miRNAs在雷公藤甲素抗肝癌效应中的作用及机制研究
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摘要
一、研究背景及目的：
在临床上，原发性肝癌作为最常见的恶性肿瘤之一，具有早期诊断困难，病程进展快，治疗效果差，病死率高等特点因此，被称为“癌中之王”。全球每年有超过600000人死于肝癌，死亡人数居所有肿瘤相关死亡第三位。研究表明，肝癌的发生与慢性病毒性肝炎、酒精性肝病及黄曲霉素摄入有密切关系。我国有数目庞大的慢性乙型肝炎患者及乙肝病毒携带者，肝癌发病率居全球第一位，给我国带来了沉重的疾病负担，造成了巨大的经济损失。手术切除是目前肝癌最有效的治疗手段，但是在我国，大部分患者在确诊时病程就已进入了中晚期，失去了手术治疗的机会。中晚期肝癌患者对常规放、化疗不敏感，而以索拉菲尼为代表的新型靶向治疗药物价格又过于昂贵且对患者预后改善不明显，使得这些治疗手段的应用受到限制，严重影响了患者的预后。因此，研发价格低廉的新型有效药物对于提高我国肝癌的治疗水平，减轻全社会的疾病负担具有重大的现实意义。
微小RNA（miRNA）是一种内源性非编码小RNA，大小约17-25个核苷酸（nt），在生物界广泛存在。miRNA的生物合成比较复杂，首先由RNA聚合酶II催化合成miRNA的初始转录子（pri-miRNA），然后pri-miRNA在细胞核中被核酸酶RNase Ⅲ Drosha切割成为前体miRNA (pre-miRNA)。经过进一步剪切后，pre-miRNA在转运蛋白exportin-5的作用下由细胞核内转移到细胞质中，最后由另一种核酸酶RNase ⅢDicer进一步切割产生具有一定生物学功能的成熟体miRNA。miRNA通过与目标信使RNA (mRNA)非完全配对，导致目标mRNA降解或者翻译受阻，从而调控靶基因的表达。miRNA对生物体的生长和发育起重要调节作用,其表达异常与多种疾病发生发展密切相关。大量研究发现，miRNA在很多肿瘤中异常转录，与肿瘤细胞的异常增殖、侵袭、转移以及拮抗凋亡等多种生物学特性联系密切。肝癌的发生发展也与miRNA异常表达密切相关。那么，调节异常表达的miRNA使其恢复至正常水平，是否有可能逆转肿瘤的恶性增殖、侵袭和转移等生物学特性呢？ Kota J等的研究首度为我们揭示了这种可能。他们发现miR-26a在HCC中异常低表达，在免疫缺陷鼠肝癌模型中人为高表达miR-26a,可以特异性的诱导肿瘤细胞凋亡，明显延缓肿瘤进展。该研究证明以miRNA为肿瘤治疗靶点可以通过诱导肿瘤细胞凋亡等途径，取得良好的治疗效果，宣告了以miRNA为治疗靶点的分子靶向治疗新时代的来临。
甲素提取自中药雷公藤，是一种二萜内酯类化合物，其具有抗炎，抗风湿，抗肿瘤以及抗免疫排斥，等多种生物学活性，具有极其重要的研究价值和应用前景。甲素自1972年被首次分离和鉴定以来，与其多种生物活性的相关分子机制及作用靶点尚不明确，多年来一直是研究人员的研究热点。既往的研究表明，甲素的多种生物学活性与其转录抑制效应密切相关，比如在免疫调节作用方面，核心机制是甲素通过抑制核转录因子NF-κB的转录活性减少炎症因子产生，从而减弱相关免疫反应；在抗瘤作用方面，甲素的诱导肿瘤细胞凋亡作用亦与其对NF-κB的转录抑制效应密切相关。另外，甲素对转录因子HSF-1和AP-1的抑制作用也是其抗瘤效应的重要机制。由此我们可以得出一个初步结论，即甲素的转录抑制作用是其产生相应生物学效应的基础。
越来越多文献表明，甲素具有非常广泛的转录抑制活性。McCallum等研究证实甲素能够抑制肿瘤细胞核糖体RNA(rRNA)的转录;Crews证实甲素通过抑制RNA聚合酶II（RNA polymeraseII）的Ser2磷酸化位点的磷酸化，从而抑制其的转录活性;StéphaneVispé等的研究进一步证实甲素能够明显抑制肿瘤细胞内RNA聚合酶II催化的信使RNA(mRNA)的转录。Liu等人进一步研究表明，甲素能够与通用转录因子TFIIH的核心亚单位ERCC3(又称XPB)特异性结合，从而抑制后者的ATP酶活性，导致TFIIH参与的、由RNA聚合酶II介导的基因转录受阻。那么甲素是否能够抑制人RNA组中另外一个重要组成部分——微小RNA(miRNA)的转录呢？遗憾的是国内外关于这个问题的研究尚处于空白，无法找到现成答案。我们知道miRNA是一种内源性非编码小RNA，大小约17-25个核苷酸（nt），在生物界广泛存在。miRNA通过与目标mRNA互补配对，导致目标mRNA降解或者翻译受阻，从而调控靶基因的表达，调节生物体的生长和发育。研究表明，miRNA的初始转录子（pri-miRNA）由RNA聚合酶II催化合成。既然miRNA由RNA聚合酶II负责转录，而甲素能够抑制RNA聚合酶II的催化活性，那么我们可以合理假设甲素也能够抑制miRNA的转录。
本课题拟针对甲素也能够抑制miRNA的转录这一设想探讨，研究甲素抗肝癌效应相关的分子机制。主要研究包括：（1）明确甲素对人肝癌细胞的生长抑制效应和诱导凋亡作用；（2）明确甲素对miRNA的转录调节作用及该作用对甲素诱导肝癌细胞凋亡效应的影响，探索其中的分子机制；（3）明确甲素对裸鼠荷瘤模型的体内抑制效应，并验证相关分子机制。
二、研究方法：
1、CCK-8细胞增殖实验检测甲素对多种肝癌细胞增殖抑制作用，获得抑制剂量及作用时间曲线，流式细胞仪检测不同浓度甲素不同作用时间对HepG2细胞的诱导凋亡作用，PI单染法流式细胞仪检测甲素对肝癌细胞周期分布的影响，免疫印迹、qRT-PCR及报告基因方法检测甲素刺激后人肝癌细胞HepG2内凋亡及生长增殖相关信号通路的变化，并使用基因过表达或者基因干扰的手段上调或者抑制相应蛋白的表达以调控该信号通路，观察相关信号通路对甲素诱导凋亡作用的影响，明确相应的信号通路与甲素诱导凋亡效应的关系，揭示甲素抑制肿瘤作用的分子机制。
2、建立能稳定表达荧光素酶的肝癌细胞株HepG2-luc，利用该细胞株建立裸鼠肝癌种植瘤模型，随机将裸鼠分为阴性对照组（DMSO）、甲素给药组（0.2mg/kg/day）、阳性对照组（1mg/kg/day），经腹腔注射途径连续给药14天，利用小动物活体成像技术及时监测各组间肿瘤组织内荧光信号强度，并观察有无远处转移情况。给药结束后获取肝癌组织样本和血液标本，TUNEL法检测裸鼠肝癌组织内细胞凋亡情况，IHC法检测相应蛋白表达情况，并检测血清内相应生化指标以评估甲素对裸鼠肝肾功能的影响。
3、miRNA芯片检测甲素刺激后HepG2细胞内miRNA表达谱的改变，生物信息技术筛选差异miRNA及所调控的信号蛋白和通路，qRT-PCR验证miRNA芯片的准确性；ChIP技术检测转录因子c-Myc与miRNA编码基因5’端序列特异性结合情况，结合基因过表达和干扰技术，评估c-Myc对miRNA的直接转录激活作用；基因过表达和干扰技术观察甲素重要靶基因与ERCC3与c-Myc之间的相互调控作用。
4、收集30例新鲜人肝癌组织样本及临床资料，qRT-PCR技术检测人肝癌组织内c-Myc、miR-17及miR-93的表达情况，后两者分别是miRNA-17-92和miRNA-106b-25的代表性成员，分析c-Myc表达水平是否与miR-17、miR-93的表达具有相关性；根据miR-17或者miR-93的表达水平将肝癌病人分为高表达组和低表达组，比较两组患者间的无复发生存期及总生存期是否有差异，以明确这两种miRNA表达异常是否影响肝癌患者的预后。
三、研究结果及结论：
1、甲素能够显著抑制多种肝癌细胞的增殖，并且该效应呈时间和剂量依赖性。
2、甲素能够在体外显著诱导多种肝癌细胞发生凋亡，且该效应亦呈剂量依赖性。甲素能够诱导HepG2细胞内caspase-3及PARP的活化，上调Bax/Bcl-2比例，激活线粒体相关凋亡途径，同时能提高P53的表达和磷酸化水平，抑制Akt的表达和磷酸化。甲素对肝癌细胞的周期分布无显著影响。
3、甲素能够显著抑制裸鼠肝癌皮下种植瘤的生长，且对裸鼠的肝肾功能无显著影响。我们利用HepG2-luc细胞建立了裸鼠荷瘤模型，将裸鼠分为阴性对照组、甲素给药组和阳性对照组。相对于阴性对照，甲素和顺铂给药组的肿瘤生长速度及体积显著降低，且甲素给药组降低更明显。TUNEL实验表明，甲素给药组和顺铂给药组裸鼠瘤体内均发生了显著的细胞凋亡。
4、甲素能够通过抑制转录因子c-Myc的转录活性，同时下调miR-17-92及miR-106b-25簇内多个成员的表达。我们首先利用miRNA芯片检测甲素对HepG2细胞内miRNA表达谱的改变，发现200nM甲素刺激24h后HepG2细胞内miRNA表达谱显著改变，超过80%的miRNA表达下降。我们进一步利用生物信息学方法筛选出显著性差异miRNA，在前十位高丰度差异miRNA中，有2种miRNA上调，8种下调，在8种下调的miRNA中有5种分别属于miRNA17-92及miR-106b-25簇。我们证明c-Myc能够直接与miR-106b-25宿主基因MCM-7的5’端上游序列CACGTG特异性结合，从而促进miR-106b-25的转录。另外已知c-Myc也能够与miR-17-92上游调控序列结合促进其表达，我们进一步证明甲素通过下调c-Myc的转录活性同时下调miR-17-92及miR-106b-25簇内多个成员的表达，导致其共同的靶基因PTEN及BIM表达升高，而后二者均具有较强的促凋亡作用，最终促进HepG2细胞发生凋亡。我们又分别构建miR-17-92及miR-106b-25真核表达质粒，同时转染至HePG2细胞内，发现二者过表达后能够显著拮抗甲素的诱导凋亡作用，证明甲素对miR-17-92和miR-106b-25的抑制作用能够促进肝癌细胞的凋亡。
5、甲素通过抑制靶分子ERCC3的表达抑制c-Myc的表达和转录活性。Western-blot及qRT-PCR实验结果表明，甲素能够显著下调HepG2细胞内ERCC3的转录和蛋白表达水平。报告基因检测结果表明，甲素在较低浓度下（25nM）即能显著抑制c-Myc的转录活性。我们构建ERCC3过表达质粒并转染HepG2细胞，发现过表达ERCC3后能够显著逆转甲素对c-Myc的抑制作用，证明甲素至少部分通过抑制靶分子ERCC3的表达而下调c-Myc的表达。更为有趣的是，我们分别过表达或者干扰ERCC3和c-Myc，发现过表达c-Myc后ERCC3表达上调，干扰c-Myc后ERCC3表达下调，反之亦然。由此，我们猜测c-Myc和ERCC3能够相互促进对方的表达，二者构成一个正反馈系统。
6、miR-17和miR-93在人肝癌组织内异常高表达，二者的表达水平高低与患者的无复发生存期及总生存期呈负相关。我们收集了30例新鲜人肝癌组织样本及临床资料，利用qRT-PCR技术检测人肝癌组织内c-Myc、miR-17及miR-93的表达情况。结果表明，c-Myc表达水平与miR-17、miR-93的表达具有正相关性，三者均在大部分肝癌组织中呈高表达状态；我们根据miR-17或者miR-93的表达水平将肝癌病人分为高表达组和低表达组，发现miR-17或者miR-93高表达组患者无复发生存期显著缩短，两组间差异具有统计学意义(P<0.05)。
结论：综上所述，我们发现甲素在体内外具有显著的抗肝癌效应，且该效应与甲素对肝癌细胞内c-Myc/miRNA簇/靶基因的调节作用有关。
Background&Aims: At present Hepatocellular carcinoma (HCC) is the third leading causeof cancer death worldwide with annual death exceeding600000. HCC is associated with avery poor prognosis because of its aggressive growth, metastasis, and resistance to the mostof current therapeutic approaches. Therefore, there is an urgent need to develop effectivetherapeutic strategies for the large number of HCC patients. MicroRNAs (miRNAs) areendogenous～23nt RNAs that negatively regulate gene expression by pairing to the mRNAsof protein-coding genes to direct their posttranscriptional repression. As key negativeregulators in gene expression, miRNAs play an important role in many cellular processes,such as differentiation, proliferation, and apoptosis. Importantly, a large body of evidence hasshown that miRNAs regulate molecular pathways in cancer by targeting various oncogenesand tumor suppressors, thus forming central nodal points in cancer development andprogression. Not surprisingly, miRNAs have also been discovered to be aberrantly expressedin HCC and some of them are functionally implicated in hepatocarcinogenesis andprogression. Combinations of genomic analyses and functional studies have identified somemiRNAs, such as miR-17-92, miR-21, and miR-221, function as oncogenes in initiation andmaintenance of HCC. In contrast, some miRNAs, including let-7, miR-122and miR-26, havebeen identified as tumor suppressors. Therefore, targeting oncogenic miRNAs and restoringtumor-suppressive miRNAs would be a reasonable therapeutic strategy for HCC patients.Triptolide is a structurally unique diterpene triepoxide isolated from Tripterygium wilfordiiHook F, a Chinese medicinal plant used for treating a wide range of diseases for centuries. Triptolide has been shown to possess potent anti-inflammatory, immunosuppressive andanticancer activity. The antitumor activity appears quite broad in that triptolide is capable ofkilling cancer cells originated from different tissues, including blood, colon, breast, brain,ovary, kidney and prostate, with IC50values in the low nanomolar range. The mechanismsresponsible for antitumor activity of triptolide have been extensively investigated in the pastfew decades. Triptolide is demonstrated to cause transcriptional inhibition, initially believedto target specific transcription factors but recently revealed to cause global transcriptioninhibition via targeting RNA polymerase I and II. More recently, Titov et al. reported thattriptolide covalently bound to a human90kD protein ERCC3(also known as XPB), andinhibited its DNA-dependent ATPase activity, which led to the inhibition of RNA polymeraseII–mediated transcription. It seems that the transcription inhibition accounts for most of theaforementioned biological activities of triptolide. However, besides downregulating theexpression of a lot of genes, triptolide has also been revealed to increase the mRNA or proteinlevels of several molecules, including p53, RIZ1and HIF-1a, and such an increase has beenshown to contribute to the anticancer activity of triptolide.
To explain this discrepancy, we hypothesized that triptolide inhibits the transcription of someoncogenic miRNAs, which in turn increase the protein level of their target genes. We usedmiRNA microarrays and observed that the miRNAs expression profiles are significantlyaltered by the treatment of triptolide. Among the modulated miRNAs, up to94%aredown-regulated. Two oncogenic miRNA cluster, mir-17-92and mir-106b-25, aredownregulated by the treatment of triptolide simultaneously. On the contrary, the putative target genes of these miRNA clusters are upregulated by triptolide. We further proved thatboth of the two miRNA clusters are directly transactivated by c-Myc, and triptolidedownregulates the expression of these miRNA clusters in a c-Myc dependent manner.Importantly, the modulation of c-Myc/miRNA clusters/target genes cascade contributes totriptolide-induced cell death.
METHODS: Cells were treated with triptolide, and the anti-HCC activity of triptolide wasevaluated using flow cytometry, western blot, and xenograft studies. microRNA microarray andquantitative reverse-transcription polymerase chain reaction was used to identify differentialmicroRNAs induced by triptolide. Chromatin immunoprecipitation assay was employed tostudy the interaction between c-Myc and genomic regions of miR106b-25. microRNAsoverexpression and knockdown experiments was performed to determine the role of thesemicroRNAs in triptolide-induced apoptosis.
RESULTS: Triptolide induces cell proliferation inhibition and marked apoptosis in multipleHCC cell lines with different p53status. Several signaling molecules belonging to differentpathways are altered by the treatment of triptolide. Xenograft tumor volume was significantlydecreased in triptolide-treated group when compared with vehicle control group. Two miRNAclusters, miR-17-92and miR-106b-25, were significantly repressed by triptolide, resulting inthe upregulation of their common target genes, including BIM, PTEN, and p21. In HCCsamples, high levels of these miRNA clusters correlated with shorter recurrence free survival.Triptolide inhibits the expression of theses miRNAs in a c-Myc-dependent manner, whichenhances triptolide-induced cell death. We further show that triptolide downregulates the expression of c-Myc through targeting ERCC3, a newly indentified triptolide-binding protein.
CONCLUDSIONS: The triptolide-induced modulation of c-Myc/miRNA clusters/targetgenes axis enhances its potent antitumor activity, thereby making triptolide an attractivechemotherapeutic agent against HCC.
在临床上，原发性肝癌作为最常见的恶性肿瘤之一，具有早期诊断困难，病程进展快，治疗效果差，病死率高等特点因此，被称为“癌中之王”。全球每年有超过600000人死于肝癌，死亡人数居所有肿瘤相关死亡第三位。研究表明，肝癌的发生与慢性病毒性肝炎、酒精性肝病及黄曲霉素摄入有密切关系。我国有数目庞大的慢性乙型肝炎患者及乙肝病毒携带者，肝癌发病率居全球第一位，给我国带来了沉重的疾病负担，造成了巨大的经济损失。手术切除是目前肝癌最有效的治疗手段，但是在我国，大部分患者在确诊时病程就已进入了中晚期，失去了手术治疗的机会。中晚期肝癌患者对常规放、化疗不敏感，而以索拉菲尼为代表的新型靶向治疗药物价格又过于昂贵且对患者预后改善不明显，使得这些治疗手段的应用受到限制，严重影响了患者的预后。因此，研发价格低廉的新型有效药物对于提高我国肝癌的治疗水平，减轻全社会的疾病负担具有重大的现实意义。
微小RNA（miRNA）是一种内源性非编码小RNA，大小约17-25个核苷酸（nt），在生物界广泛存在。miRNA的生物合成比较复杂，首先由RNA聚合酶II催化合成miRNA的初始转录子（pri-miRNA），然后pri-miRNA在细胞核中被核酸酶RNase Ⅲ Drosha切割成为前体miRNA (pre-miRNA)。经过进一步剪切后，pre-miRNA在转运蛋白exportin-5的作用下由细胞核内转移到细胞质中，最后由另一种核酸酶RNase ⅢDicer进一步切割产生具有一定生物学功能的成熟体miRNA。miRNA通过与目标信使RNA (mRNA)非完全配对，导致目标mRNA降解或者翻译受阻，从而调控靶基因的表达。miRNA对生物体的生长和发育起重要调节作用,其表达异常与多种疾病发生发展密切相关。大量研究发现，miRNA在很多肿瘤中异常转录，与肿瘤细胞的异常增殖、侵袭、转移以及拮抗凋亡等多种生物学特性联系密切。肝癌的发生发展也与miRNA异常表达密切相关。那么，调节异常表达的miRNA使其恢复至正常水平，是否有可能逆转肿瘤的恶性增殖、侵袭和转移等生物学特性呢？ Kota J等的研究首度为我们揭示了这种可能。他们发现miR-26a在HCC中异常低表达，在免疫缺陷鼠肝癌模型中人为高表达miR-26a,可以特异性的诱导肿瘤细胞凋亡，明显延缓肿瘤进展。该研究证明以miRNA为肿瘤治疗靶点可以通过诱导肿瘤细胞凋亡等途径，取得良好的治疗效果，宣告了以miRNA为治疗靶点的分子靶向治疗新时代的来临。
甲素提取自中药雷公藤，是一种二萜内酯类化合物，其具有抗炎，抗风湿，抗肿瘤以及抗免疫排斥，等多种生物学活性，具有极其重要的研究价值和应用前景。甲素自1972年被首次分离和鉴定以来，与其多种生物活性的相关分子机制及作用靶点尚不明确，多年来一直是研究人员的研究热点。既往的研究表明，甲素的多种生物学活性与其转录抑制效应密切相关，比如在免疫调节作用方面，核心机制是甲素通过抑制核转录因子NF-κB的转录活性减少炎症因子产生，从而减弱相关免疫反应；在抗瘤作用方面，甲素的诱导肿瘤细胞凋亡作用亦与其对NF-κB的转录抑制效应密切相关。另外，甲素对转录因子HSF-1和AP-1的抑制作用也是其抗瘤效应的重要机制。由此我们可以得出一个初步结论，即甲素的转录抑制作用是其产生相应生物学效应的基础。
越来越多文献表明，甲素具有非常广泛的转录抑制活性。McCallum等研究证实甲素能够抑制肿瘤细胞核糖体RNA(rRNA)的转录;Crews证实甲素通过抑制RNA聚合酶II（RNA polymeraseII）的Ser2磷酸化位点的磷酸化，从而抑制其的转录活性;StéphaneVispé等的研究进一步证实甲素能够明显抑制肿瘤细胞内RNA聚合酶II催化的信使RNA(mRNA)的转录。Liu等人进一步研究表明，甲素能够与通用转录因子TFIIH的核心亚单位ERCC3(又称XPB)特异性结合，从而抑制后者的ATP酶活性，导致TFIIH参与的、由RNA聚合酶II介导的基因转录受阻。那么甲素是否能够抑制人RNA组中另外一个重要组成部分——微小RNA(miRNA)的转录呢？遗憾的是国内外关于这个问题的研究尚处于空白，无法找到现成答案。我们知道miRNA是一种内源性非编码小RNA，大小约17-25个核苷酸（nt），在生物界广泛存在。miRNA通过与目标mRNA互补配对，导致目标mRNA降解或者翻译受阻，从而调控靶基因的表达，调节生物体的生长和发育。研究表明，miRNA的初始转录子（pri-miRNA）由RNA聚合酶II催化合成。既然miRNA由RNA聚合酶II负责转录，而甲素能够抑制RNA聚合酶II的催化活性，那么我们可以合理假设甲素也能够抑制miRNA的转录。
本课题拟针对甲素也能够抑制miRNA的转录这一设想探讨，研究甲素抗肝癌效应相关的分子机制。主要研究包括：（1）明确甲素对人肝癌细胞的生长抑制效应和诱导凋亡作用；（2）明确甲素对miRNA的转录调节作用及该作用对甲素诱导肝癌细胞凋亡效应的影响，探索其中的分子机制；（3）明确甲素对裸鼠荷瘤模型的体内抑制效应，并验证相关分子机制。
二、研究方法：
1、CCK-8细胞增殖实验检测甲素对多种肝癌细胞增殖抑制作用，获得抑制剂量及作用时间曲线，流式细胞仪检测不同浓度甲素不同作用时间对HepG2细胞的诱导凋亡作用，PI单染法流式细胞仪检测甲素对肝癌细胞周期分布的影响，免疫印迹、qRT-PCR及报告基因方法检测甲素刺激后人肝癌细胞HepG2内凋亡及生长增殖相关信号通路的变化，并使用基因过表达或者基因干扰的手段上调或者抑制相应蛋白的表达以调控该信号通路，观察相关信号通路对甲素诱导凋亡作用的影响，明确相应的信号通路与甲素诱导凋亡效应的关系，揭示甲素抑制肿瘤作用的分子机制。
2、建立能稳定表达荧光素酶的肝癌细胞株HepG2-luc，利用该细胞株建立裸鼠肝癌种植瘤模型，随机将裸鼠分为阴性对照组（DMSO）、甲素给药组（0.2mg/kg/day）、阳性对照组（1mg/kg/day），经腹腔注射途径连续给药14天，利用小动物活体成像技术及时监测各组间肿瘤组织内荧光信号强度，并观察有无远处转移情况。给药结束后获取肝癌组织样本和血液标本，TUNEL法检测裸鼠肝癌组织内细胞凋亡情况，IHC法检测相应蛋白表达情况，并检测血清内相应生化指标以评估甲素对裸鼠肝肾功能的影响。
3、miRNA芯片检测甲素刺激后HepG2细胞内miRNA表达谱的改变，生物信息技术筛选差异miRNA及所调控的信号蛋白和通路，qRT-PCR验证miRNA芯片的准确性；ChIP技术检测转录因子c-Myc与miRNA编码基因5’端序列特异性结合情况，结合基因过表达和干扰技术，评估c-Myc对miRNA的直接转录激活作用；基因过表达和干扰技术观察甲素重要靶基因与ERCC3与c-Myc之间的相互调控作用。
4、收集30例新鲜人肝癌组织样本及临床资料，qRT-PCR技术检测人肝癌组织内c-Myc、miR-17及miR-93的表达情况，后两者分别是miRNA-17-92和miRNA-106b-25的代表性成员，分析c-Myc表达水平是否与miR-17、miR-93的表达具有相关性；根据miR-17或者miR-93的表达水平将肝癌病人分为高表达组和低表达组，比较两组患者间的无复发生存期及总生存期是否有差异，以明确这两种miRNA表达异常是否影响肝癌患者的预后。
三、研究结果及结论：
1、甲素能够显著抑制多种肝癌细胞的增殖，并且该效应呈时间和剂量依赖性。
2、甲素能够在体外显著诱导多种肝癌细胞发生凋亡，且该效应亦呈剂量依赖性。甲素能够诱导HepG2细胞内caspase-3及PARP的活化，上调Bax/Bcl-2比例，激活线粒体相关凋亡途径，同时能提高P53的表达和磷酸化水平，抑制Akt的表达和磷酸化。甲素对肝癌细胞的周期分布无显著影响。
3、甲素能够显著抑制裸鼠肝癌皮下种植瘤的生长，且对裸鼠的肝肾功能无显著影响。我们利用HepG2-luc细胞建立了裸鼠荷瘤模型，将裸鼠分为阴性对照组、甲素给药组和阳性对照组。相对于阴性对照，甲素和顺铂给药组的肿瘤生长速度及体积显著降低，且甲素给药组降低更明显。TUNEL实验表明，甲素给药组和顺铂给药组裸鼠瘤体内均发生了显著的细胞凋亡。
4、甲素能够通过抑制转录因子c-Myc的转录活性，同时下调miR-17-92及miR-106b-25簇内多个成员的表达。我们首先利用miRNA芯片检测甲素对HepG2细胞内miRNA表达谱的改变，发现200nM甲素刺激24h后HepG2细胞内miRNA表达谱显著改变，超过80%的miRNA表达下降。我们进一步利用生物信息学方法筛选出显著性差异miRNA，在前十位高丰度差异miRNA中，有2种miRNA上调，8种下调，在8种下调的miRNA中有5种分别属于miRNA17-92及miR-106b-25簇。我们证明c-Myc能够直接与miR-106b-25宿主基因MCM-7的5’端上游序列CACGTG特异性结合，从而促进miR-106b-25的转录。另外已知c-Myc也能够与miR-17-92上游调控序列结合促进其表达，我们进一步证明甲素通过下调c-Myc的转录活性同时下调miR-17-92及miR-106b-25簇内多个成员的表达，导致其共同的靶基因PTEN及BIM表达升高，而后二者均具有较强的促凋亡作用，最终促进HepG2细胞发生凋亡。我们又分别构建miR-17-92及miR-106b-25真核表达质粒，同时转染至HePG2细胞内，发现二者过表达后能够显著拮抗甲素的诱导凋亡作用，证明甲素对miR-17-92和miR-106b-25的抑制作用能够促进肝癌细胞的凋亡。
5、甲素通过抑制靶分子ERCC3的表达抑制c-Myc的表达和转录活性。Western-blot及qRT-PCR实验结果表明，甲素能够显著下调HepG2细胞内ERCC3的转录和蛋白表达水平。报告基因检测结果表明，甲素在较低浓度下（25nM）即能显著抑制c-Myc的转录活性。我们构建ERCC3过表达质粒并转染HepG2细胞，发现过表达ERCC3后能够显著逆转甲素对c-Myc的抑制作用，证明甲素至少部分通过抑制靶分子ERCC3的表达而下调c-Myc的表达。更为有趣的是，我们分别过表达或者干扰ERCC3和c-Myc，发现过表达c-Myc后ERCC3表达上调，干扰c-Myc后ERCC3表达下调，反之亦然。由此，我们猜测c-Myc和ERCC3能够相互促进对方的表达，二者构成一个正反馈系统。
6、miR-17和miR-93在人肝癌组织内异常高表达，二者的表达水平高低与患者的无复发生存期及总生存期呈负相关。我们收集了30例新鲜人肝癌组织样本及临床资料，利用qRT-PCR技术检测人肝癌组织内c-Myc、miR-17及miR-93的表达情况。结果表明，c-Myc表达水平与miR-17、miR-93的表达具有正相关性，三者均在大部分肝癌组织中呈高表达状态；我们根据miR-17或者miR-93的表达水平将肝癌病人分为高表达组和低表达组，发现miR-17或者miR-93高表达组患者无复发生存期显著缩短，两组间差异具有统计学意义(P<0.05)。
结论：综上所述，我们发现甲素在体内外具有显著的抗肝癌效应，且该效应与甲素对肝癌细胞内c-Myc/miRNA簇/靶基因的调节作用有关。
Background&Aims: At present Hepatocellular carcinoma (HCC) is the third leading causeof cancer death worldwide with annual death exceeding600000. HCC is associated with avery poor prognosis because of its aggressive growth, metastasis, and resistance to the mostof current therapeutic approaches. Therefore, there is an urgent need to develop effectivetherapeutic strategies for the large number of HCC patients. MicroRNAs (miRNAs) areendogenous～23nt RNAs that negatively regulate gene expression by pairing to the mRNAsof protein-coding genes to direct their posttranscriptional repression. As key negativeregulators in gene expression, miRNAs play an important role in many cellular processes,such as differentiation, proliferation, and apoptosis. Importantly, a large body of evidence hasshown that miRNAs regulate molecular pathways in cancer by targeting various oncogenesand tumor suppressors, thus forming central nodal points in cancer development andprogression. Not surprisingly, miRNAs have also been discovered to be aberrantly expressedin HCC and some of them are functionally implicated in hepatocarcinogenesis andprogression. Combinations of genomic analyses and functional studies have identified somemiRNAs, such as miR-17-92, miR-21, and miR-221, function as oncogenes in initiation andmaintenance of HCC. In contrast, some miRNAs, including let-7, miR-122and miR-26, havebeen identified as tumor suppressors. Therefore, targeting oncogenic miRNAs and restoringtumor-suppressive miRNAs would be a reasonable therapeutic strategy for HCC patients.Triptolide is a structurally unique diterpene triepoxide isolated from Tripterygium wilfordiiHook F, a Chinese medicinal plant used for treating a wide range of diseases for centuries. Triptolide has been shown to possess potent anti-inflammatory, immunosuppressive andanticancer activity. The antitumor activity appears quite broad in that triptolide is capable ofkilling cancer cells originated from different tissues, including blood, colon, breast, brain,ovary, kidney and prostate, with IC50values in the low nanomolar range. The mechanismsresponsible for antitumor activity of triptolide have been extensively investigated in the pastfew decades. Triptolide is demonstrated to cause transcriptional inhibition, initially believedto target specific transcription factors but recently revealed to cause global transcriptioninhibition via targeting RNA polymerase I and II. More recently, Titov et al. reported thattriptolide covalently bound to a human90kD protein ERCC3(also known as XPB), andinhibited its DNA-dependent ATPase activity, which led to the inhibition of RNA polymeraseII–mediated transcription. It seems that the transcription inhibition accounts for most of theaforementioned biological activities of triptolide. However, besides downregulating theexpression of a lot of genes, triptolide has also been revealed to increase the mRNA or proteinlevels of several molecules, including p53, RIZ1and HIF-1a, and such an increase has beenshown to contribute to the anticancer activity of triptolide.
To explain this discrepancy, we hypothesized that triptolide inhibits the transcription of someoncogenic miRNAs, which in turn increase the protein level of their target genes. We usedmiRNA microarrays and observed that the miRNAs expression profiles are significantlyaltered by the treatment of triptolide. Among the modulated miRNAs, up to94%aredown-regulated. Two oncogenic miRNA cluster, mir-17-92and mir-106b-25, aredownregulated by the treatment of triptolide simultaneously. On the contrary, the putative target genes of these miRNA clusters are upregulated by triptolide. We further proved thatboth of the two miRNA clusters are directly transactivated by c-Myc, and triptolidedownregulates the expression of these miRNA clusters in a c-Myc dependent manner.Importantly, the modulation of c-Myc/miRNA clusters/target genes cascade contributes totriptolide-induced cell death.
METHODS: Cells were treated with triptolide, and the anti-HCC activity of triptolide wasevaluated using flow cytometry, western blot, and xenograft studies. microRNA microarray andquantitative reverse-transcription polymerase chain reaction was used to identify differentialmicroRNAs induced by triptolide. Chromatin immunoprecipitation assay was employed tostudy the interaction between c-Myc and genomic regions of miR106b-25. microRNAsoverexpression and knockdown experiments was performed to determine the role of thesemicroRNAs in triptolide-induced apoptosis.
RESULTS: Triptolide induces cell proliferation inhibition and marked apoptosis in multipleHCC cell lines with different p53status. Several signaling molecules belonging to differentpathways are altered by the treatment of triptolide. Xenograft tumor volume was significantlydecreased in triptolide-treated group when compared with vehicle control group. Two miRNAclusters, miR-17-92and miR-106b-25, were significantly repressed by triptolide, resulting inthe upregulation of their common target genes, including BIM, PTEN, and p21. In HCCsamples, high levels of these miRNA clusters correlated with shorter recurrence free survival.Triptolide inhibits the expression of theses miRNAs in a c-Myc-dependent manner, whichenhances triptolide-induced cell death. We further show that triptolide downregulates the expression of c-Myc through targeting ERCC3, a newly indentified triptolide-binding protein.
CONCLUDSIONS: The triptolide-induced modulation of c-Myc/miRNA clusters/targetgenes axis enhances its potent antitumor activity, thereby making triptolide an attractivechemotherapeutic agent against HCC.
引文
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[18] Vispé S, DeVries L, Créancier L, Besse J, Bréand S, Hobson DJ, Svejstrup JQ, Annereau JP, Cussac D,Dumontet C, Guilbaud N, Barret JM, Bailly C.Triptolide is an inhibitor of RNA polymerase I andII-dependent transcription leading predominantly to down-regulation of short-lived mRNA. MolCancer Ther,2009,8(10):2780-90
[19] Carthew RW, Sontheimer EJ.Origins and Mechanisms of miRNAs and siRNAs. Cell,2009,136(4):642-55
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[24] Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T.MicroRNA-21regulatesexpression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology,2007,133(2):647-58
[25] Zhang X, Liu S, Hu T, Liu S, He Y, Sun S. Up-regulated microRNA-143transcribed by nuclear factorkappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression. Hepatology,2009,50(2):490-9
[26] Pineau P, Volinia S, McJunkin K, Marchio A, Battiston C, Terris B, Mazzaferro V, Lowe SW, CroceCM, Dejean A.miR-221overexpression contributes to liver tumorigenesis. Proc Natl Acad Sci U S A,2010,107(1):264-9
[27] Kota J, Chivukula RR, O'Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, Chang TC,Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT.Therapeutic microRNA deliverysuppresses tumorigenesis in a murine liver cancer model. Cell,2009,137(6):1005-17
[28] Rossi JJ. New hope for a microRNA therapy for liver cancer. Cell,2009,137(6):990-2
[29] Alisi A, Balsano C. Enhancing the efficacy of hepatocellular carcinoma chemotherapeutics withnatural anticancer agents.Nutr Rev,2007,65:550-3
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[5] Schafer DF, Sorrell MF. Hepatocellular carcinoma. Lancet,1999,353:1253-7
[6] Arzumanyan A, Reis HM, Feitelson MA. Pathogenic mechanisms in HBV-and HCV-associatedhepatocellular carcinoma. Nat Rev Cancer,2013,13:123-35
[7] Koo HS, Kang SD, Lee JH. Triptolide Inhibits the Proliferation of Immortalized HT22HippocampalCells Via Persistent Activation of Extracellular Signal-Regulated Kinase-1/2by Down-RegulatingMitogen-Activated Protein Kinase Phosphatase-1Expression. Journal of Korean NeurosurgicalSociety,2009,46:389-96
[8] Liu Q. Triptolide and its expanding multiple pharmacological functions. InternationalImmunopharmacology,2011,11:377-83
[9] Lou YJ, Jin J. Triptolide down-regulates bcr-abl expression and induces apoptosis in chronicmyelogenous leukemia cells. Leuk Lymphoma,2004,45:373-6
[10] Wang X, Matta R, Shen G. Mechanism of triptolide-induced apoptosis: Effect on caspase activationand Bid cleavage and essentiality of the hydroxyl group of triptolide. J Mol Med,2006,84:405-15
[11] Soundararajan R, Sayat R, Robertson GS. Triptolide: An inhibitor of a disintegrin andmetalloproteinase10(ADAM10) in cancer cells. Cancer Biol Ther,2009,8:2054-62
[12] Zhou ZL, Yang YX, Ding J. Triptolide: structural modifications, structure-activity relationships,bioactivities, clinical development and mechanisms. Nat Prod Rep,2012,29:457-75
[13] Chang HJ, Kim MH, Baek MK. Triptolide inhibits tumor promoter-induced uPAR expression viablocking NF-kappaB signaling in human gastric AGS cells. Anticancer Res,2007,27:3411-7
[14] Xu B, Guo X, Mathew S. Triptolide simultaneously induces reactive oxygen species, inhibitsNF-kappaB activity and sensitizes5-fluorouracil in colorectal cancer cell lines. CancerLett,2010,291:200-8
[15] Geng Y, Fang M, Wang J. Triptolide down-regulates COX-2expression and PGE2release bysuppressing the activity of NF-kappaB and MAP kinases in lipopolysaccharide-treated PC12cells.Phytother Res,2012,26:337-43
[16] Matsui Y, Watanabe J, Ikegawa M. Cancer-specific enhancement of cisplatin-induced cytotoxicitywith triptolide through an interaction of inactivated glycogen synthase kinase-3beta with p53.Oncogene,2008,27:4603-14
[17] Basanez G, Soane L, Hardwick JM. A new view of the lethal apoptotic pore. PLoS Biol,2012,10:e1001399
[18] Galluzzi L, Kepp O, Kroemer G. Caspase-3and prostaglandins signal for tumor regrowth in cancertherapy. Oncogene,2012,31:2805-8
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[22] Lee YS, Dutta A. MicroRNAs in cancer.Annu Rev Pathol,2009,4:199-227
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[24] Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T.MicroRNA-21regulatesexpression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology,2007,133(2):647-58
[25] Zhang X, Liu S, Hu T, Liu S, He Y, Sun S. Up-regulated microRNA-143transcribed by nuclear factorkappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression. Hepatology,2009,50(2):490-9
[26] Pineau P, Volinia S, McJunkin K, Marchio A, Battiston C, Terris B, Mazzaferro V, Lowe SW, CroceCM, Dejean A.miR-221overexpression contributes to liver tumorigenesis. Proc Natl Acad Sci U S A,2010,107(1):264-9
[27] Kota J, Chivukula RR, O'Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, Chang TC,Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT.Therapeutic microRNA deliverysuppresses tumorigenesis in a murine liver cancer model. Cell,2009,137(6):1005-17
[28] Rossi JJ. New hope for a microRNA therapy for liver cancer. Cell,2009,137(6):990-2
[29] Alisi A, Balsano C. Enhancing the efficacy of hepatocellular carcinoma chemotherapeutics withnatural anticancer agents.Nutr Rev,2007,65:550-3
[1]1.Poon D, Anderson BO, Chen LT, Tanaka K, Lau WY, Van Cutsem E, Singh H, ChowWC, Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet,2003,362:1907-17
[2] Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet,2012,379:1245-55
[3] Schuppan D, Afdhal NH. Liver cirrhosis. Lancet,2008,371:838-51
[4] Okuda K. Hepatocellular carcinoma. J Hepatol,2000,32:225-37
[5] Schafer DF, Sorrell MF. Hepatocellular carcinoma. Lancet,1999,353:1253-7
[6] Arzumanyan A, Reis HM, Feitelson MA. Pathogenic mechanisms in HBV-and HCV-associatedhepatocellular carcinoma. Nat Rev Cancer,2013,13:123-35
[7] Koo HS, Kang SD, Lee JH. Triptolide Inhibits the Proliferation of Immortalized HT22HippocampalCells Via Persistent Activation of Extracellular Signal-Regulated Kinase-1/2by Down-RegulatingMitogen-Activated Protein Kinase Phosphatase-1Expression. Journal of Korean NeurosurgicalSociety,2009,46:389-96
[8] Liu Q. Triptolide and its expanding multiple pharmacological functions. InternationalImmunopharmacology,2011,11:377-83
[9] Lou YJ, Jin J. Triptolide down-regulates bcr-abl expression and induces apoptosis in chronicmyelogenous leukemia cells. Leuk Lymphoma,2004,45:373-6
[10] Wang X, Matta R, Shen G. Mechanism of triptolide-induced apoptosis: Effect on caspase activationand Bid cleavage and essentiality of the hydroxyl group of triptolide. J Mol Med,2006,84:405-15
[11] Soundararajan R, Sayat R, Robertson GS. Triptolide: An inhibitor of a disintegrin andmetalloproteinase10(ADAM10) in cancer cells. Cancer Biol Ther,2009,8:2054-62
[12] Zhou ZL, Yang YX, Ding J. Triptolide: structural modifications, structure-activity relationships,bioactivities, clinical development and mechanisms. Nat Prod Rep,2012,29:457-75
[13] Chang HJ, Kim MH, Baek MK. Triptolide inhibits tumor promoter-induced uPAR expression viablocking NF-kappaB signaling in human gastric AGS cells. Anticancer Res,2007,27:3411-7
[14] Xu B, Guo X, Mathew S. Triptolide simultaneously induces reactive oxygen species, inhibitsNF-kappaB activity and sensitizes5-fluorouracil in colorectal cancer cell lines. CancerLett,2010,291:200-8
[15] Geng Y, Fang M, Wang J. Triptolide down-regulates COX-2expression and PGE2release bysuppressing the activity of NF-kappaB and MAP kinases in lipopolysaccharide-treated PC12cells.Phytother Res,2012,26:337-43
[16] Matsui Y, Watanabe J, Ikegawa M. Cancer-specific enhancement of cisplatin-induced cytotoxicitywith triptolide through an interaction of inactivated glycogen synthase kinase-3beta with p53.Oncogene,2008,27:4603-14
[17] Basanez G, Soane L, Hardwick JM. A new view of the lethal apoptotic pore. PLoS Biol,2012,10:e1001399
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