当前位置: 首页>>特色栏目>>营养课堂>>正文
【双语课堂】RNA干扰 - 细胞之主宰
2010-06-29 14:04 丁香园 biofish  发布范围:公开

Master of the Cell
by Judy Lieberman

RNA interference, with its powerful promise of therapy for many diseases, may also act as a mater regulator of most – if not all – cellular process.

One of the biggest surprises in biology in the past decades was the discovery that humans have about the same number of protein coding genes as a worm. That puzzling finding began to make sense when we realized that we were missing a big part of the picture: a lot of DNA is transcribed into RNA but never into proteins. The more we learn about these RNAs, the more we realize how much complexity they add. Some of these noncoding RNAs, called microRNAs because of their small size, interfere with protein expression by chopping up protein coding transcripts or inhibiting their translation into proteins. Their effect on cell fate and function is far wider than we initially thought. In recent years, it has become clear that microRNAs can act as master switches by regulating large networks of genes.
I came to work on microRNAs by a circuitous path. I started as a theoretical high-energy particle physicist, but after 8 years decided to go to medical school to do work that more directly helped people. As part of my medical training in hematology and oncology, I began a postdoc at MIT in the lab of Herman Eisen in the early eighties when molecular biology was just coming into its own: The T-cell receptor had just been discovered (work to which the Eisen lab contributed), and HIV was about to be identified as the cause of AIDS. With no therapy available then, AIDS patients died a truly gruesome death. The Eisen lab studied the cytotoxic T cells that were supposed to protect us against viral infections like HIV.
本人(作者Judy Lieberman)选择研究microRNA工作可谓是一波三折。起先我是一名高能粒子理论物理学家,但8年后决定改学医学,这样可更直接的帮助人们。在学习血液学和肿瘤学期间,我开始在麻省理工学院Herman Eisen实验室做博士后。这是在(20世纪)80年代早期,分子生物学开始独立成科:刚刚发现了T细胞受体(Eisen实验室也参与了),同时,HIV也即将被确认为AIDS的病原体。由于没有有效的治疗方法,AIDS患者死的很惨。Eisen实验室研究了细胞毒T细胞,它们被认为可以保护人类免受病毒的感染,如HIV。
After my postdoc, I was offered a job at Tufts–New England Medical Center that combined clinical work in hematology with running a lab. I decided that my new lab would work on understanding the T-cell response to HIV and why these cells fail to control the infection, with an eye towards developing immune-based therapy. We also investigated how cytotoxic T cells activate programmed cell death (apoptosis) in virally infected cells.
I was immersed in HIV and T-cell immunology work in 1998 when I read the Fire and Mello paper describing one of the first examples of RNA interference (RNAi) in C. elegans. I was intrigued and perplexed by the paper: how could a double-stranded RNA possibly silence gene expression? I would periodically ask a colleague working on worms, Keith Blackwell, if there was an explanation for this strange phenomenon.
1998年,当我还埋头于HIV和T细胞免疫的工作中时,读到了Fire和Mello的文章,他们首次描述了线虫中RNAi的现象。我被深深地吸引了,并且困惑不解:一段双链RNA怎么可能沉默一个基因的表达呢?于是,我经常询问一个研究蠕虫的同事——Keith Blackwell,期望他能解释这一奇怪的现象。
My curiosity was thus piqued when 3 years later Carl Novina, a postdoc in Phillip Sharp’s lab whom I had known when he was a graduate student at Tufts, came to me with the still-unpublished news that RNA silencing also functions in mammalian cells. Tom Tuschl had found that genes could be silenced by introducing small double-stranded RNAs into a cell. Carl wanted to find out if this method could be used to inhibit HIV infection of immune cells. The prospect was tantalizing. What if we could use small interfering RNA to block HIV infection in humans?
3年后,当Carl Novina(Phillip Sharp实验室的博士后,我在Tufts时认识他,当时他还是一个研究生)跑来给我看他准备发表的新闻(即哺乳动物细胞中也存在类似的RNA干扰现象)时,我的好奇心得到了极大的满足。Tom Tuschl已经发现细胞内一些基因可以被导入的一小段双链RNA沉默。Carl想探究这一方法能否被用于抑制HIV感染免疫细胞。想到我们如果能用这些小干扰RNA来阻断HIV感染人类,这前景太诱人了!
Because T cells—the natural target of HIV in the body—are difficult to transfect, we started by trying to block infection in an epithelial cell line that was engineered to express CD4 and CCR5, two receptors required for HIV infection. Transfecting the cells with a small interfering RNA (siRNA) designed to degrade the messenger RNA (mRNA) for CD4 indeed blocked HIV infection by 4- to 10-fold. Encouraged by these results, we used RNA silencing to target an HIV gene that encodes for the viral capsid, and found that we could knock down both the host mRNA and the viral mRNA within the host cell. Knocking down either gene could stop the spread of the infection in cell culture. It was no small achievement. Our paper was one of the first in the field to show the potential of RNAi in treating human disease.2 Of course, at the time, no one understood that RNAi is actually a very basic antiviral mechanism. Organisms, like plants and more primitive animals that don’t have adaptive immune systems, use RNAi to attack and degrade viral mRNAs. In retrospect, it made a lot of sense that our HIV experiment would work.
因为T细胞(HIV天然的靶细胞)很难被转染,起先我们利用改造的上皮细胞(能表达CD4和CCR5,HIV感染必需的受体)来尝试阻断效果,当转染一段针对CD4 mRNA的小干扰RNA后,HIV感染率真的下降了4~10倍!于是我们再接再厉,设计了针对编码HIV衣壳基因的siRNA,结果宿主和病毒的mRNA都下调了,并且,任何一个基因的下调均能阻止HIV在培养细胞中传播。这是一个很大的进展,这是首次发现siRNA有用于治疗人类疾病的潜力。当然,当时无人明白RNAi实际上是最基本的一种反病毒机制。一些没有适应性免疫系统的生物,如植物和那些更低等的动物,利用RNAi来攻击和降解病毒的mRNA。现在回想起来,我们做的这些HIV实验真是太有意义了!


RNA interference is much more than just a cell’s antiviral technique. This mechanism acts as a master regulator of gene expression, directing a cell’s response to developmental and environmental cues.
I was excited about translating our promising in vitro results into therapies, but in 2001 there were still no good small animal models of HIV infection. Without my knowledge, my postdocs, led by Erwei Song, decided to test the concept of using RNAi to protect against a different disease in mice—hepatitis.3 Two groups had shown that rapid intravenous injection of siRNAs in a large volume (so-called “hydrodynamic injection”) in mice was able to knock down expression of a simultaneously injected luciferase transgene in some organs in the mouse. The most effective knockdown was in liver cells. Using RNAi to prevent hepatitis might work. Erwei and his friends in China, who were skilled at performing the exceedingly tricky hydrodynamic injections, tried to silence a transcript for one of the caspases. This enzyme triggers programmed cell death in liver cells in virtually all forms of hepatitis. However, it didn’t work and all the mice died. When Erwei finally told me what had happened, I thought the approach was promising, but suggested trying to knock down a different target.
我非常希望我们那前景诱人的体外实验结果能转化成治疗方法,但在2001年,还没有理想的小动物作为HIV感染模型。在我不知情的情况下,我的博士后在Erwei Song领导下,决定先检验RNAi能否保护小鼠不感染肝炎病毒。两组小鼠在快速大量静脉注射(所谓“动力进样”)siRNA后,一些器官中带有荧光素酶的转基因出现下调。下调效果最明显的是肝细胞,表明RNAi可能真的能起保护作用。Erwei和他非常善于动力进样的中国朋友试图沉默一个caspase的转录本,实际上所有的嗜肝病毒都能利用这个酶启动肝细胞发生凋亡,但是,这次RNAi没有效果,且全部的小鼠都死亡了。最后当Erwei告诉我这些情况时,我肯定了他们的方法,但建议换一个靶基因。
Because of my research on apoptosis in the immune system and antiviral immunity, I knew that liver cell death in hepatitis, no matter what the cause, is triggered by activating a death receptor called Fas on liver cells. Infection with hepatitis B and C viruses, for example, does not kill liver cells directly. Rather, the inflammation they cause induces Fas expression on liver cells and attracts killer lymphocytes bearing the counter-receptor for Fas (called Fas ligand) to infiltrate the liver, where they attack Fas-bearing liver cells (see graphic above). Triggering Fas is the common pathway for liver damage. Therefore knocking down Fas at the beginning of the pathway seemed like a good idea.
After hydrodynamic injection of siRNAs to knock down expression of the Fas receptor, Fas expression was reduced by 80% throughout the liver. As a consequence, there was a dramatic reduction in liver-cell damage. The technique could prevent liver damage not only in models of chronic hepatitis, but also in an acute liver damage model, in which all mice normally die within 3 days.4 After knocking down Fas in the liver, most mice survived the lethal challenge and recovered. It was clear that knocking down the Fas receptor could potentially block damage from any kind of hepatitis insult. What was more impressive was how easy it was to get these experiments to work. Once we started looking at Fas, we finished all of the experiments in the study in a month or two. When things work that well, it gives you the sense that you’re looking at a really fundamental process, rather than a curious side pathway. I was very excited and optimistic that small RNAs could be the basis for a new type of drug.
Research soon emerged showing that developing RNAi drugs wouldn’t be quite so easy. The active small RNAs, called small interfering RNAs or siRNAs, mediate RNAi silence genes by binding to a matching messenger RNA (mRNA) sequence and cutting it. But researchers found that a single siRNA could silence other mRNAs—not just the ones being targeted. These off-target effects could arise from one of two mechanisms: siRNAs were either hitting unintended genes that share partial sequence complementarity, or they were triggering the intracellular immune sensors that recognize viral double-stranded RNAs, causing inflammation and widespread immune stimulation. These potential problems were rapidly addressed by others who found that chemical modifications of the siRNA sugar backbone could block most off-target effects without jeopardizing gene knockdown. The other obstacle, which is still a major problem, was the incredible difficulty getting cells to take up naked RNAs. Hepatocytes were relatively easy because the liver is the filtering organ of the body, with a rich blood supply that routinely takes up particulates.
We tried to address some of these issues while working on an RNAi-based microbicide to prevent sexual transmission of viral infection (and ultimately HIV) in mice. Because of the lack of a small animal model for HIV transmission at the time, we decided to first try to block herpes transmission in mice. We developed a way of getting siRNAs into epithelial cells by either mixing them with a transfection lipid used to introduce exogenous nucleic acids into cells in the lab or by adding a cholesterol tag to the end of the RNA sequence that allowed the RNA to be taken up into cells. The result: effective gene silencing of an epithelial cell receptor that the herpes virus uses to enter the cell. The method could actually protect mice from a lethal vaginal dose of HSV-2 without causing immune recognition of the siRNA.5 However, neither of these methods was effective at transducing the T cells that HIV infects; we are still testing ways to modify siRNAs that could prevent HIV transmission, with some promising leads.
Erwei Song finished his postdoc in my lab in 2004, and returned to China to work as a breast cancer surgeon. When I visited China in 2005 for the annual meeting of a US–Sino comprehensive HIV research program in Beijing that I had helped organize, Song came to see me with exciting news. He had been working at Sun Yat Sen University in Guangzhou on his own projects and told me that he had discovered a method for culturing cancer stem cells from breast tumors. At the time, the cancer stem-cell hypothesis, which posits that breast cancer is initiated by a rare population of cancer stem cells, was controversial (and still is), in part because these cells are hard to identify and because the mouse models for human cancer might not accurately reflect how cancers originate in a human. These cells are relatively resistant to current chemotherapy and radiation therapy. They survive after cancer therapy and replenish the cancerous mass, leading to relapse. Although some tumors may be formed by initiating cells that do not resemble stem cells, it is likely that stem cell–like tumor cells are more aggressive at forming tumors, with a higher likelihood of relapse and metastasis.
2004年,Erwei Song完成了在我实验室的博士后工作回到中国,成为一名乳腺外科医生。2005年,我来到北京参加中美HIV综合性研究计划的年会(本人也参与了组织工作),Song来见我并带来了令人兴奋的消息。他一直在广州中山大学做自己的项目并发明了培养乳腺肿瘤干细胞的方法。当时,肿瘤干细胞学说认为乳腺癌源自极小一部分肿瘤干细胞,这一提法至今还存在争论,其中部分是因为这些(肿瘤干)细胞很难鉴别,而且人类肿瘤的小鼠模型不能准确反应人类肿瘤起源的真实情况。相对来说,这些(肿瘤干)细胞对现今的化疗和放疗具有更强的抵抗能力,当化疗和放疗把普通的肿瘤细胞杀死后,这些(肿瘤干)细胞能存活下来,补充“空缺”,从而导致肿瘤复发。虽然某些肿瘤的起始细胞并不像干细胞,但具有干细胞样的肿瘤细胞有更强的侵袭性、更高的复发和转移率。

With his success at culturing these elusive cells, I couldn’t help but wonder what their microRNA expression profile looked like in comparison to other cells. microRNAs, the small endogenous RNAs that mediate RNAi naturally, were first identified in seminal studies by Victor Ambros and Gary Ruvkun in 1993, 5 years before the Fire and Mello paper. Their work suggested that small RNAs are instrumental in regulating development, as would later be confirmed by studies in several model organisms. I had a hunch based on this work that microRNA expression would be different in breast cancer stem cells than in more differentiated tumor cells or normal tissue and that it would change as the stem cells differentiated to form a tumor.
看到Song成功培养了这些难以捉摸的细胞(注:Song认为是肿瘤干细胞,但作者比较谨慎),我不禁想知道这些细胞的microRNA表达谱与其它细胞有何不同。microRNA是一类内源性的小RNA,能天然的介导RNA干扰,由Victor Ambros 和 Gary Ruvkun在1993年研究精液时首次鉴定出来,比Fire 和 Mello发现RNAi早了5年。Victor Ambros 和 Gary Ruvkun的研究提示小RNA在调节发育上是能起作用的,后来在其它模式生物中也确认了这一点。据此,我预感到乳腺肿瘤干细胞的microRNA表达谱将不同于分化更高的肿瘤细胞和正常组织细胞,并且这一表达谱在肿瘤干细胞发生分化、形成肿瘤的过程中会发生改变。
Our collaborative effort revealed that the breast cancer stem cells expressed far fewer microRNAs than their more differentiated counterparts. One family of microRNAs stood out: the let-7 family containing 11 related sequences in humans. let-7 is one of the most evolutionarily ancient microRNAs that Ruvkun’s lab had shown regulates the larval-to-adult transition in worms. The more we looked at this microRNA in functional assays, the more interesting it became. let-7 was not expressed in cancer stem cells, but its expression increased as the cell differentiated. When we infected cancer stem cells with a lentivirus expressing let-7, we could force their differentiation into treatment-susceptible cancer cells. Surprisingly, forced expression of let-7 also reduced the number of tumors formed in the mouse and reduced their metastases.
It appeared that we had found not only a potential therapeutic mediator, but also a factor that controlled cancer cell “stemness.” In fact, let-7 controlled a number of stem cell properties, including the ability to self-renew and differentiate into different cell types (or “multipotency”). It accomplished this task by regulating the expression of more than one gene. Frank Slack had previously identified the oncogene RAS as a target of let-7 and David Bartel’s group had identified another oncogene, HMGA2, as a target. We found that let-7 regulation of RAS contributed to loss of self-renewal, while knockdown of HMGA2 led to loss of multipotency. Our study suggested that let-7 might be a master regulator of defining cancer stem cell properties.6 Together with the earlier studies in worms, this suggested that let-7 might be a master regulator of “stemness” more generally. At the time, researchers regarded small RNAs as rheostats that fine tune gene expression and cellular function; they were thought to only make small adjustments in expression. When I wrote the let-7 paper and called it a “master regulator,” controlling the very identity of a stem cell, I was asked to change the wording. However, I was convinced that these small RNAs are more powerful than the field had acknowledged.
似乎我们不仅发现了一种潜在的治疗载体,而且发现了控制肿瘤细胞“干细胞化”的因子。实际上,let-7控制着一定数量的干细胞属性,包括自我更新和分化成不同细胞类型(多潜能分化)的能力,要做到这些,必须调控不止一个的靶基因。Frank Slack先前将癌基因RAS鉴定为let-7的靶基因,David Bartel鉴定了另外一个靶基因,也是癌基因,HMGA2。我们发现,let-7对RAS的调控使干细胞丧失自我更新能力,对HMGA2的调控促进多潜能分化。我们的研究提示let-7可能是一个肿瘤干细胞属性最主要的调控者。综合以前的蠕虫研究成果,let-7是“干细胞化”最主要调控者的结论可能更具有普遍意义,而当时研究人员认为小RNA只是对基因表达和细胞功能进行精细的调整(注:“量”的水平,作者的提法属于“质”的水平)。当我在一篇关于let-7的文章中将其称为控制干细胞本质属性的“主要调控者”时,我被要求改变这一称谓,但是,我确信这些小RNA的作用远远超出了我们当时对它的了解。
While we were examining microRNAs in breast cancer stem cells, we were also looking at their role in blood cell differentiation from immature progenitor cells—somewhat more familiar territory for me. We became especially interested in one microRNA—miR-24—that stood out because it is upregulated as multipotent blood cells differentiate into a wide variety of mature blood cells. These mature cells are no longer capable of proliferating. Introducing miR-24 into proliferating normal and tumor cells also stopped them from further cell division. To understand how miR-24 worked, we wanted to identify the genes it regulated. It was a challenge, as microRNAs are only ~22 nucleotides long and bind to their targets by matching their sequence to a sequence in the target mRNA. But it’s a loose match at best—not every base pair matches its complementary nucleic acid. The algorithms used to predict which mRNA targets will be regulated by a particular microRNA are based on sequence matching. This often identifies thousands of potential targets and sometimes misses important ones like the oncogene RAS as a target of let-7. The algorithms place a lot of emphasis on target mRNAs that contain an exact 7 or 8 nucleotide match in their 3′ untranslated region (UTR) to residues 2–8 of the miRNA, called the “seed” region. Instead, we looked at all mRNAs that were downregulated when miR-24 was expressed in cells that don’t normally express it. We found 248 downregulated mRNAs, which did not overlap much with those found by the prediction algorithms.
当我们在乳腺肿瘤干细胞中检测这些microRNA时,我也关注它们在血细胞分化过程中的作用,比较这是我的老本行(血液学)。我们逐渐对一个microRNA产生了特别的兴趣,miR-24,它之所以突出是因为它在多潜能血细胞分化成各种成熟细胞的过程中表达上调了。这些成熟细胞是没有增殖能力的,当把miR-24导入增殖中的正常细胞和肿瘤细胞时,它们都停止了分裂。为了弄清miR-24的作用机制,必须鉴定出它的靶基因,这是一个挑战。因为microRNA仅有22nt,它们与靶基因mRNA上的互补序列结合并不严格(容许有几个碱基不匹配),这样,基于序列匹配的预测microRNA靶基因的算法会产生数千个潜在靶基因(假阳性),并且有时会漏掉一些重要的靶基因(假阴性),如预测let-7的靶基因时就漏掉了RAS。这些算法特别重视靶mRNA 3’UTR区的7~8个碱基序列,它们与microRNA中2~8个碱基匹配,也称“种子”序列。相反,我们在细胞(平时不表达miR-24)中过表达miR-24后观察了所有microRNA,发现有248个下调的mRNA,与软件预测的靶基因比,并不重叠(注:实际的与预测的吻合性不好)
To make sense of the large list of potential targets and choose a small number of genes to test experimentally, we collaborated with Winston Hide, a bioinformatician at the Harvard School of Public Health. Not only did miR-24 suppress major transcription factors that regulate the cell cycle—E2F2 and MYC—it micromanaged the expression of many of the transcripts that E2F2 and MYC activated (see graphic above). Most of the downstream genes we looked at experimentally were regulated by miR-24 recognition of “seedless” complementary sequences.7 We now think that miR-24 is another example of a “master regulator” of the cell, which acts by directly suppressing the expression of many genes that act in interconnected pathways.
为了弄明白这一大堆的靶基因,必须先有目的的挑选几个来做实验,我们开始了与哈佛公共卫生学院从事生物信息学的Winston Hide合作。我们发现miR-24不仅抑制重要的调节细胞周期的转录因子——E2F2和MYC,也能对E2F2和MYC活化后许多转录本的表达进行微调。我们在实验中关注的下游基因供miR-24识别的是非“种子序列”。现在,我们认为miR-24是说明小RNA是细胞最主要调控者的又一个例子,能直接抑制信号互联网络中许多基因的表达。
Introducing microRNAs, such as let-7 or miR-24, that force cancer stem cells to differentiate or cause cells to stop dividing could be used for cancer therapy. Let-7 could make tumors more susceptible to standard cancer chemotherapy or radiation. Targeting cancer stem cells, especially, might address this highly malignant and refractory source of recurrent tumors. This is an approach we are now working on.
Jumping into RNAi research as it was just beginning has been extraordinarily rewarding. As I move into new fields, however, I’ve never given up on trying to understand the questions that I asked when I started my lab. Although I completely abandoned theoretical particle physics, I am still deeply involved in understanding how HIV manipulates and overcomes antiviral immunity, and how antiviral killer lymphocytes destroy their targets. As a physician, I hope that solving these questions and understanding how microRNAs work can be used to improve treatments for HIV and cancer. In my work on RNAi, and indeed during the 25 years I have been engaged in biomedical research, I have had one foot in basic research and the other in translational work, seeking to apply new understanding of biology to improving patient treatment. I am optimistic that RNAi will be harnessed to produce a new class of drugs to treat many diseases.



 联系我们 | 网站地图 | 返回首页 

第四军医大学军事预防医学系营养与食品卫生学教研室  地址:陕西西安长乐西路17号