WO2014015824A1 - 具有转录和转录后双水平基因沉默功能的microRNA的筛选方法 - Google Patents

具有转录和转录后双水平基因沉默功能的microRNA的筛选方法 Download PDF

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WO2014015824A1
WO2014015824A1 PCT/CN2013/080160 CN2013080160W WO2014015824A1 WO 2014015824 A1 WO2014015824 A1 WO 2014015824A1 CN 2013080160 W CN2013080160 W CN 2013080160W WO 2014015824 A1 WO2014015824 A1 WO 2014015824A1
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microrna
gene
transcription
target gene
expression
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任进
王以政
苗玲玲
宫丽崑
戚新明
李成刚
邢国振
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中国科学院上海药物研究所
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a method for screening microRNAs having both transcriptional and post-transcriptional bi-level gene silencing functions, by the present invention
  • the method of screening for microRNAs has a mechanism for simultaneous regulation of gene expression at both transcriptional and post-transcriptional levels (dual regulation).
  • BACKGROUND OF THE INVENTION Gene silencing is a broad concept, an epigenetic regulation process of gene expression, usually refers to a phenomenon in which a specific gene in an organism is not expressed or expressed below normal levels, but is not caused by a change in the genetic material itself. .
  • This phenomenon is widespread in the biological world, is a gene expression regulation mechanism in the body of the organism, and is also one of the important techniques for inhibiting gene expression in biological research. Reducing the expression level of a gene and observing the phenomenon is a very effective means of studying the function of this gene.
  • nucleic acids as drug targets, a variety of drugs have been developed that silence specific genes.
  • oligonucleotides have been used in clinical trials, such as antisense oligonucleotides, small interfering RNA (Si NA), aptamers, and mirror-isomerized microRNAs.
  • Gene silencing can be achieved in a number of ways, and can occur at the transcriptional or post-transcriptional level.
  • Transcriptional level gene silencing includes histone modifications, genomic imprinting, paramutation, transposon silencing, transgene silencing, positional effects, RNA-mediated DNA methylation, and the like.
  • Post-transcriptional gene silencing can be achieved by RNA interference, nonsense strand-mediated RNA decay, and the like.
  • RNA interference (RNAi) is the most commonly used post-transcriptional gene silencing technology. It introduces small fragments of exogenous double-stranded RNA (dsRNA) or small hairpin RNA (shRNA) that are homologous to endogenous mRNA.
  • dsRNA or shRNA is finally processed by Dicer into a functional double-stranded small RNA (siRNA) of 21 bases in length.
  • RNAi technology has been widely used in scientific research of gene function. It is the most commonly used gene silencing technology in current research, and siRNA is used in the treatment and drug development of various diseases (such as viral infection, tumor, etc.). Both have important implications, but RNAi technology has its limitations:
  • RNAi does not completely block the expression of genes, especially genes with high expression; (2) RNAi is not suitable for all genes, such as dsRNA for some in nerve cells. Gene-suppressing effects are not obvious; (3) RNAi has an off-target effect; (4) SiRNA activates interferon-responsive genes in the body, causing side effects.
  • MicroRNAs are small-molecule non-coding RNAs that are newly discovered by endogenously produced regulatory genes in recent years. The effect of regulation is usually to suppress gene expression. Recent studies have found that at least 60% of the genes are regulated by microRNAs, which are involved in almost all life processes such as cell proliferation, differentiation, apoptosis and metabolism, in cardiovascular diseases, nervous system diseases, hematopoietic diseases, diabetes and The pathological process of tumors also plays an important role. As an endogenous small RNA molecule with good adaptability and specificity and significant inhibitory effect, the development of microRNA as a gene silencing drug has received great attention. A number of microRNAs and their modified sequences are currently in clinical development as drugs.
  • microRNA gene silencing mechanisms are generally post-transcriptional regulatory mechanisms.
  • the target site of miRNA is mostly located in the 3'-untranslated region (3'-UT) of mRNA, and the functional single-stranded mature miRNA is integrated into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • gene expression is inhibited at the post-transcriptional level by mRNA cleavage, mRNA adenosine or inhibition of translation.
  • This mode of regulation of microRNAs relies on the complementary pairing of the miRNA seed region sequence (bases 2-7) with the 3' untranslated region sequence of the destination mRNA.
  • miRNAs bind to the promoter region of genes in a complementary pairing manner, and by enriching the binding of RNA polymerase, the enrichment of histone methyltransferase or DNA methyltransferase in this region leads to The methylation of histones or DNA, thereby inhibiting gene transcription at the transcriptional level, does not depend on sequence matching of the miRNA seed region, and can inhibit gene expression at lower doses and for a longer period of time. Thus, miRNAs can cause silencing of a gene of interest by transcription or post-transcriptional inhibition.
  • the present inventors have conducted extensive experiments and verification that the selected miRNA can simultaneously act on the target gene by two methods (transcriptional inhibition and post-transcriptional translational inhibition), and the exogenously administered miRNA can simultaneously inhibit the target gene.
  • the way in which transcription and translation thus effectively inhibit the expression of the gene of interest indicates that this "transcriptional and post-transcriptional two-pronged" regulation of the target gene by miRNA can be an effective means of gene silencing. Accordingly, it is an object of the present invention to provide a method for screening microRNAs having both transcriptional and post-transcriptional bi-level gene silencing functions.
  • Another object of the present invention is to provide a microRNA which is screened by the above method. It is still another object of the present invention to provide a use of the microRNA screened by the above method.
  • Yet another object of the invention is to provide a method of gene silencing.
  • a method of screening a microRNA having both transcriptional and post-transcriptional bi-level gene silencing functions comprising:
  • Step 3) chemically synthesizing the microRNA obtained in the above steps 1) and 2), and detecting the effect of the microRNA on the expression of the target gene protein, thereby screening for a microRNA capable of down-regulating the expression of the target gene protein;
  • Step 4) detecting the effect of the microRNA capable of down-regulating the expression of the target gene protein in the step 3) on the expression of the target gene mRNA, thereby screening for microRNA capable of simultaneously down-regulating the mRNA and protein levels of the target gene;
  • Step 5 The microRNA transcription and post-transcriptional dual gene silencing ability was verified by gene-directed mutagenesis and luciferase reporter gene method to screen for microRNAs with transcriptional and post-transcriptional dual-level gene silencing.
  • step 1) the miRBase database is http://www.mirbase.org/search.shtml.
  • the TargetScan database is http://www.targetscan.org
  • the above database is the most widely used database in the field of scientific research and drug development.
  • the use of the above database of the present invention can ensure the accuracy and reliability of prediction.
  • the method for detecting the effect of the microRNA on the expression of the target gene protein includes lipofection and Western blotting.
  • the method for detecting the effect of the microRNA capable of down-regulating the expression of the target gene protein on the expression of the mRNA of the target gene obtained in the step 3) includes total RNA extraction, cDNA reverse transcription and real-time fluorescent quantitative PCR.
  • step 5 the method further comprises the following steps:
  • microRNA mimics obtained in step 4 were simultaneously transferred into the cells, and the microRNA mimics obtained in step 4) were added to detect the effect of the 5' flanking region microRNA binding site mutation on renilla luciferase activity, confirming microRNA A binding site to the 5' flanking region of the gene of interest is used to determine whether the microRNA has a transcriptional level of regulatory ability, thereby screening for a microRNA having a transcriptional level of regulatory ability.
  • TargetScan database prediction b) using TargetScan database prediction, combined with artificial alignment, confirming the binding site of the selected microRNA to the 3' untranslated region of the target gene, using the gene site-directed mutagenesis technique to mutate the binding site on the 3' untranslated region, and then The wild type and the mutant 3' untranslated region were inserted into the firefly luciferase vector psiCHECK2, and the renilla luciferase carried by the vector itself was used as an internal reference, and the wild type and mutant psiCHECK2 vectors were respectively transferred into the cells.
  • microRNA mimics were added to detect the effect of the microRNA binding site mutation in the 3' untranslated region on firefly luciferase activity, and the microRNA with the transcriptional level of regulatory ability obtained in step a) was confirmed to be 3' untranslated.
  • the binding site of the region ultimately determines whether the microRNA has a post-transcriptional (translational) level of regulatory ability, thereby screening for a microRNA having both transcriptional and post-transcriptional dual-level gene silencing functions.
  • the manual comparison refers to using the Vector NTI software (Invitrogen), NAhybrid online software (http: ⁇ bibiserv.techfak.uni-bielefeld.de/) based on the prediction of the above database (miRbase, TargetScan). Mahvbrid/ ) and manual lookup of nucleic acid sequence fragments (Excel, Microsoft) to investigate the ability of microRNAs to bind to the gene of interest and pairing sequences.
  • Vector NTI software Invitrogen
  • NAhybrid online software http: ⁇ bibiserv.techfak.uni-bielefeld.de/
  • Mahvbrid/ manual lookup of nucleic acid sequence fragments
  • the cell may be a PLC/PRF/5 liver cell, a 293 cell, or a 786-O kidney cell line.
  • the microRNA screened by the above method can down-regulate the mRNA expression of the target gene (having transcriptional resilience) by binding to a specific site of the 5' flanking region, and can pass Binding to the 3' untranslated region-specific site to down-regulate the expression of the gene of interest protein (with post-transcriptional/translational inhibition).
  • step 4 we screened microRNAs that can simultaneously down-regulate mRNA and protein levels of the target gene. Down-regulation of mRNA levels may be related to the transcriptional level of microRNAs (binding to the promoter or 5' flanking region), and protein levels may be down-regulated.
  • mRNA levels may be related to the transcriptional level of microRNAs (binding to the promoter or 5' flanking region), and protein levels may be down-regulated.
  • the TargetScan database uses the number of base pairs of the microRNA seed region and the 3' untranslated region of the target gene (generally requires a number of pairs greater than 6) to screen the prediction results, but due to the complexity of the RNA secondary structure, the number of paired bases is less than 6
  • microRNAs may also bind efficiently to the 3' untranslated region of the gene of interest, thereby downregulating the expression of the gene of interest. Therefore, in this case, the data can be better filtered using a certain degree of manual alignment.
  • TargetScan database prediction combined with artificial alignment, confirm the binding site of the selected microRNA to the 3' untranslated region of the target gene, and use the gene site-directed mutagenesis technique to mutate the binding site on the 3' untranslated region, and then The wild type and mutant 3' untranslated regions were inserted into the luciferase vector, respectively, and the wild-type and mutant vectors were respectively transferred into the cells, and microRNA mimics were added to detect the 3' untranslated region microRNA binding site.
  • the effect of the mutation on luciferase activity confirms the binding site of the microRNA to the 3' untranslated region of the gene of interest, and ultimately determines whether the microRNA has a post-transcriptional (translational) level of regulatory ability.
  • the gene of interest may be any gene without limitation.
  • the gene of interest is a cytochrome P450 2E1 (CYP2E1) gene or a type I heme oxygenase (Haem oxygenase). ⁇ , ⁇ ) Gene.
  • a microRNA screened by the above method is provided.
  • microRNA 552 miR-552
  • microRNA 1254 miR-1254
  • type I heme oxygenase gene as a gene.
  • the microRNA of the present invention has utility in the development and screening of drugs for the treatment of various diseases.
  • a method of gene silencing comprising gene silencing using a microRNA screened by the above method.
  • microRNA is an endogenous small RNA molecule with good adaptability and specificity and significant inhibition effect.
  • the microRNAs screened in the present invention have better gene silencing effects by using the microRNAs screened by the method of the present invention than the previously reported microRNAs of the same gene of interest (Fig. 9); the present invention provides a new and effective method.
  • the microRNA screening method can obtain a good gene silencing effect; the present invention provides a new design idea for the gene silencing technology commonly used in scientific research; the present invention also provides a new idea for the development and design of microRNA drugs for diseases.
  • Fig. 1 is a flow chart schematically showing a screening method of micro NAs having transcriptional and post-transcriptional bilevel gene silencing functions of the present invention.
  • Figure 2 is a graph showing the microRNA obtained by predicting the 5' flanking region of CYP2E1 using the miRBase database and its binding site on the 5' flanking region. Among them, 5TR refers to the 5' flanking area.
  • Figure 3 is a graph showing the microRNA obtained by predicting the 3' untranslated region of CYP2E1 using the TargetScan database and its binding site on the 3' untranslated region. Among them, 3'UTR refers to the 3' untranslated area.
  • Figure 4 is a graph showing the effect of predicted microRNAs on CYP2E1 protein expression. among them:
  • Figure 5 is a graph showing the inhibitory effect of miR-552 on the expression level of CYP2E1 m NA.
  • CYP2E1 protein expression was down-regulated in a dose-dependent manner with miR-552 analogs, whereas downregulation of mRNA levels did not.
  • the mRNA expression level of CYP2E1 was detected using a Rotor Gene real-time quantitative PCR instrument using Takara's fluorescent quantitative PC reagent, and the mRNA level of the internal reference gene GAPDH was also detected.
  • the mRNA expression level of CYP2E1 is based on the expression level of GAPDH, and the expression level of CYP2E1 mRNA is normalized by nc-1 to obtain the normalized expression level of CYP2E1 mRNA.
  • * indicates p ⁇ 0.05 vs nc (relative to nc), * * indicates ⁇ ⁇ 0 ⁇ 01 vs nc (relative to nc).
  • Figure 6 is a graph showing the effect of mutations in the miR-552 binding site of the 5' flanking region of CYP2E1 on miR-552 transcriptional repression. among them:
  • the nucleotides 9-21 of miR-552 can be complementary to the nucleotide sequence of 313-326 before the transcription initiation site of the CYP2E1 promoter region, wherein the lowercase letter is the CYP2E1 promoter region corresponding to miR-552 binding.
  • FIG. 7 is a graph showing the effect of mutation of the CYP2E1 3' untranslated region miR-552 binding site on the post-transcriptional inhibition ability of miR-552. among them:
  • the CYP2E1 m NA 3,-UTR region is partially complementary to the miR-552 seed sequence region, in which the lower case letter represents the nucleotide sequence following the mutation of the possible binding site of miR-552 in the 3'-UT region of CYP2E1 mRNA.
  • Luciferase reporter assay showed that miR-552 inhibited CYP2E1 3'-UTR activity, while 3'-UTR, which has a possible binding site for miR-552, could not inhibit.
  • Normalized luciferase activity refers to normalization of firefly luciferase activity by nc of 1, based on internal renilla luciferase activity, to obtain normalized luciferase activity. ** indicates p ⁇ 0.01 vs nc
  • Figure 8 is a graph showing the inhibitory effect of miR-1254 on HMOX1 mRNA and protein expression. among them:
  • HMOX1 protein and mRNA expression was down-regulated in a dose-dependent manner with the miR-1254 analog.
  • the mRNA expression level of HMOX1 was detected using a Rotor Gene real-time quantitative PCR instrument using Takara's real-time PCR reagent, and the mRNA level of the internal reference gene GAPDH was also detected.
  • the normalized expression level of HMOX1 mRNA was based on the expression level of GAPDH, and the expression level of CYP2E1 mRNA was normalized by nc-1 to obtain the normalized expression level of HMOX1 mRNA. ** means p ⁇ 0.01 vs nc (relative to nc), *** means p ⁇ 0.001 vs nc (relative to nc).
  • Figure 9 is a graph showing that the miR-552 screened has a better CYP2E1 silencing effect than the previously reported miR-378. among them:
  • GAPDH was used as a sample load control. In the figure, the number is the name of the microRNA, and nc is the negative control.
  • the plasmids used (PGL4.10, PGL4.73 and psiCHECK2) were purchased from Promega;
  • Luciferase activity assay kit purchased from Promega;
  • Cell culture fluid purchased from Invitrogen;
  • CYP2E1 antibody for western blot detection purchased from Millipore;
  • HMOX1 Western blot antibody purchased from Santa Cruz.
  • MicroRNA 552 (miR-552) with dual inhibitory ability was screened by the following procedure using a gene encoding cytochrome P450 isoform 2E1 (Cytochrome P450 2E1, CYP2E1).
  • Step 1) Using the miRBase database to predict the microRNA binding site of the 5' flanking region of the lkb before the CYP2E1 transcription initiation site (the possible location of the promoter), and obtain a series of microRNAs that may bind to the promoter region of the target gene ( See Figure 2);
  • Step 2) Using the TargetScan database to perform microRNA binding site prediction on the 3' untranslated region of CYP2E1, a series of microRNAs that may bind to the 3' untranslated region of CYP2E1 were obtained (see Figure 3);
  • Step 3) Using chemically synthesized microRNA mimics (mimics) to detect the effect of the microRNA obtained in step 1) and step 2) on the expression of the target gene protein, and screening for microRNA 552 which can significantly down-regulate the expression of the target gene protein (see Figure 4);
  • Step 4) Using chemically synthesized miR-552 mimics to detect the effect of miR-552 on CYP2E1 m NA expression, miR-552 was found to down-regulate CYP2E1 m NA levels ( see Figure 5);
  • Step 5 Using miRbase and TargetScan database predictions, combined with manual alignment, find the binding site of miR-552 in the 5' flanking region and 3' untranslated region of CYP2E1 (see Figure 2, Figure 7A). The binding site of the 5' flanking region and the 3' untranslated region of CYP2E1 to miR-552 was mutated using a gene-directed mutagenesis technique (see Figure 6A, Figure 7A). Wild type 5' flanking zone and 3' untranslated zone location and sequence:
  • the wild type 5' flanking region is located in the human genome, 1000 bases upstream of the transcription start point of the CYP2E1 gene, chromosome 10, position: 1352354624-1352355623;
  • the wild type 3' untranslated region sequence is located at the mature mRNA end of CYP2E1, 1755-1912, for a total of 158 bases.
  • the wild type and mutant 5' flanking regions and 3' untranslated regions were inserted into the luciferase reporter gene, respectively (5' flanking region insertion position: PGL4.10 plasmid cloning site; 3' untranslated region insertion position: The cloning site of psiCHECK2 plasmid, (1) miR-552, by binding to the 5' flanking region-specific site, down-regulates mRNA expression of the target gene, demonstrating that miR-552 has transcriptional repression (see Figure 6B). (2) miR-552 binds to the 3' untranslated region-specific site and down-regulates the expression of the target gene protein, demonstrating that miR-552 has translational inhibition (see Figure 7B);
  • Step 6) Combining the data of steps 3), 4) and 5), miR-552 can be screened to significantly down-regulate CYP2E1 mRNA and protein levels with dual transcriptional and post-transcriptional inhibition.
  • Example 1 The inhibitory effect of miR-552 on the expression of CYP2E1 mRNA and protein was examined in human hepatoma cells PLC PRF/5 cells.
  • CYP2E1 25nM, 50nM, 100nM, 200nM miR-552 were transfected into human hepatoma cell PLC/PRF/5 cells, and the expression of CYP2E1 protein was detected by Western blotting. CYP2E1 was detected by reverse transcription-real-time quantitative PCR. Changes in mRNA expression. As shown in Figures 4 and 5, miR-552 dose-dependently decreased the expression of CYP2E1 protein ( Figure 5), and the CYP2E1 mRNA level decreased slightly with no dose-related ( Figure 5).
  • Example 2 miR-552 inhibits its transcriptional activity by acting on the CYP2E1 gene promoter region through a non-seed sequence region.
  • the human CYP2E1 gene promoter region (5' flanking region) wild type (wt) and miR-552 binding site mutant (mt) were cloned into the PGL3 luciferase reporter system for co-transfection of miR- Effect of 552 and its various mutants on luciferase activity.
  • MiR-552 significantly inhibited the CYP2E1 promoter activity without reducing the mutant promoter activity (see Figure 6B).
  • Example 3 miR-552 exerts post-transcriptional inhibition of its expression by the CYP2E1 mRNA 3,-untranslated region.
  • the miR-1254 of 50 ⁇ and ⁇ were transfected into human kidney 293 cells respectively.
  • the protein expression of HMOX1 was detected by Western blotting.
  • the expression of HMOX1 mRNA was detected by reverse transcription-real-time quantitative PCR.
  • miR-1254 can be dose-dependently induced HMOX1 protein level expression was significantly decreased (Fig. 8A, C), and HMOX1 mRNA levels were significantly decreased (Fig. 8B).

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Abstract

提供了一种具有转录和转录后双水平基因沉默功能的microRNA的筛选方法。使用该筛选方法筛选得到的microRNA既能够通过结合于目的基因的5'侧翼区特异位点从而下调目的基因mRNA的表达，也能够通过结合于目的基因3'非翻译区特异位点从而下调目的基因蛋白表达。还提供了使用该方法筛选得到的microRNA以及该microRNA用于基因沉默的用途和方法。

Description

具有转录和转录后双水平基因沉默功能的 microRNA的筛选方法 技术领域 本发明属于生物技术领域， 更具体而言， 涉及一种具有转录和转录后双 水平基因沉默功能的 microRNA 的筛选方法， 由本发明的方法筛选得到的 microRNA具有同时在转录与转录后水平调控基因表达的机制 （双重调控）。 背景技术 基因沉默是一个广义的概念， 是基因表达的表观遗传调控过程， 通常是 指生物体中特定基因不表达或表达低于正常水平的现象， 但并不是由于遗传 物质本身的改变所引起。 这一现象广泛存在于生物界， 是生物体内源存在的 基因表达调控机制， 同时也是生物学研究中抑制基因表达的重要技术之一。 降低某基因的表达水平， 进而观察所引起的现象是研究此基因功能非常有效 的手段。 以核酸为药物靶点， 已开发了对特定基因沉默的多种药物。 目前已 有多种形式的寡核苷酸被应用于临床试验， 如反义寡核苷酸、 小干扰 RNA (Small interfering RNA, si NA), 核酸适体 （aptamers) 、 镜像异构微小 RNA
( spiegelmers microRNA ) 等。
基因沉默可通过多种方式实现， 可以发生在转录或转录后水平。 转录水 平的基因沉默包括组蛋白修饰、 基因组印迹、 副突变、 转座子沉默、 转基因 沉默、 位置效应、 RNA介导的 DNA甲基化等。 转录后基因沉默可通过 RNA 干扰、 无义链介导的 RNA衰变等方式实现。 RNA干扰 （RNA interference, RNAi)是目前最常用的转录后基因沉默技术， 将与内源 mRNA同源的小片段 外源双链 RNA(dsRNA)或小发夹 RNA(small hairpin RNA, shRNA)导入细胞内 可以诱导该 mRNA降解， 从而阻断相应基因表达。 无论 dsRNA或 shRNA最后 都要被 Dicer加工成 21个碱基长度的功能性双链小 RNA (siRNA)。
目前， siRNA介导的基因沉默技术已被广泛应用于基因功能的科学研究， 是目前研究中最常用的基因沉默技术， 同时 siRNA在多种疾病（如病毒感染、 肿瘤等） 的治疗与药物研发中均具有重要的意义， 但 RNAi技术有其局限性：
( 1 ) 在哺乳动物细胞中， RNAi并不能完全阻断基因的表达， 特别是表达高 的基因； （2) RNAi并不适用于所有的基因， 如 dsRNA对一些在神经细胞中 发挥功能的基因抑制作用不明显； （3 ) RNAi的脱靶效应 （off-target) 明显； (4) SiRNA会激活体内的干扰素反应基因， 引起副作用。
微小 RNA(microRNA， miRNA)是近年新发现的细胞内源产生的调节基 因表达的小分子非编码 RNA， 其调节的效应通常是抑制基因表达。到目前的 研究发现， 至少 60%以上基因的表达受 microRNA调控， 它们参与了细胞增 殖、 分化、 凋亡和代谢等几乎所有生命过程， 在心血管疾病、 神经系统疾病、 造血系统疾病、 糖尿病及各种肿瘤的病理过程中也发挥了重要作用。 作为一 种内源的、 具有良好的适应性与特异性、 抑制效果显著的小 RNA 分子， microRNA 作为基因沉默药物的开发受到了极大的关注。 目前已有多个 microRNA及其修饰序列作为药物处于临床开发阶段。
microRNA作用机制
目前已知的 microRNA基因沉默机制一般为转录后调控机制。 miRNA的 靶位点大多位于 mRNA的 3'非翻译区域（3 '-untranslated region, 3'-UT ),功 能性的单链成熟 miRNA 被整合到 RNA 诱导沉默复合体 （RNA-induced silencing complex, RISC) 中， 与靶 mRNA结合， 通过 mRNA剪切、 mRNA 去腺苷或抑制翻译等方式在转录后水平抑制基因表达。 microRNA 的这种调 节方式依赖于 miRNA种子区 （Seed region) 序列 （第 2〜7位碱基） 与目的 mRNA 3'非翻译区序列的互补配对。
近年来， 也有报道表明 miRNA 以互补配对的方式结合到基因的启动子 区， 通过影响 RNA聚合酶的结合， 弓 I起组蛋白甲基转移酶或 DNA甲基转移 酶在该区域的富集导致组蛋白或 DNA 的甲基化， 从而在转录水平上抑制基 因转录， 这种调节方式并不依赖于 miRNA种子区的序列匹配， 能够在较低 剂量、较长时间来抑制基因表达。 因此， miRNA可以通过转录或转录后抑制 的方式引起目的基因沉默。 发明内容 本发明人经过大量的实验与验证， 筛选得到的 miRNA可以同时通过两 种方式 （转录抑制和转录后的翻译抑制） 作用于目的基因， 这种外源给予的 miRNA 可以通过同时抑制目的基因的转录和翻译从而有效抑制目的基因表 达的方式说明 miRNA对靶基因的这种"转录和转录后双管齐下"的调节方式 可以成为基因沉默的有效手段。 因此， 本发明的一个目的是提供一种具有转录和转录后双水平基因沉默 功能的 microRNA的筛选方法。
本发明的另一个目的是提供一种由上述方法筛选得到的 microRNA。 本发明的再一个目的是提供一种由上述方法筛选得到的 microRNA的用 途。
本发明的又一个目的是提供一种基因沉默的方法。
在本发明的一个方面中， 提供了一种具有转录和转录后双水平基因沉默 功能的 microRNA的筛选方法， 所述方法包括：
步骤 1 ) :使用 miRBase数据库对目的基因的转录起始位点前 l-2kb的 5' 侧翼区（ 5 '-flanking region, 5 '-FR) (启动子可能存在的位置）进行 microRNA 结合位点预测， 得到能够结合于目的基因启动子区域的 microRNA;
步骤 2) :使用 TargetScan数据库对目的基因的 3'非翻译区进行 microRNA 结合位点预测， 得到能够结合于目的基因 3 '非翻译区的 microRNA;
步骤 3 ) : 化学合成上述步骤 1 )和步骤 2) 中得到的 microRNA, 并检测 microRNA对目的基因蛋白表达的影响， 由此筛选得到能够下调目的基因蛋 白表达的 microRNA;
步骤 4 ) :检测步骤 3 )中得到的能够下调目的基因蛋白表达的 microRNA 对目的基因 mRNA表达的影响，由此筛选得到能够同时下调目的基因 mRNA 和蛋白水平的 microRNA；
步骤 5 ) : 使用基因定点突变与萤光素酶报告基因的方法验证 microRNA 转录与转录后双重基因沉默能力， 从而筛选得到具有转录和转录后双水平基 因沉默功能的 microRNA。
本发 明 方法 中 ， 步骤 1 ) 中 ， 所述 miRBase 数据库为 http ://www.mirbase.org/search. shtml。
本发明方法中， 步骤 2 ) 中， 所述 TargetScan 数据库为 http://www.targetscan.org
上述数据库是目前应用最广泛， 在科研领域、 药物开发领域认可程度 最高的数据库。 本发明使用上述数据库可以保证预测的准确性与可靠性。
本发明方法中， 步骤 3 ) 中， 检测 microRNA对目的基因蛋白表达的影 响的方法包括脂质体转染与蛋白免疫印迹等。 本发明方法中， 步骤 4 ) 中， 检测步骤 3 ) 中得到的能够下调目的基因蛋 白表达的 microRNA对目的基因 mRNA表达的影响的方法包括总 RNA抽提， cDNA反转录以及实时荧光定量 PCR。
本发明方法中， 步骤 5 ) 中， 进一步包括以下步骤：
a)使用 miRbase数据库预测，结合人工比对，确认筛选得到的 microRNA 与目的基因 5'侧翼区的结合位点， 使用基因定点突变技术， 将 5'侧翼区上的 结合位点突变， 然后将野生型与突变型的 5'侧翼区分别插入携带 renilla (海 肾） 萤光素酶的载体 （PGL4.10，Promega)， 用携带 firefly (萤火虫） 萤光素 酶的载体 PGL4.73作为内参，将 PGL4.10与 PGL4.73这两个载体同时转入细 胞中， 加入步骤 4 ) 中得到的 microRNA模拟物， 检测 5'侧翼区 microRNA 结合位点突变对 renilla萤光素酶活性的影响， 确认 microRNA与目的基因 5' 侧翼区的结合位点， 最终确定该 microRNA是否具有转录水平的调控能力， 由此筛选得到具有转录水平的调控能力的 microRNA。
b ) 使用 TargetScan 数据库预测， 结合人工比对， 确认筛选得到的 microRNA与目的基因 3'非翻译区的结合位点， 使用基因定点突变技术， 将 3 '非翻译区上的结合位点突变， 然后将野生型与突变型的 3'非翻译区分别插 入 firefly萤光素酶载体 psiCHECK2，以载体自身携带的 renilla萤光素酶为内 参， 将分别携带野生型与突变型的 psiCHECK2 载体分别转入细胞中， 加入 microRNA模拟物， 检测 3'非翻译区 microRNA结合位点突变对 firefly萤光 素酶活性的影响， 确认步骤 a ) 中得到的具有转录水平的调控能力的 microRNA与目的基因 3'非翻译区的结合位点， 最终确定该 microRNA是否 具有转录后 （翻译） 水平的调控能力， 由此筛选得到具有转录和转录后双水 平基因沉默功能的 microRNA。
本发明中， 所述人工比对， 是指在上述数据库 （miRbase, TargetScan) 预测的基础上， 使用 Vector NTI 软件 （ Invitrogen )， NAhybrid在线软件 ( http：〃 bibiserv.techfak.uni-bielefeld.de/mahvbrid/ ) 以及核酸序列片段的手工 查找（Excel, Microsoft) ,考察 microRNA与目的基因的结合能力与配对序列。
其中， 所述细胞可为 PLC/PRF/5 肝脏细胞， 也可以为 293细胞， 也可以 为 786-O肾脏细胞系。
因此， 通过上述方法筛选得到的 microRNA既能够通过结合于 5'侧翼区 特异位点从而下调目的基因 mRNA表达 （具有转录抑制能力）， 又能够通过 结合于 3'非翻译区特异位点从而下调目的基因蛋白表达的作用 （具有转录后 /翻译抑制能力）。
在步骤 4) 中， 我们筛选得到了可以同时下调目的基因 mRNA与蛋白水 平的 microRNA, mRNA水平下调可能与 microRNA的转录水平 （结合于启 动子或 5'侧翼区） 调控机制相关， 蛋白水平下调可能与 microRNA的转录后 调控机制相关， 因此， 基于下列原因， 我们应用基因定点突变与萤光素酶报 告基因的方法验证 microRNA的作用机制。
① 使用 miRbase与 TargetScan数据库预测 microRNA与目的基因的结合 位点时有一定的局限性。 例如 TargetScan数据库会使用 microRNA种子区域 与目的基因 3'非翻译区碱基配对数量 （一般要求配对数量大于 6) 来筛选预 测结果， 但由于 RNA二级结构的复杂性， 配对碱基数量少于 6， 或存在 U-G 配对时， microRNA也可能与目的基因 3'非翻译区进行有效结合， 从而起到 下调目的基因表达的作用。 因此， 在这种情况下， 使用一定程度的人工比对 的方式可以对数据进行更好的筛选。
② 使用 miRbase数据库预测，结合人工比对，确认筛选得到的 microRNA 与目的基因 5'侧翼区的结合位点， 使用基因定点突变技术， 将 5'侧翼区上的 结合位点突变， 然后将野生型与突变型的 5 '侧翼区分别插入萤光素酶载体， 并将载体转入细胞中， 加入 microRNA模拟物， 检测 5'侧翼区 microRNA结 合位点突变对萤光素酶活性的影响， 确认 microRNA与目的基因 5'侧翼区的 结合位点， 最终确定该 microRNA是否具有转录水平的调控能力。
③ 使用 TargetScan 数据库预测， 结合人工比对， 确认筛选得到的 microRNA与目的基因 3'非翻译区的结合位点， 使用基因定点突变技术， 将 3 '非翻译区上的结合位点突变， 然后将野生型与突变型的 3'非翻译区分别插 入萤光素酶载体， 将分别携带野生型与突变型的载体分别转入细胞中， 加入 microRNA模拟物， 检测 3'非翻译区 microRNA结合位点突变对萤光素酶活 性的影响， 确认 microRNA与目的基因 3'非翻译区的结合位点， 最终确定该 microRNA是否具有转录后 （翻译） 水平的调控能力。
综合①-③的实验结果， 即可得到可以同时在转录与转录后（翻译）水平 调控目的基因表达的 microRNA。 本发明中， 所述目的基因可为任何基因而不受限制， 优选地， 所述目的 基因为细胞色素 P450酶亚型 2E1 (Cytochrome P450 2E1, CYP2E1 )基因或 I 型血红素氧化酶 （Haem oxygenase Ι, ΗΜΟΧΙ ) 基因。
在本发明的另一个方面中， 提供了由上述方法筛选得到的 microRNA。 优选地， 本发明中， 例如， 以细胞色素 P450酶亚型 2E1基因为目的基 因， 通过上述方法， 筛选得到具有转录和转录后双水平基因沉默功能的 microRNA 552 (miR-552)。 或者， 例如， 以 I型血红素氧化酶基因为目的基 因， 通过上述方法， 筛选得到具有转录和转录后双水平基因沉默功能的 microRNA 1254 (miR-1254)。
在本发明的另一个方面中， 提供了由上述方法筛选得到的 microRNA的 用于使基因沉默的用途。 因此， 本发明的 microRNA具有在开发与筛选用于 治疗各种疾病的药物中的用途。
在本发明的另一个方面中， 提供了基因沉默的方法， 所述方法包括使用 由上述方法筛选得到的 microRNA进行基因沉默。
与 siRNA相比， microRNA是一种内源的、具有良好的适应性与特异性、 抑制效果显著的小 RNA分子。 本发明筛选得到 microRNA与前人报道的针 对同一目的基因的 microRNA相比，应用本发明的方法筛选得到的 microRNA 具有更好的基因沉默效果 （图 9); 本发明提供了一种新的有效的 microRNA 筛选方法， 可以得到良好的基因沉默效果； 本发明为科学研究中常用的基因 沉默技术提供了新的设计思路； 本发明也为针对疾病的 microRNA药物开发 设计提供了新的思路。 附图说明 下面结合附图与实施例对本发明做进一步的详细说明， 其仅仅是对本发 明的描述而不是限定。
图 1 为示意性显示本发明的具有转录和转录后双水平基因沉默功能的 micro NAs的筛选方法的流程图。
图 2为显示使用 miRBase数据库预测 CYP2E1 5'侧翼区所得的 microRNA 及其在 5'侧翼区上的结合位点的图。 其中， 5TR即指 5'侧翼区。 图 3 为显示使用 TargetScan 数据库预测 CYP2E1 3'非翻译区所得的 microRNA及其在 3'非翻译区上的结合位点的图。 其中， 3'UTR即指 3'非翻 译区。
图 4为显示预测所得的 microRNA对 CYP2E1蛋白表达的影响的图。 其 中：
A. PLC/P F/5细胞中转染各 miRNA后检测 CYP2E1表达的蛋白印迹图。 GAPDH作为上样量对照。 图中数字为 microRNA的名字， nc是指阴性对照
( negative control ) 。
B.图 4A对应的统计学结果。 其中， 使用 ImageQuant Solutions软件对 CYP2E1与内参基因 GAPDH的条带进行灰度检测， 然后依据 GAPDH的灰 度值， 以 nc为 1， 对 CYP2E1的蛋白表达水平进行归一化处理， 从而得到归 一化 CYP2E1蛋白表达水平。
图 5 为显示 miR-552 对 CYP2E1 m NA表达水平的抑制作用的图。 CYP2E1蛋白表达随 miR-552 类似物剂量依赖性下调，而 mRNA水平的下调 无此特点。 使用 Rotor Gene荧光实时定量 PCR仪， 使用 Takara的荧光定量 PC 试剂， 检测 CYP2E1 的 mRNA表达水平， 同时检测内参基因 GAPDH 的 mRNA水平。 归一化 CYP2E1的 mRNA表达水平是指依据 GAPDH的表 达水平， 以 nc为 1， 对 CYP2E1 的 mRNA表达水平进行归一化处理， 从而 得到归一化 CYP2E1的 mRNA表达水平。 *表示 p< 0.05 vs nc (相对于 nc) ， * *表示 ρ< 0·01 vs nc (相对于 nc) 。
图 6为显示 CYP2E1 5'侧翼区 miR-552结合位点突变对 miR-552转录抑 制能力的影响的图。 其中：
A. miR-552第 9-21位核苷酸能够与 CYP2E1启动子区转录起始位点前 第 313-326位核苷酸序列互补配对， 其中小写字母为 CYP2E1启动子区对应 miR-552结合位点突变后的核苷酸序列。
B. 荧光素酶报告基因实验显示 miR-552抑制野生型 CYP2E1启动子活 性， 而对于将 miR-552可能的结合位点突变了的 CYP2E1启动子， 则不能抑 制其活性。归一化萤光素酶活性指依据内参 firefly萤光素酶活性，以 nc为 1， 对 renilla萤光素酶活性进行归一化处理， 从而得到归一化萤光素酶活性。 * 表示 p< 0.05 vs nc (相对于 nc) 。 图 7为显示 CYP2E1 3'非翻译区 miR-552结合位点突变对 miR-552转录 后抑制能力的影响的图。 其中：
A. CYP2E1 m NA 3，-UTR区与 miR-552种子序列区部分互补配对， 其 中， 小写字母代表 CYP2E1 mRNA 3'-UT 区中 miR-552可能的结合位点突 变后的核苷酸序列。
B. 荧光素酶报告基因实验显示 miR-552抑制 CYP2E1 3'-UTR活性， 而 对 miR-552可能的结合位点突变了的 3'-UTR不能发挥抑制作用。 归一化萤 光素酶活性指依据内参 renilla萤光素酶活性， 以 nc为 1， 对 firefly萤光素酶 活性进行归一化处理， 从而得到归一化萤光素酶活性。 **表示 p< 0.01 vs nc
(相对于 nc) 。
图 8为显示 miR-1254对 HMOXl mRNA与蛋白表达的抑制作用的图。 其中：
A. 293细胞中转染 miR-1254类似物后检测 HMOX1表达的蛋白印迹图。 Beta-actin作为上样量对照。 nc是指阴性对照。
B. HMOX1蛋白与 mRNA表达随 miR-1254类似物剂量依赖性下调。使 用 Rotor Gene 荧光实时定量 PCR仪，使用 Takara的荧光定量 PCR试剂，检 测 HMOX1的 mRNA表达水平，同时检测内参基因 GAPDH的 mRNA水平。 归一化 HMOX1的 mRNA表达水平是指依据 GAPDH的表达水平， 以 nc为 1，对 CYP2E1的 mRNA表达水平进行归一化处理，从而得到归一化 HMOX1 的 mRNA表达水平。 ** 表示 p< 0.01vs nc (相对于 nc)， ***表示 p< 0.001 vs nc (相对于 nc) 。
C. 图 8A对应的统计学结果。 其中， 使用 ImageQuant Solutions软件对 HMOXl与内参基因 beta-actin的条带进行灰度检测， 然后依据 actin的灰度 值， 以 nc为 1， 对 HMOX1的蛋白表达水平进行归一化处理， 从而得到归一 化 HMOX1蛋白表达水平。
图 9 为显示筛选所得 miR-552 比前人报道的 miR-378 具有更好的 CYP2E1沉默效果的图。 其中：
A. PLC/P F/5细胞中转染 miR-552与 miR-378模拟物后检测 CYP2E1 表达的蛋白印迹图。 GAPDH作为上样量对照。 图中数字为 microRNA的名 字， nc是指阴性对照 ( negative control ) B. 图 9A对应的统计学结果。 其中， 使用 ImageQuant Solutions软件对 CYP2E1与内参基因 GAPDH的条带进行灰度检测， 然后依据 GAPDH的灰 度值， 以 nc为 1， 对 CYP2E1的蛋白表达水平进行归一化处理， 从而得到归 一化 CYP2E1蛋白表达水平。 ** 表示 p< 0.01vs nc (相对于 nc) 。
具体实施方式 实施例中所使用的试剂、 仪器来源：
人肝癌细胞 PLC/PRF/5细胞， 293细胞： 中科院上海生命科学院生化与 细胞研究所细胞库；
所用质粒 （PGL4.10, PGL4.73与 psiCHECK2) 均购自 Promega;
萤光素酶活性检测试剂盒： 购于 Promega;
转染试齐1 J: lipofectamine 2000购于 Invitrogen;
细胞培养液： 购于 Invitrogen;
蛋白免疫印迹仪器： 购于 BioRad;
基因定点突变试剂盒： 购于 Merck;
实时定量 PCR仪： QIAGEN;
实时定量 PCR试剂： 上海皓嘉；
CYP2E1蛋白印迹检测用抗体： 购于 Millipore;
HMOX1蛋白印迹检测用抗体： 购于 Santa Cruz。
除特别指定外， 本发明中所使用的方法均为本领域中的常规方法。 制备实施例
制备实施例 1 microRNA 552 (miR-552) 的制备
以细胞色素 P450酶亚型 2E1 (Cytochrome P450 2E1, CYP2E1 ) 为巨的 基因，通过下述步骤筛选得到具有双重抑制能力的 microRNA 552(miR-552)。
步骤 1 ): 使用 miRBase数据库对 CYP2E1转录起始位点前 lkb的 5'侧 翼区 （启动子可能存在的位置） 进行 microRNA结合位点预测， 得到一系列 可能结合于目的基因启动子区域的 microRNA (见图 2) ; 步骤 2):使用 TargetScan数据库对 CYP2E1的 3'非翻译区进行 microRNA 结合位点预测，得到一系列可能结合于 CYP2E1 3'非翻译区的 microRNA (见 图 3 ) ;
步骤 3 ) : 使用化学合成的 microRNA模拟物 （mimics) 检测步骤 1 ) 与 步骤 2) 所得 microRNA对目的基因蛋白表达的影响， 筛选得到可以显著下 调目的基因蛋白表达的 microRNA 552 (见图 4);
步骤 4): 使用化学合成的 miR-552模拟物 （mimics) 检测 miR-552对 CYP2E1 m NA表达的影响，发现 miR-552可以下调 CYP2E1 m NA水平（见 图 5 ) ;
步骤 5 ): 使用 miRbase与 TargetScan数据库预测， 结合人工比对， 找到 miR-552在 CYP2E1的 5'侧翼区和 3'非翻译区的结合位点（见图 2， 图 7A)。 使用基因定点突变技术，将 CYP2E1的 5'侧翼区和 3'非翻译区与 miR-552的 结合位点突变 （见图 6A， 图 7A)。 野生型 5'侧翼区与 3'非翻译区位置与序 列：
野生型 5'侧翼区位于人类基因组， CYP2E1 基因转录起始点上游 1000 个碱基， 10号染色体， 位置： 1352354624-1352355623；
野生型 3'非翻译区序列位于 CYP2E1成熟 mRNA末端， 1755-1912， 共 计 158个碱基。
将野生型与突变型 5'侧翼区与 3'非翻译区分别插入萤光素酶报告基因中 ( 5'侧翼区插入位置： PGL4.10 质粒多克隆位点； 3'非翻译区插入位置： psiCHECK2质粒多克隆位点）， 实验证明 （1 ) miR-552是通过结合于 5'侧翼 区特异位点，起到下调目的基因 mRNA表达的作用，证明 miR-552具有转录 抑制能力 （见图 6B); (2) miR-552是通过结合于 3'非翻译区特异位点， 起 到下调目的基因蛋白表达的作用， 证明 miR-552 具有翻译抑制能力 （见图 7B) ;
步骤 6) : 结合步骤 3 )、 4) 和 5 ) 的数据， 筛选得到可以同时显著下调 CYP2E1 mRNA与蛋白水平， 具有转录与转录后双重抑制能力的 miR-552。
制备实施例 2 microRNA 1254 (miR-1254) 的制备
按照前述的方法，我们选择目的基因 I型血红素氧化酶（Haemoxygenase Ι , ΗΜΟΧΙ ) , 使用同样的计算机预测与实验手段得到同时具有转录和转录后 双重基因沉默能力的 microRNA 1254 (mi -1254) (见图 8 )， 这一数据说明 本发明方法的有效性。 实验实施例
实施例 1 在人肝癌细胞 PLC PRF/5细胞中检测 miR-552对 CYP2E1 mRNA和蛋白表达水平的抑制作用。
在人肝癌细胞 PLC/PRF/5细胞中分别转染 25nM、 50nM、 100nM、 200nM 的 miR-552， 用蛋白印迹实验检测细胞内 CYP2E1的蛋白表达变化， 用逆转 录-实时定量 PCR的方法检测 CYP2E1 mRNA表达变化。 如图 4和 5所示， miR-552可剂量依赖地引起 CYP2E1蛋白水平表达下降 (图 5)，CYP2E1 mRNA 水平轻微下降， 无剂量相关性 (图 5)。 实施例 2 miR-552通过非种子序列区作用于 CYP2E1基因启动子区抑 制其转录活性。
将人 CYP2E1基因启动子区 （5'侧翼区） 野生型 (wt)和 miR-552结合位 点突变型 (mt) (见图 6A)克隆入 PGL3荧光素酶报告系统，检测共转染 miR-552 及其各种突变体对荧光素酶活性的影响。 如图 6所示， MiR-552可以明显抑 制 CYP2E1启动子活性， 而不降低突变的启动子活性 (见图 6B)。 实施例 3 miR-552通过种子序列区作用 CYP2E1 mRNA 3，-非翻译区而 对其表达进行转录后抑制。
将人 CYP2E1 3'非翻译区野生型 (wt)和 miR-552结合位点突变型 (mt)克隆 入双荧光素酶报告基因检测系统， 检测共转染 miR-552对荧光素酶活性的影 响。 如图 7所示， miR-552可以明显抑制 CYP2E1 3'非翻译区荧光素酶活性， 而对突变型 3'非翻译区荧光素酶活性抑制程度较小 (见图 7B)。 实施例 4 在人肾脏 293细胞中检测 miR-1254对 HMOX1 mRNA和蛋 白表达水平的抑制作用。
在人肾脏 293细胞中分别转染 50ηΜ、 ΙΟΟηΜ的 miR-1254， 用蛋白印迹 实验检测细胞内 HMOX1 的蛋白表达变化， 用逆转录-实时定量 PCR的方法 检测 HMOX1 mRNA表达变化。 如图 8所示， miR-1254可剂量依赖地引起 HMOXl蛋白水平表达显著下降 (图 8A， C), HMOXl mRNA水平显著下降 (图 8B)。

Claims

权 利 要 求
1、 一种具有转录和转录后双水平基因沉默功能的 microRNA 的筛选方 法， 所述方法包括：
步骤 1 ) :使用 miRBase数据库对目的基因的转录起始位点前 l-2kb的 5' 侧翼区进行 microRNA结合位点预测， 得到能够结合于目的基因启动子区域 的 microRNA;
步骤 2) :使用 TargetScan数据库对目的基因的 3'非翻译区进行 microRNA 结合位点预测， 得到能够结合于目的基因 3 '非翻译区的 microRNA;
步骤 3 ) : 化学合成上述步骤 1 )和步骤 2) 中得到的 microRNA, 并检测 microRNA对目的基因蛋白表达的影响， 由此筛选得到能够下调目的基因蛋 白表达的 microRNA;
步骤 4 ) :检测步骤 3 )中得到的能够下调目的基因蛋白表达的 microRNA 对目的基因 mRNA表达的影响，由此筛选得到能够同时下调目的基因 mRNA 和蛋白水平的 microRNA；
步骤 5 ) : 使用基因定点突变与萤光素酶报告基因的方法验证 microRNA 转录与转录后双重基因沉默能力， 从而筛选得到具有转录和转录后双水平基 因沉默功能的 microRNA。
2、 根据权利要求 1所述的方法， 其中， 步骤 3 ) 中， 检测 microRNA对 目的基因蛋白表达的影响的方法包括脂质体转染与蛋白免疫印迹。
3、 根据权利要求 1所述的方法， 其中， 步骤 4 ) 中， 检测步骤 3 ) 中得 到的能够下调目的基因蛋白表达的 microRNA对目的基因 mRNA表达的影响 的方法包括总 RNA抽提， cDNA反转录以及实时荧光定量 PCR。
4、 根据权利要求 1所述的方法， 其中， 步骤 5 ) 中， 进一步包括以下步 骤：
a)使用 miRbase数据库预测，结合人工比对，确认筛选得到的 microRNA 与目的基因 5'侧翼区的结合位点， 使用基因定点突变技术， 将 5'侧翼区上的 结合位点突变， 然后将野生型与突变型的 5'侧翼区分别插入携带海肾萤光素 酶的载体， 用携带萤火虫萤光素酶的载体作为内参， 将所述两个载体同时转 入细胞中， 加入步骤 4)中得到的 microRNA, 检测 5'侧翼区 microRNA结合 位点突变对海肾萤光素酶活性的影响， 确认 microRNA与目的基因 5 '侧翼区 的结合位点， 最终确定该 microRNA是否具有转录水平的调控能力， 由此筛 选得到具有转录水平的调控能力的 microRNA;
b ) 使用 TargetScan 数据库预测， 结合人工比对， 确认筛选得到的 microRNA与目的基因 3'非翻译区的结合位点， 使用基因定点突变技术， 将 3'非翻译区上的结合位点突变， 然后将野生型与突变型的 3'非翻译区分别插 入萤火虫萤光素酶载体， 以载体自身携带的海肾萤光素酶为内参， 将分别携 带野生型与突变型的载体分别转入细胞中， 加入 microRNA模拟物， 检测 3' 非翻译区 microRNA结合位点突变对萤火虫萤光素酶活性的影响， 确认步骤 a)中得到的具有转录水平的调控能力的 microRNA与目的基因 3'非翻译区的 结合位点， 最终确定该 microRNA是否具有转录后水平的调控能力， 由此筛 选得到具有转录和转录后双水平基因沉默功能的 microRNA。
5、 根据权利要求 4所述的方法， 其中， 所述细胞为 PLC/PRF/5肝脏细 胞、 293细胞或 786-O肾脏细胞系。
6、 根据权利要求 1所述的方法， 其中， 所述目的基因为细胞色素 P450 酶亚型 2E1基因或 I型血红素氧化酶基因。
7、 一种具有转录和转录后双水平基因沉默功能的 microRNA, 其由权利 要求 1〜6中任一项所述的方法筛选得到。
8、由权利要求 1〜6中任一项所述的方法筛选得到 microRNA用于基因沉 默的用途。
9、 一种基因沉默的方法， 所述方法包括使用由权利要求 1〜6 中任一项 所述的方法筛选得到 microRNA进行基因沉默。
PCT/CN2013/080160 2012-07-26 2013-07-26 具有转录和转录后双水平基因沉默功能的microRNA的筛选方法 WO2014015824A1 (zh)

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