博文
我的第一篇英文演讲稿（RNAi）
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Good morning, everyone.
Today I will show you RNAi and its therapeutic applications.
RNAi is a potent gene-silencing tool which is triggered by dsRNA. It is ubiquitous in Eukaryotes, but recent evidence implies that there is an RNAi-like mechanism in prokaryotes which is used in a defence system.
We all know Nobel Prize for Physiology or Medicine in 2006 is awarded to two American Scientists who invented RNAi. The two guys are showed in this picture，yes，they are young and RNAi is younger。Once a biotechnology is awarded Nobel Prize，it must be tested through several decades of years，such as gene knock-out。However，RNAi is an exception. It is so strong that nearly everyone in the world focuses on it. In both 2000 and 2002, RNAi is chosen as one of ten most significant biotechnologies by Science magazine. And now, RNAi technology has been used in many fields and all over the world. Before introducing the applications, let’s begin with the discovery of RNAi.
In 1990, Napoli wanted to deepen petunias’ flower pigmentation through introducing exogenous transgenes called CHS. But in fact it didn’t work. Instead, flowers showed variegated pigmentation as a result of corresponding gene-silencing. This phenomenon was termed as “co-suppression”. Later, similar phenomena were discovered in worms, fruit fly, yeast and mammals.
RNAi was proposed in C. elegans by Fire and Mello in1998. Let’s see this picture, progeny of worms with GFP-reporter were injected ds-control RNA or ds-gfpG RNA, leading to adult body having fully active GFP or only entire vulval musculature expressing active GFP. Yes, RNAi nearly made gfp gene silent. Fire and Mello also found that the power of dsRNA-induced RNAi was much bigger than antisense RNA-based strategies for inhibiting gene expression. Their works were very novel and significant, so they were well-deserved for the Nobel Prize. The paper published in Nature in 1998 played a very important role as a footstone, the authors of which included a Chinese name Xu Siqun.
Next, let’s give RNAi a definition. RNAi or RNA interference is a potent and ubiquitous gene-silencing mechanism in Eukaryotes, but recent evidence implied that there is a RNAi-like machnism in prokaryotes. RNAi is an evolutionarily conserved cellular defence process. it is of great importance for the regulation of gene expression, defending against viral infections, and keeping jumping genes under control. RNAi is a post-transcriptional gene silencing mechanism,besides; Histone methylaiton and heterochromatinization belong to pre-transcriptional gene silencing pathways.
This picture shows us a general model of RNAi pathway. Double-stranded RNA binds to a protein complex, Dicer, which belongs to RNaseIII and cleaves it into fragments that have about 21-23 nucleotides. Don’t ignore that Dicer is ATP-dependent. Then, these fragments are bound to another complex, RISC, which is RNAi-induced silencing-complex. Based on its potential helicase activity, the dsRNA fragment is unwound. One is eliminated but the other remains bound to the RISC complex and serves as a probe to detect mRNA molecules. When it pairs with a complementary mRNA, this mRNA will be degraded by RISC, and corresponding gene is silenced.
Here is another RNAi mechanism induced by miRNA. Long primary miRNA transcript is cleaved by Drosha (also RNaseIII) to 60-70nt stem loop or hairpin precursors .The Hairpin precursor is also simply called hpRNA. Then, hpRNA is exported to cytoplasm where Dicer chops the loop out to give the mature miRNA. The next step is common to siRNA, and once incorporated into RISC, miRNA will repress productive translation by pairing to partial complementary site of target mRNA. Please notice it, the mRNA is not degraded but translation is blocked. Compared with siRNA, miRNA-mediated gene-silencing is normally endogenous
After class, I have a short video on RNAi to show you, which will introduce RNAi more vividly.
After understanding what RNAi is, we should begin to discuss how to use RNAi. Till now, RNAi has been used in many fields, including crops quality and yield improvement, plant breeding, animal husbandry, gene function study, drug target determination, and so on. Japanese scientists had decreased the caffeine content in coffee through RNAi in 2003. Especially, RNAi play a very important role in functional genomics. High-throughput RNAi screen in mammalian cells will tell us more about functions of proteins in our bodies. This picture shows us a simple method to study the function of a special gene and determine the target of a special drug. Here we mainly present RNAi applications on gene therapy, in spite of which are now still in course of testing on mice and other mammals.
In 2006, an article in Science magazine reported that gene therapist Mark Kay of Stanford University in California, who wanted to treat hepatitis with RNAi technology, but ended in the deaths of dozens of mice, so it alarm us that we should be very careful when looking to RNAi as a treatment for HIV, cancer, neurological diseases, and more.
Then, let’s see the details.
HIV-1 utilizes the CD4 receptor and the chemokine receptors CCR5/CXCR4 for entry. Chemokine is a component which is necessary for immunization. Each of these receptors is a potential RNAi target to block viral entry. If HIV virus enter cell, it will integrate to host genome and replicate through reverse transcription. Instead of targeting the receptor or co-receptor of mRNAs, incoming genomic viral RNA is a potential target, as well as all classes of viral transcripts. RISC interacts with one of the siRNA and recognize complementary target RNA. After cleaving the target RNA, the RISC complex will recycle. But in fact, not all HIV viruses can be silenced, some will escape.
Before discuss RNAi in Cancer therapy, we first see one of the most important signaling pathways to control growth and proliferation of our cells called MAPK pathway, MAPK means mitogen-activated protein kinase. Through this pathway, growth factors can activate a tyrosine kinase receptor; this phosphorylates the membrane-associated SH2 domain-containing adapter GRB, making it a docking site for the GTP/GDP exchange factor SOS. SOS converts RAS-GDP to RAS-GTP, and this in turn activates Raf and the downstream enzymes, which culminates in the production of transcription factors. Nox1, NADPH oxidase1, indirectly stimulated by Ras, generates ROS that disturbs the growth control contributing to cell transformation. Ras is mutated to an oncogenic form in about 15% of human cancer, so if we use virus-mediated RNAi to inhibit this pathway in Ras level, Raf level or Nox1 level, the tumor can be suppressed stably.
This table shows us some putative siRNA targets against cancer. Melanoma is an aggressive skin cancer. The oncogene B-raf was found to be mutated in 59% of melanoma cell lines. In this context, retrovirus and lentivirus siRNAs have been designed to knock down the activating mutation B-raf in more than 60% of malignant melanomas. Besides, Cyclin E can be the target of liver cancer. bcl-2livin and survivin function as antiapoptosis, they can be the targets of adenocarcinoma. On the other hand, we can use RNAi to inhibit the growth of new blood vessels, in order to cut off the blood to tumors and kill them.
The possible therapeutic applications of RNAi for neurological diseases are broad, ranging form acquired diseases, such as viral infections, to purely genetic disorders. Of them, dominantly inherited neurodegenerative diseases, including Huntington’s disease and related polyglutamine disorders, familial Alzheimer’s disease, and so on. This picture show us three ways to inhibiting polyglutamine-protein expression with RNAi. One of them was done by Xia Haibin who now is in Shaanxi Normal University. Left: cells are transfected with preformed siRNA duplexes, generally in combination with a transfection reagent. The transfected siRNA becomes incorporated into the RISC following after entry, leading to degradation of target gene expression. Middle: transfected plasmid vectors must reach the nucleus for transcription of shRNA cassette (hpRNA). The shRNA is processed and exported to the cytoplasm where it is incorporated into the RISC for directed gene silencing. Right: viral vectors encoding siRNA or shRNA bind to the target cell via a receptor, followed by receptor-mediated endosmosis. Uncoating of the viral protein coat or fusion of the viral envelope with cellular membrane releases viral genome and allows trafficking to the nucleus.
To conclude, I want say that in America there were at least 6 biocompanies which are exploring RNAi for medicine before Nobel Prize was awarded to Fire and Mello in 2006. Sirna, in San Francisco, is the most famous biocompany, mainly focusing on RNAi medicine. Now, Sirna has been purchased by Merck in Germany. Yes, RNAi has a big potential in gene therapy. We can believe it but RNAi is still not fully safe. Human cancer can be treated used RNAi technology in the long run.
Thank you!
https://blog.sciencenet.cn/blog-200233-201608.html
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