肝细胞特异性敲除Dicer小鼠肝脏中细胞素P450酶的异常表达 苗玲玲，任进（中科院上海药物研究所药物安全评价研究中心，上海201203）
摘要：目的研究肝细胞特异性敲除Dicer小鼠肝脏中细胞素P450酶(CYP)的表达变化，发现可能被microRNA调控的CYP，进而研究microRNA对于CYP相关生理或病理过程的影响。方法Alb promoter-driven Cre recombinase转基因小鼠与含Floxed allele of Dicer的小鼠杂交得到肝细胞特异性敲除Dicer的小鼠。取3,4,5,6周龄小鼠肝脏，匀浆后提取蛋白质或抽提总RNA进行相关实验。人肝癌细胞系PLC/PRF/5中用基因沉默的方法敲减DICER后，检测CYP的表达变化。结果Cyp2e1在所检测各年龄段肝细胞特异性敲除Dicer小鼠肝脏中表达均下调，而Cyp1a2和Cyp3a11仅在5-6周龄肝细胞特异性敲除Dicer小鼠肝脏中表达下调，Cyp2b10在5-6周龄肝细胞特异性敲除Dicer小鼠肝脏中表达上调。人肝癌细胞PLC/PRF/5中DICER基因沉默后CYP2E1表达下调。结论DICER表达下调后几种CYP 表达均有不同程度变化，体内外结果均显示CYP2E1表达被下调，因DICER是microRNA成熟过程中重要的核酸内切酶，故而CYP2E1基因表达可能受到microRNA调控。CYP2E1是肝脏内重要的代谢酶，乙醇对CYP2E1的诱导是酒精致肝毒性的重要环节，CYP2E1的表达量和肿瘤易感性、糖尿病等也密切相关。microRNA可能成为CYP2E1的重要调节因素，从而参与到CYP2E1相关的生理或病理过程。 关键词：Dicer,CYP,MicroRNA
Altered Cytochrome P450Expression in Hepatocyte-specific Dicer Knockout
Mouse Livers
Lingling Miao,Jin Ren(Center for Drug Safety Evaluation and Research,State Key Laboratory of New Drug Research,Shanghai Institute of Materia Medica,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai201203)
ABSTRACT
OBJECTIVE CYP enzymes exhibit high interindividual variability that is not completely explained by known environmental and genetic factors.To further understand this variability,we hypothesized that microRNAs may regulate CYP expression and use hepatocyte-specific Dicer knockout mice as a model to investigate the potential role of microRNA in CYP regulation. METHODS Dicer,an enzyme essential for the processing of microRNAs,was disrupted in hepatocytes using a conditional knockout mouse model to elucidate the consequences of loss of microRNAs.Albumin-Cre and Dicer1loxP/loxP mice were crossed to obtain hepatocyte-specific Dicer knockout mice(Albumin-Cre;Dicer1loxP/loxP mice).Human hepatoma PLC/PRF/5cells which endogenously expressed CYP2E1were used for the in vitro experiments.CYP mRNA and protein expression were detected by
real-time quantitative reverse transcription-PCR and western blot.
RESULTS The mRNA and protein expression levels of four Cyps(Cyp2e1,Cyp1a2,Cyp3a11, Cyp2b10)were changed in Dicer-deficient mouse livers.Cyp2e1mRNA and protein levels were reduced progressively with age in Dicer-deficient mouse livers.Cyp1a2and cyp3a11expression were decreased in5and6-week-old Dicer-deficient mouse livers.Cyp2b10expression was
increased in5and6-week-old Dicer-deficient mouse livers.CYP2E1expression was also decreased in DICER knockdown human hepatoma cells.
CONCLUSION CYP2E1could be regulated by DICER.DICER deficiency lead to down-regulation of CYP2E1,and it’s well known that DICER deficiency lead to aberrant expression of mature microRNA,so we expected that CYP2E1might be positively regulated by a microRNA that could be processed by DICER,or negatively regulated by a microRNA which was up-regulated by DICER deficiency.MicroRNA might be involved in the physiological or pathological process mediated by CYP2E1.
Key Words:Dicer,CYP,MicroRNA
细胞素P450酶(CYP)，又称混合功能氧化酶或单加氧酶，属于亚铁血红蛋白超家族，主要存在于肝微粒体中，在药物等外源性化合物和内源性物质如脂肪酸、胆固醇、胆汁酸及类固醇激素的生物转化中发挥了非常重要的作用[1,2]。
CYP作为机体内重要的代谢酶，其基因表达调控也受到广泛关注。调节CYP表达的因素非常多，CYP具有明显的组织分布特异性，主要分布在肝脏，部分亚型在肝外组织如肾脏，肺，脑等也有分布；一些CYP亚型的表达还具有明显的雌雄差异，如cyp2b9,cyp2b10在成年鼠中仅在雌性表达。体内的激素，如胰岛素、糖原、生长激素、瘦素，以及各种性激素能够调节很多CYP亚型的表达。部分CYP的底物同时也是其诱导剂或抑制剂。另外，很多CYP具有基因多态性，造成了人中显著的个体差异。sony cr33
CYP基因表达调控的分子机制较复杂，在转录、转录后和翻译后水平都可被调节。在转录水平，肝脏表达较丰富的转录因子和核受体可以直接结合到基因启动子区，调节CYP 基因的转录，如HNF1α,HNF4α,albumin D-site binding protein(DBP), CCAAT/enhancer-binding proteins(C/EBP),pregnane X receptor(PXR),constitutive androstane receptor(CAR),farnesol X receptor(FXR)[3].另外，其他转录因子如Sp-1,PKC信号通路, NFκB也可调节其表达[4-6].在转录后水平，CYP也受到了广泛调节，如NFκB可诱导血红素加氧酶从而影响CYP蛋白的稳定性[6]，CYP2E1mRNA的5’-非翻译区和mRNA中一段16个核苷酸的序列均能够影响其翻译的效率和mRNA的稳定性[7,8]。在翻译后水平，部分CYP 的底物同CYP形成加合物可影响其蛋白的稳定性。
近年来，细胞内源产生调节基因表达的一类小分子非编码RNA受到普遍关注，因其平均长度只有22个核苷酸，故被命名为microRNA.在人类中表达的microRNA有一千多种，人体中60%的基因都可能被其调节。MicroRNA参与了发育、细胞分化与增殖、凋亡等一系列生物过程，在肿瘤、代谢紊乱等人类疾病的产生和发展过程中发挥了重要作用[9]。
MicroRNA参与了染质结构、基因转录、mRNA加工、mRNA稳定性调节、蛋白翻译等多个过程的调控，其调节的效应通常是抑制基因表达。真核生物基因组中microRNA的编码序列广泛存在，RNA聚合酶II负责其转录，产生转录本pri-microRNA.后续加工过程的第一步反应发生在细胞核中，pri-microRNA被microprocessor complex Drosha–DGCR8 (pasha)剪切产生pre-microRNA.Pre-microRNA被Exportin-5-Ran-GTP转运到胞浆中，核酸酶Dicer协同双链RNA结合蛋白TRBP切除pre-microRNA hairpin生成mature length的双链microRNA.其功能性的单链（mature microRNA）与Ago蛋白被整合到RNA-induced silencing complex(RISC)中，与target mRNA结合，通过mRNA剪切、mRNA去腺苷或抑制翻译等方式抑制基因表达[10.11]。
microRNA的靶基因极为广泛，已有的报道表明部分CYP也是其靶基因，如CYP1B1, CYP2E1,CYP3A4能够直接或间接被microRNA调控[12-14]，microRNA对P450酶的调控可能成为P450酶表达与活性个体差异的原因之一。由于microRNA的作用在很大程度上取决
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后退跑于碱基互补配对，从而决定了一个microRNA可能有若干靶基因，而同一基因也能够被若
干microRNA调控，为筛选最有可能被microRNA调控的CYP，我们利用肝细胞特异敲除的金钱的魔力教学设计
Dicer小鼠作为模型,Dicer功能性敲除后大部分microRNA的成熟受到影响，利用此模型
筛选可能受microRNA调控的CYP.
吃菜养心1.材料方法
1.1肝细胞特异性敲除Dicer的小鼠
Alb promoter-driven Cre recombinase转基因小鼠与含Floxed allele of Dicer的小鼠杂交可以得到肝细胞特异性敲除Dicer的小鼠。取3,4,5,6周龄小鼠肝脏，匀浆后提取蛋白质或抽
提总RNA进行相关实验。
1.2细胞培养与转染
人肝癌细胞系PLC/PRF/5从American Type Culture Collection购得。细胞均培养于Dulbecco’s modified Eagle medium，添加10%胎牛血清和antibiotics，培养于37。C含5%CO2
的培养箱中。细胞的消化采用含0.5mM EDTA的0.25％胰酶。细胞接种24h后依据说明
书用lipofectamine2000(Invitrogen)转染。转染前细胞换至低血清培液。
1.3总RNA抽提，反转录与实时定量PCR
总RNA用Trizol（Invitrogen）抽提。第一链cDNA合成为20μL体系中加入5μg RNA, random hexamer,dNTP和M-MuLV reverse trancriptase.实时定量PCR用SYBR Premix Ex
Taq Kit，在Rotor-gene Q PCR仪上进行。
1.4Western印迹检测
细胞总蛋白用Sodium dodecyl sulfate lysis buffer提取，94℃加热10min，蛋白变性。
组织或细胞蛋白样品用SDS-8%PAGE胶电泳分离后，湿转法转移印迹至PVDF膜上。一
抗孵育，4℃过夜。二抗孵育，室温2小时。化学发光显（ECL），用ImageQuant分析
软件对显影条带进行光密度分析，目的蛋白质同内参的光密度比值作为目的蛋白的相对表达
形象进度定量。
2.结果
敦煌学十八讲2.1Cyp2e1在所检测各年龄段肝细胞特异性敲除Dicer小鼠肝脏中表达均下调。
核酸内切酶Dicer在microRNA产生过程中剪切pre-microRNA生成mature microRNA.
肝细胞特异敲除Dicer的小鼠(KO小鼠)在出生后三周，肝细胞Dicer的功能已显著丧失，
microRNA的成熟被抑制，大部分mature microRNA的表达下降[15,16]。取不同年龄Dicer肝
脏特异敲除小鼠的肝脏，检测几种Cyps(Cyp2e1,Cyp1a2,Cyp3a11,Cyp2b10)的表达量。
Fig.1表明，与杂合子对照组相比，纯合子KO小鼠Cyp2e1蛋白(A)和mRNA(B)表达在较
早的年龄（3
周）即有明显下降。且随着年龄增长，变化越明显。
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∞①巧∞仄《一①一Fig.1Cyp2e1mRNA and protein levels were reduced progressively with age in Dicer-deficient mouse livers.(A)Immunoblots of total lysates from Dicer-deficient(-/-)and control(+/-,wt)mouse livers probed with anti-Cyp2e1antibodies.Tubu1a or Canx served as the protein loading control.The age of the mice at the time of sacrifice was indicated by3,4,5,6W(3, 4,5,6weeks).The mouse number was indicated by the labeling closest to the panels.(B)Cyp2e1 mRNA level determined by qRT-PCR,normalized toβ-actin mRNA level.
2.2Cyp1a2和3a11在5-6周龄肝细胞特异性敲除Dicer小鼠肝脏中表达下调。
Fig.2表明，与杂合子对照组相比，纯合子KO小鼠Cyp1a2蛋白(A)和mRNA(B)和Cyp3a11蛋白(C)和mRNA(D)在较早的年龄（3,4周）无明显变化，而在5-6周龄肝细胞特异性敲除Dicer
小鼠肝脏中表达下调。
Fig.2Cyp1a2and cyp3a11expression were decreased in5and6-week-old Dicer-deficient mouse livers.
(A)and(C)Immunoblots of total lysates from Dicer-deficient(-/-)and control(+/-, wt)mouse livers probed with anti-Cyp1a2or anti-Cyp3a11antibodies.Tubu1a or Canx served as the protein loading control.The age of the mice at the time of sacrifice was indicated by3,4,5, 6W(3,4,5,6weeks).The mouse number was indicated by the labeling closest to the panels.(B) and(D)Cyp1a2and Cyp3a11mRNA levels determined by qRT-PCR,normalized toβ-actin mRNA level.
2.3Cyp2b10在5-6周龄肝细胞特异性敲除Dicer小鼠肝脏中表达上调。
Fig.3表明，与杂合子对照组相比，纯合子KO小鼠Cyp2b10蛋白(A)和mRNA(B)在较早的年龄（3,4周）无明显变化，而在5-6周龄小鼠中，杂合子对照组小鼠肝脏Cyp2b10表达量极低，KO小鼠肝脏中Cyp2b10
表达显著上调。
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仄《一①一Fig.3Cyp2b10expression was increased in 5and 6-week-old Dicer -deficient mouse livers.(A)Immunoblots of total lysates from Dicer -deficient (-/-)and control (+/-,wt)mouse livers probed with anti-Cyp2b10antibodies.Tubu1a or Canx served as the protein loading control.The age of the mice at the time of sacrifice was indicated by 3,4,5,6W (3,4,5,6weeks).The mouse number was indicated by the labeling closest to the panels.（B ）Cyp2b10mRNA level determined
by qRT-PCR,normalized to β-actin mRNA level.
2.4人肝癌细胞PLC/PRF/5中DICER 基因沉默后CYP2E1表达下调。
小鼠肝细胞中Dicer 被敲除后，肝脏产生明显的脂肪变性和糖原耗损，细胞生长相关的基因表达增加，胚胎期特异表达的基因异常表达，在肝脏内细胞的过度增殖与大量凋亡并存，Dicer 敲除的肝细胞凋亡增加，而部分逃脱cre 酶作用的肝细胞再生侵袭至整个肝脏。突变小鼠中的三分之二在出生后一年会产生自发性肝细胞癌变。肝细胞Dicer 敲除小鼠的肝脏在出生三周后就有形态学的变化，KO 小鼠与对照小鼠相比表面较苍白；出生后六周肝脏呈现土黄，其上散布正常颜的斑点；出生后12周正常颜的斑点扩展至整个肝脏[15]。
基于肝细胞Dicer 敲除小鼠5-6周龄时已出现明显的肝脏病变，在此年龄KO 小鼠中检测到的CYP 表达变化可能是Dicer 敲除后mature microRNA 表达降低所致，也可能是肝脏病变的继发性反应。为排除后一种可能性，我们在人肝癌细胞系PLC/PRF/5中用基因沉默的方法knock down Dicer 后，检测CYP 的表达变化，Fig.4表明CYP2E1的蛋白(A)和mRNA (B)表达水平在敲减Dicer
后同样下降，与小鼠体内的结果一致。Fig.4CYP2E1mRNA and protein expression levels were decreased in DICER knockdown
human PLC/PRF/5cells.(A)PLC/PRF/5cells were transfected with nonsense or DICER specific siRNA oligos for 72h and analyzed for CYP protein expression by immunoblotting.CANX served as loading control.(B)Bar chart corresponds to (A).(C)CYP mRNA level determined by qRT-PCR,normalized to β-actin mRNA level.
3讨论
细胞素P450酶的基因表达水平受到多种因素影响，其基因调控机制复杂，在转录、转录后、翻译后水平都可受到调节。很多CYP 基因表达具有明显的个体差异，其机制多是基因的多形性，但此机制不能完全解释此现象。我们推测具有广泛调控作用的非编码小
RNA
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