中科院两PI接连发表JBC，PLoS文章

　　

生物通报道：来自中科院上海生科院生化与细胞所的季红斌研究组与刘新垣研究组，接连在JBC，以及Plos One杂志上发表文章，分别解析了肿瘤细胞中miR-143-HK2表达失调的分子新机制，以及K-ras突变体在肺癌发生、发展及存活中的重要作用。

在第一篇“miR-143 regulates cancer glycolysis via targeting hexokinase 2”文章中，研究人员揭示了miR-143通过靶向己糖激酶2（HK2）参与调控肿瘤细胞的糖代谢，并阐明了肿瘤细胞中miR-143-HK2表达失调的分子机制。

肿瘤细胞即使在有充足氧气的情况下，仍倾向于通过糖酵解途径提供能量，这就是普遍存在于肿瘤细胞中的“沃伯格效应”，而系统性地寻找并鉴定参与调控这一效应的miRNA还未有报道。由于miRNA对靶基因的调控存在降解mRNA和抑制蛋白翻译两种途径，目前常规的生物信息学分析方法无法利用基因表达谱数据发现那些主要在蛋白翻译水平被miRNA调控的基因。

为解决这一问题，研究人员基于前期工作中构建的一个功能相关基因的信息网络（gNET），通过特定基因“活性”的变化来代替该基因的表达变化，来整合性地分析肿瘤miRNA和基因表达谱数据，发现了一系列在肿瘤中失调的“miRNA-糖酵解酶基因”配对，包括糖酵解中第一个限速酶HK2与miR-143。

有意思的是，在肿瘤细胞株以及临床肺癌样本中的研究结果都表明miR-143对HK2的表达调控主要发生在蛋白水平，而对其mRNA的影响较小，验证了这一生物信息学新方法的优越性；miR-143可以通过下调HK2的表达抑制肿瘤细胞葡萄糖代谢，细胞增殖，锚定非依赖生长和裸鼠皮下成瘤的能力；进一步研究还发现，miR-143在mTOR信号通路下游介导了其对HK2的调控。该研究不仅为寻找肿瘤中表达失调的miRNA-靶基因配对提供了新的预测方法，而且深化了我们对于miRNA生物学功能的认识。

另外一篇文章（Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele）则是由这两个研究组与美国哈佛大学Dr. Kwok-Kin Wong合作完成。

肺癌是严重危害我国人民生命健康的重大疾病之一，揭示其中关键的致病基因在肺癌发生发展及存活中的作用将为临床上肺癌的“个体化”分子靶向治疗提供潜在的药靶和新的策略；而可时空调控基因表达的小鼠模型能够更好地促进肺癌发病分子机理的研究。目前国际上应用最广泛的肺癌动物模型就是 LSL-K-rasG12D小鼠模型，可以通过鼻腔滴入携带Cre表达基因的腺病毒或与肺上皮细胞特异性的Cre转基因小鼠杂交来实现K-ras突变体的激活，从而导致肺癌的发生；而该模型唯一的缺点就是K-ras突变体一旦激活，就无法调控其表达和活性。

在这篇文章中，研究人员在LSL-K-rasG12D小鼠模型基础上，构建了一个可时空调控K-ras突变体表达的小鼠模型LSL-ER-K-rasG12D；该小鼠的ER-KrasG12D的表达可被CRE诱导， 而ER-K-rasG12D蛋白的激活可通过Tamoxifen来调控。在小鼠胚胎成纤维细胞中的研究表明Tamoxifen处理可以诱导ER-K-rasG12D的激活促进细胞异常增殖，恶性转化以及侵袭和浸润；而Tamoxifen 去除后，小鼠胚胎成纤维细胞增殖、恶性转化及侵袭能力可基本恢复到正常水平；动物体内研究发现通过腹腔注射小鼠Tamoxifen可持续激活K-rasG12D并促进小鼠肺部肿瘤的早期发生，而Tamoxifen撤掉后大部分的肺部肿瘤会发生细胞凋亡。

这在很大程度上提高了我们对K-ras突变体在肺癌发生、发展及存活中作用的认识，并为将来深入研究肺癌发病分子机理提供一个较为理想的研究体系，对新型肺癌小鼠模型的建立和发展具有重要意义。

（生物通：万纹）

原文摘要：

miR-143 regulates cancer glycolysis via targeting hexokinase 2

High glycolysis, well-known as "Warburg effect", is frequently observed in a variety of cancers. Whether the deregulation of miRNAs contributes to the "Warburg effect" remains largely unknown. Since miRNA regulates gene expression at both mRNA and protein levels, we constructed a gene functional association network (gNET), which allows us to detect the gene activity instead of gene expression, to integratively analyze the microarray data for gene expression and miRNA expression profiling and identify glycolysis related gene-miRNA pairs deregulated in cancer. Hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis, is among the top list of genes predicted and potentially regulated by multiple miRNAs including miR-143. Interestingly, miR-143 expression was inversely associated with HK2 protein level but not mRNA level in human lung cancer samples. MiR-143, downregulated by mammalian target of rapamycin (mTOR) activation, reduces glucose metabolism and inhibits cancer cell proliferation and tumor formation through targeting HK2. Collectively, we have not only established a novel methodology for gene-miRNA pair prediction but also identified miR-143 as an essential regulator of cancer glycolysis via targeting HK2.

Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-rasG12D (ER-K-rasG12D) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-rasG12D independent of the gender status. Furthermore, tamoxifen-inducible activation of K-rasG12D promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-rasG12D in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.