櫚酸=2∶1, 0.5 mmol/L)誘導(dǎo)24 h。采用油紅O染色及甘油三酯(Triglyceride,TG)、丙二醛(Malonyldialdehyde,MDA)檢測,觀察肝細(xì)胞的脂肪變性及脂質(zhì)過氧化。以實時熒光定量PCR(Real-Time PCR)法檢測circRNA-0046366在脂肪變性前后的表達(dá)改變。在circBase數(shù)據(jù)庫搜索、circRNA-miRNA互補(bǔ)序列分析的基礎(chǔ)上,預(yù)測circRNA-0046366的靶miRNA及其下游基因。 通過Spearman相關(guān)性分析,揭示circRNA-0046366水平與靶miRNA表達(dá)、下游mRNA表達(dá)、肝細(xì)胞脂肪變性及脂質(zhì)過氧化指標(biāo)的相關(guān)性。結(jié)果 與正常對照組相比,脂肪變性模型組的肝細(xì)胞內(nèi)脂滴、TG[(24.4±2.4) μmol/gvs(263.7±17.5) μmol/g,P<0.05]及MDA[(0.6±0.1) μmol/gvs(1.8±0.2) μmol/g,P<0.05]含量明顯升高。circRNA-0046366表達(dá)水平在脂肪變性的肝細(xì)胞中則顯著降低(P<0.05)。circRNA-0046366可與過氧化物酶體增殖物激活受體α(peroxisome proliferator-activated receptor α, PPARα)競爭性結(jié)合miR-34a,從而阻斷miR-34a對PPARα翻譯抑制作用。相關(guān)性分析顯示,肝細(xì)胞的circRNA-0046366與PPARα表達(dá)水平呈顯著正相關(guān)(r=0.78,P<0.05),與miR-34a表達(dá)水平(r=-0.83,P<0.05)、TG(r=-0.72,P<0.05)及MDA(r=-0.69,P<0.05)含量則呈顯著負(fù)相關(guān)。結(jié)論 肝細(xì)胞的circRNA-0046366水平與脂肪變性及脂質(zhì)過氧化呈負(fù)相關(guān)。其機(jī)制可能與靶向結(jié)合miR-34a,從而解除PPARα的表達(dá)抑制相關(guān)。
環(huán)狀RNA;肝細(xì)胞;脂肪變性;微小RNA;過氧化物酶體增殖激活受體α
近年研究[1-2]顯示,miRNA與肝細(xì)胞脂肪變性密切相關(guān),miR-181d、miR-10b等11種miRNA均可靶向調(diào)節(jié)脂質(zhì)代謝相關(guān)基因的表達(dá)水平,從而干預(yù)細(xì)胞內(nèi)甘油三脂(Triglyceride,TG)的轉(zhuǎn)運、合成、氧化,在此基礎(chǔ)上影響肝細(xì)胞的脂肪變程度。 然而迄今為止,對肝細(xì)胞脂肪變相關(guān)miRNA的調(diào)控機(jī)制仍不明確。
20世紀(jì)70年代在RNA病毒中發(fā)現(xiàn)一類非編碼RNA—環(huán)狀RNA(circular RNA,circRNA)[3],近年來高通量測序結(jié)合轉(zhuǎn)錄組分析發(fā)現(xiàn),circRNA大量存在于真核細(xì)胞中[4-6]。circRNA可通過堿基互補(bǔ)充當(dāng)miRNA海綿(miRNA sponge)[7-8],與miRNA發(fā)生競爭性結(jié)合,進(jìn)而解除其對下游靶基因的轉(zhuǎn)錄抑制作用。這提示circRNA可能成為miRNA調(diào)節(jié)因子,在多種病理生理過程發(fā)揮重要作用。
為探討circRNA與肝細(xì)胞脂肪變性的關(guān)聯(lián),本研究采用油酸/棕櫚酸誘導(dǎo)肝細(xì)胞脂肪變性模型。結(jié)合circBase[9]數(shù)據(jù)庫搜索及circRNA-miRNA互補(bǔ)序列分析,預(yù)測對肝細(xì)胞脂肪變相關(guān)性miRNA具有調(diào)控作用的circRNA。 通過實時定量PCR檢測TG及丙二醛(Malonyldia-ldehyde,MDA)水平定量,驗證circRNA表達(dá)水平與肝細(xì)胞脂肪變性、脂質(zhì)過氧化指標(biāo)的相關(guān)性。 在miRNA “種子”序列與circRNA、mRNA互補(bǔ)配對的基礎(chǔ)上,提出circRNA-miRNA-mRNA級聯(lián)調(diào)控的可能作用機(jī)制。
1.1 材料 HepG2細(xì)胞購自上海細(xì)胞庫;棕櫚酸(PA,P9767)、油酸(OA,07501)均購自SIGMA公司;TG檢測試劑盒(北京普利萊基因技術(shù)有限公司);BCA蛋白濃度測定試劑盒(碧云天生物公司);MDA檢測試劑盒(碧云天生物公司);Cell Counting Kit-8(CCK-8)試劑盒(碧云天生物技術(shù)公司);FastQuant RT Kit逆轉(zhuǎn)錄試劑盒(大連寶生物工程有限公司);HieffTMqPCR SYBR@Green Master Mix(上海翊圣生物科技有限公司));PCR儀(ABI,美國);酶標(biāo)儀(Thermo,芬蘭);紫外分光光度計(Eppendorf,德國)。
1.2 方法
1.2.1 建立肝細(xì)胞脂肪變性模型:將HepG2細(xì)胞隨機(jī)分為正常對照組(n=3)、脂肪變性模型組(n=3)。各組細(xì)胞采用含100 g/L的高糖DMEM培養(yǎng)72 h后,脂肪變性模型組改為含有0.5 mmol/L游離脂肪酸(free fatty acid, FFA)混合物(OA∶PA=2∶1)的高脂培養(yǎng)基,經(jīng)高脂誘導(dǎo)24 h,收集細(xì)胞待用。
1.2.2 油紅O染色:采用70%的油紅O溶液,浸染10~15 min,60%異丙醇/三蒸水沖洗,蘇木素復(fù)染,鏡檢。
1.2.3 細(xì)胞活力檢測:按上述分組方法,于96孔板中接種HepG2細(xì)胞(1×104個/孔)。分別采用正?;蚋咧囵B(yǎng)基誘導(dǎo)24 h后,加入CCK-8溶液(10 μl/孔),37 ℃孵育4 h,采用酶標(biāo)儀測定450 nm處吸光度值。
1.2.4 TG、MDA含量檢測:收集正常對照組、脂肪變性模型組細(xì)胞,分別采用BCA法作蛋白定量,TG酶法作TG定量,TBA比色法作MDA定量。
1.2.5 circRNA-0046366下游靶miRNA、靶mRNA預(yù)測:通過circRNA與miRNA序列互補(bǔ)分析,并結(jié)合circBase數(shù)據(jù)庫,以預(yù)測與circRNA-0046366靶向結(jié)合的miRNA。利用miRNA靶點預(yù)測工具TargetScan,發(fā)現(xiàn)miR-34a的下游靶基因。
1.2.6 circRNA-0046366、miR-34a及PPARα mRNA含量檢測:分別提取各組細(xì)胞Total RNA,合成 cDNA第一鏈。設(shè)計并合成引物,引物序列如下(見表1),miR-34a qPCR引物由大連寶生物工程有限公司提供。 配制反應(yīng)液,設(shè)置反應(yīng)程序,應(yīng)用ABI7500實時PCR儀獲得目的基因擴(kuò)增曲線及CT值,采用 2-ΔΔCT法對各組CT值進(jìn)行結(jié)果分析。
1.2.7 相關(guān)性分析:采用Spearman法分析miR-34a、過氧化物酶體增殖激活受體α(peroxisome proliferation activated receptor α,PPARα)與circRNA-0046366的相關(guān)性,及TG、MDA與circRNA-0046366的相關(guān)性。
表1 目的基因引物序列
Tab 1 Target gene primer sequence
目的基因基因序列(5′?3′)GAPDHF:CTCCAAAATCAAGTGGGGCR:TGGTTCACACCCATGACGAAU6F:CTCGCTTCGGCAGCACAR:AACGCTTCACGAATTTGCGTcircRNA?0046366F:CATCCAGGGATGGGCACACR:GCTTCACAGCCTCATCGGAGPPARαF:CGGCGAGGATAGTTCTGGAR:GGGACCACAGGATAAGTCACC
2.1 油紅O染色結(jié)果 正常對照組HepG2細(xì)胞為不規(guī)則形梭形,胞質(zhì)無明顯紅染或淡染。脂肪變性模型組細(xì)胞廣泛染色,胞質(zhì)含大量紅色脂滴(見圖1)。CCK-8結(jié)果顯示兩組細(xì)胞生長均良好, 細(xì)胞活力無明顯差異。
圖1 肝細(xì)胞油紅O染色(200×) A:正常對照組;B:脂肪變性模型組
Fig 1 Oil Red O staining of hepatocytes (200×) A: normal control group; B: steatosis model group
2.2 TG含量、MDA水平比較 正常對照組和脂肪變性模型組的TG含量分別為(24.4±2.4) μmol/g 和(263.7±17.5) μmol/g。與正常對照組相比,脂肪變性模型組的TG含量明顯升高(t=13.6,P<0.05)。正常對照組和脂肪變性模型組的MDA含量分別為(0.6±0.1) μmol/g 和(1.8±0.2) μmol/g,兩者比較,差異有統(tǒng)計學(xué)意義(t=5.2,P<0.05,見表2)。
2.3 circRNA-0046366的靶miRNA、靶mRNA 生物信息學(xué)分析結(jié)果顯示,miR-34a為circRNA-0046366下游靶miRNA(見圖2),PPARα為miR-34a下游靶基因(見圖3)。
圖2 circRNA-0046366下游靶miRNAFig 2 Downstream target miRNA of circRNA-0046366
圖3 miR-34a下游靶基因預(yù)測Fig 3 Predicting on downstream target gene of miR-34a
2.4 circRNA-0046366、miR-34a及PPARα mRNA的表達(dá)水平 正常對照組circRNA-0046366水平顯著高于脂肪變性模型組[(3.43±0.55)倍,P<0.05];脂肪變性模型組miR-34a水平顯著高于正常對照組[(1.92±0.30)倍,P<0.05];正常對照組PPARα mRNA水平顯著高于脂肪變性模型組(1.75±0.19)倍(P<0.05,見圖4)。
注:與正常對照組比較,*P<0.05。
圖4 兩組細(xì)胞circRNA-0046366、miR-34a及PPARα mRNA水平比較 A: circRNA-0046366; B: miR-34a; C: PPARα
Fig 4 The expression levels of circRNA-0046366, miR-34a and PPARα mRNA between two groups A: circRNA-0046366; B: miR-34a; C: PPARα mRNA
2.5 circRNA-0046366與miR-34a、PPARα相關(guān)性分析 脂肪變性模型組circRNA-0046366與miR-34a呈顯著負(fù)相關(guān)(r=-0.83,P<0.05);脂肪變性模型組 circRNA-0046366與PPARα呈顯著正相關(guān)(r=0.78,P<0.05,見圖5)。
2.6 circRNA-0046366與TG及MDA相關(guān)性分析 脂肪變性模型組circRNA-0046366與TG呈顯著負(fù)相關(guān)(r=-0.72,P<0.05);脂肪變性模型組circRNA-0046366與MDA呈顯著負(fù)相關(guān)(r=-0.69,P<0.05,見圖6)。
目前研究已表明,circRNA具有較強(qiáng)的轉(zhuǎn)錄后調(diào)控作用[7-8]。多種circRNA均攜帶miRNA應(yīng)答元件(miRNA response element,MRE),并能夠與miRNA競爭性結(jié)合,從而成為競爭性內(nèi)源RNA(competing endogenous RNA,ceRNA)。這一作用可解除miRNA對其靶基因的表達(dá)抑制,從而調(diào)控心力衰竭、骨關(guān)節(jié)炎及腫瘤等多種病理生理過程[10-14]。本研究在circBase數(shù)據(jù)庫的基礎(chǔ)上開展circRNA與miRNA“種子”序列的互補(bǔ)配對分析,發(fā)現(xiàn)脂肪酸合成酶基因的差異剪切產(chǎn)物可環(huán)化形成circRNA-0046366,且其177~183 bp序列攜帶miR-34a特異性 MRE。 Ding等[15]報道,miR-34a通過抑制PPARα的翻譯水平,可下調(diào)CPT1等脂質(zhì)氧化相關(guān)的下游基因表達(dá),導(dǎo)致肝細(xì)胞脂肪變性。由此推測,circRNA-0046366可能通過與miR-34a競爭性結(jié)合,消除其對下游靶基因的抑制作用,進(jìn)而發(fā)揮拮抗肝細(xì)胞脂肪變性的效應(yīng)。
圖5 circRNA-0046366與miR-34a、PPARα相關(guān)性分析 A: circRNA-0046366與miR-34a; B: circRNA-0046366與PPARα; 圖6 circRNA-0046366與TG、MDA含量相關(guān)性分析 A: circRNA-0046366與TG; B: circRNA-0046366與MDA
Fig 5 The correlation analysis of circRNA-0046366 level and miR-34a, PPARα expression A: circRNA-0046366 level and miR-34a expression; B: circRNA-0046366 level and PPARα expression; Fig 6 The correlation analysis of circRNA-0046366 level and TG, MDA content A: circRNA-0046366 level and TG content; B: circRNA-0046366 level and MDA cotent
為闡明circRNA-0046366與肝細(xì)胞脂肪變性的相關(guān)性,本研究采用高脂培養(yǎng)基(OA∶PA=2∶1,0.5 mmol/L)誘導(dǎo)肝細(xì)胞脂肪變性模型,并定量檢測circRNA-0046366在脂肪變性前后的表達(dá)變化。結(jié)果顯示,脂肪變性模型組肝細(xì)胞含有大量脂滴,且TG含量顯著高于正常對照組,脂肪變細(xì)胞內(nèi)的MDA水平也明顯升高。因而,高脂培養(yǎng)可誘導(dǎo)肝細(xì)胞發(fā)生脂肪變性及脂質(zhì)過氧化。Real-Time PCR檢測表明,肝細(xì)胞的circRNA-0046366表達(dá)水平伴隨脂肪變性而明顯下調(diào)。Spearman相關(guān)性分析提示,circRNA-0046366水平與肝細(xì)胞的TG、MDA含量呈顯著負(fù)相關(guān)。上述結(jié)果證實circRNA-0046366與肝細(xì)胞脂肪變性密切相關(guān),可能在脂肪變性和脂質(zhì)過氧化過程中發(fā)揮負(fù)調(diào)控作用。
近年多項研究提出,circRNA、miRNA及其下游mRNA可構(gòu)成“circRNA -miRNA-mRNA”調(diào)控網(wǎng)絡(luò),對腫瘤和阿爾茲海默癥等[16]的發(fā)生、發(fā)展具有重要意義。在circRNA-0046366特征性低表達(dá)的基礎(chǔ)上,本研究針對其靶miRNA進(jìn)行定量檢測,可見miR-34a表達(dá)水平在脂肪變性的肝細(xì)胞內(nèi)大幅度上升。利用miRNA靶點預(yù)測工具TargetScan來分析miR-34a與mRNA的序列互補(bǔ)性,發(fā)現(xiàn)PPARα mRNA 3′ 非編碼區(qū)1668~1674 bp、5080~5086 bp、8004~8010 bp均存在miR-34a 2-7位堿基的互補(bǔ)序列[17]。由于PPARα具有促進(jìn)脂肪酸及脂蛋白代謝,改善胰島素抵抗和非酒精性脂肪性肝炎(non-alcohol steatohepatitis,NASH)的作用[18],因此,高豐度miR-34a與PPARα靶向性結(jié)合能夠阻礙脂質(zhì)代謝,誘導(dǎo)肝細(xì)胞脂肪變性,這與Ding等[15]的報道相吻合。結(jié)合circRNA及miRNA的表達(dá)改變,提示circRNA-0046366在正常肝細(xì)胞中可能競爭性結(jié)合miR-34a,從而避免PPARα發(fā)生表達(dá)抑制,由此發(fā)揮拮抗脂肪變性的效應(yīng)。而circRNA-0046366在脂肪變性的肝細(xì)胞中出現(xiàn)表達(dá)缺失,阻斷了這一負(fù)向調(diào)節(jié)機(jī)制,結(jié)果導(dǎo)致miR-34a介導(dǎo)的PPARα抑制,隨之引起TG蓄積和脂質(zhì)過氧化。
綜上所述,circRNA-0046366在肝細(xì)胞脂肪變性過程中呈明顯低表達(dá),并與TG含量、脂質(zhì)過氧化水平呈顯著負(fù)相關(guān),其作用機(jī)制可能與競爭性結(jié)合miR-34a,繼而解除肝細(xì)胞內(nèi)PPARα的表達(dá)抑制相關(guān)。
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(責(zé)任編輯:王全楚)
The association and related mechanisms between human circRNA-0046366 and hepatocyte steatosis
GUO Xingya, ZHANG Ruinan, HE Chongxin, SUN Fang, FAN Jiangao, PAN Qin
Department of Gastroenterology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
Objective To investigate the association and related mechanisms between human circular RNA-0046366 (circRNA-0046366) and hepatocyte steatosis.Methods HepG2 cells were randomly divided into groups of normal control (n=3) and steatosismodel group (n=3), respectively. The normal control group was treated with DMEM, while the steatosis-model group was administrated with high-fat medium (OA∶PA=2∶1, 0.5 mmol/L) for 24 hours. Both oil red O staining and triglyceride (TG) concentration were employed to evaluate the hepatocyte steatosis. Hepatic level of malonaldehyde (MDA) reflected the lipid peroxidation, which occurred on the basis of hepatocyte steatosis. Real-time PCR exhibited the alternation of circRNA-0046366 expression during hepatic steatosis. Bioinformatic analysis demonstrated the target miRNA, and downstream mRNAs, of circRNA-0046366 by means of circBase searching and circRNA-miRNA complementation. Finally, the association among circRNA-0046366 level, miRNA, mRNAs, hepatocyte steatosis, and lipid peroxidation was assessed by Spearman correlation analysis.Results Compared with normal control group, steatosismodel group exhibited significant increase in lipid droplets, and hepatic contents TG [(24.4±2.4) μmol/gvs(263.7±17.5) μmol/g,P<0.05] and MDA [(0.6±0.1) μmol/gvs(1.8±0.2) μmol/g,P<0.05]. The expression level of circRNA-0046366 underwent statistically decrease (P<0.05) during the hepatocyte steatosis. Both circRNA-0046366 and peroxisome proliferator-activated receptor α (PPARα) shared the sequence complementary to miR-34a, which may abolish the translational inhibition of miR-34a on PPARα by binding competition. In result, the circRNA-0046366 level correlated with PPARα expression (r=0.78,P<0.05), but negatively correlated with miR-34a level (r=-0.83,P<0.05), and concentrations of TG (r=-0.72,P<0.05) and MDA (r=-0.69,P<0.05) in the steatotic HepG2 cells. Conclusion circRNA-0046366 level negatively correlates with hepatic steatosis and lipid peroxidation. Abolishment of the PPARα inhibition by miR-34a may underlie its effect.
Circular RNA; Hepatocyte; Steatosis; miRNA; PPARα
科技部“973”項目(2012CB517501);國家自然科學(xué)基金項目(81470859、81270492、81070346)
郭行雅,在讀碩士,研究方向:消化系統(tǒng)疾病的研究。E-mail: 13856041526@163.com
潘勤,副研究員,研究方向:消化系統(tǒng)疾病的研究。E-mail: pan_qin@yeah.net
10.3969/j.issn.1006-5709.2017.06.022
R575
A
1006-5709(2017)06-0713-05
2016-10-10