莱菔硫烷调控肺腺癌干细胞的miRNA特征及其机制研究

Sulforaphane (SFN) is a natural compound converted from glucoraphanin, a major glucosinolated in broccoli/broccoli sprouts. It is traditionally applied as a drug that can prevent cancer because of its function of inhibiting phase 1 metabolism enzymes that convert procarcinogens to carcinogens and inducing phase 2 metabolism enzymes that promote excretion of carcinogens. Subsequent studies revealed that SF could modulate diverse cellular activities, such as inhibiting the growth of cancer cells, inducing apoptosis and cell cycle arrest and suppressing angiogenesis and metastasis by different signaling pathway.In recent years,many researchers found that SFN could suppressed self-renewaling and proliferation of cancer stem cells in some kind of tumors,such as breast cancer and lung cancer.SFN particularly suppressed CSCs in a lower concentration,compared to that in cancer cells.More importantly,when SFN was withdrew, it maintained the role of suppression in CSCs. Our previus study also provieded the similar evidence in lung cancer.it suggested that SFN could play action by derect and inderect ways.. MicroRNAs (miRNA), 21–23 nucleotide non-protein coding RNAs, act as powerful regulators of gene expression at the post-transcriptional level by targeting specific mRNA degradation or by suppression or activation of translation. Accumulating evidences have demonstrated that miRNAs influence a wide range of biological processes including development, lifespan, metabolism, and tumorigenesis. Transcription of miRNA gene is regulated at multiple levels in response to differentiation and mitogenic signaling. Some miRNAs are characteristic of specific differentiated cell types, whereas others are specifically expressed in stem and progenitor cells during early development. Recent evidence has shown that miRNAs can function as tumor suppressors or oncogenes and were involved in the differentiation and quiescence of CSCs in various types of tumors.. In previous studies,we found that SFN via miR-214 suprressed lung cancer stem cell.But SFN as small molecular natural compound,how it lead to changes of miRNAs and targeted genes remains unknown.We supposed that SFN couled suppress cancer stem cells by up-regulating exppression of miR-214 and/or facilitating the combination of miR-214 and its targeted genes..In this research, we will use the DNA chips and miRNA chips to screem the targets of SFN and miR-214. then we constructe dual fluorescence vectors which contain different length of sequences related to miR-214 or its targeted genes. By dual florescence experiments,we will find out the potential binding sequences of SFN and miR-214, understand how SFN faciliate the binding of miR-214 and targeted sequences. By these studies, we will elucidate the interaction of SFN、miR-214 and their targeted sequences, and provide a new way to explore miRNA drugs and tumor therapy.

莱菔硫烷（SFN）是一种经典的肿瘤预防药物，其抑癌功效同样强大。研究表明，SFN不仅能抑制肿瘤细胞的增殖，还能特异地抑制肿瘤干细胞的自我更新和增殖。miRNA是细胞信号调控的关键成员。我们的研究证实，SFN可引起多种miRNA转录变化，通过miR-214抑制肺癌干细胞的增殖。目前miRNA的调控机制目前仍不清楚。我们推测SFN可能通过未知方式调控miRNA转录，从而抑制肿瘤干细胞增殖。本研究拟采用DNA芯片和miRNA芯片技术，分析SFN对miRNA的影响，使用特异性miRNA拮抗剂研究其与其他miRNA及重要干性基因的关系。分析SFN对重要miRNA启动子甲基化、p53及Lin-28等的影响。应用shRNA技术和荧光报告基因技术，分析SFN在miRNA序列中的潜在作用靶点和作用通路，阐明SFN、miRNA以及相关靶点之间的关系，为miRNA小分子药物的研究及应用提供实验依据。

莱菔硫烷（SFN）是从绿花椰菜或绿花椰菜幼芽中提取的一种天然化合物，传统上用于预防或减少肿瘤发生。文献研究表明，除了解毒、减毒功能外，莱菔硫烷通过多种信号通路调控肿瘤细胞增殖和肿瘤干细胞增殖等。同时，SFN还能引起多种miRNA的转录水平变化。本研究通过肿瘤球形成实验富集肿瘤干细胞，应用全miRNA测序、甲基化PCR技术等，分析SFN调控miRNA转录变化的特征及其潜在分子机制。结果表明，在较低浓度下SFN能有效降低A549细胞中ALDH+细胞比例，降低H460细胞肿瘤球中的侧群细胞（肿瘤干细胞）比例以及肿瘤球形成能力，同时也这两种细胞的Nanog、c-Myc、Sox-2、Oct、β-catenin等肿瘤干细胞相关信号也呈浓度依赖性的变化。进一步分析表明，SFN能诱导肺腺癌细胞株的一系列miRNAs变化。对莱菔硫烷处理A549和H460细胞前后的全系列miRNA进行了高通量测序与分析，结果如下：莱菔硫烷A549肿瘤球(TSSF)与肿瘤球空白组（TScon）比较，有39种miRNA显著增高，而38种miRNA显著下降；莱菔硫烷H460肿瘤球(TSSF)与肿瘤球空白组（TScon）比较，有39种miRNA显著增高，而38种miRNA显著下降。这些显著变化的miRNA的靶向基因涉及细胞周期、生存、肿瘤转移、干性维持等众多功能。应用RT-PCR技术验证与肺腺癌相关的一些miRNAs(miR-214、127、200c、199b、663a、545)转录变化时结果与测序结果类似。对miR-200c的变化机制分析表明，SFN可能通过抑制DNMT1和 DNMT3a，从而下调miR-200c启动子序列的甲基化水平，进而上调miR-200c的转录水平 ，达到抑制肿瘤干细胞增殖的作用。而在miR-214启动子甲基化的分析中并未发现甲基化水平变化。这些结果提示SFN除了经基因序列甲基化调节来调控miRNA转录水平外，可能存在其他调控机制，如组蛋白乙酰化。本研究提示SFN可通过多种机制调控肺腺癌肿瘤干细胞的增殖，作为一种多通路调节子，有望成为一种新型抗肿瘤药物。