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Introduction of RNA interference

• RNA interference (RNAi), as commonly defined, is a phenomenon leading to post-transcriptional gene silencing (PTGS)

• Or in other words RNAi is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.

• RNA interference most likely evolved as a mechanism for cells to eliminate unwanted foreign genes.

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Introduction of RNA interference

• It is presumed that cells employ RNAi to tightly regulate protein levels in response to various environmental stimuli.

• Although RNAi as a mechanism of PTGS was first discovered in plants and may have evolved as a cellular defense mechanism against foreign DNA and RNA, it is very clear that RNAi is widely employed in most if not all eukaryotic cells as a mechanism to regulate the expression of endogenous genes.

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A diagram showing at which stages in the DNA-mRNA-protein pathway expression can be controlled.

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History of RNAi• PTGS and RNAi were discovered in genetic

transformation studies of eukaryotic cells (principally plants and worms).

• RNAi was discovered unexpectedly, when experimental manipulation of the expression of specific genes: Inhibiting antisense in C.elegans

• In 1998, it was discovered that injection of dsRNA was much more effective for silencing of gene expression in C. elegans than was single-stranded antisense RNA.

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History of RNAi• Collectively, the studies of A. thaliana and

C.elegans showed that both TGS and PTGS can be initiated by the same RNA degradation pathway.

• TGS occurs when the dsRNA includes promoter sequences, whereas PTGS occurs when the dsRNA includes coding sequences.

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History of RNAi

• Other organisms were assayed for their capacity to induce RNAi:

• Drosophila: Kenner dell and coworkers who showed the involvement of the frizzled and frizzled2 genes in the wingless pathway after introduction of dsRNA into embryos.

RNAi is an ATP-dependent and translation-independent event where the introduced dsRNA is processed into 21–23 nucleotide fragments that guide the cleavage of endogenous transcripts.

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History of RNAi

• Yeast: (To study the functions of RNAi ) Volpe et al. deleted Argonaute, dicer, and RNA-dependent RNA polymerase homologs.

• In mammalian cells: RNAi was first employed as a tool to induce the silencing of the targeted gene.

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History of RNAi

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• RNAi mechanisms may provide explanations for various biological phenomena that were previously described, but without any understanding of the possible underlying mechanism.

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Specialized enzymes cutter

• Drosha: RNase III , Create two slices, primary miRNA with 70-65 nucleotides in length

• Dicer: Being part of the RNase III family, Dicer cleaves dsRNA and pre-microRNA

Functional domains:RNase III domainsPAZ domain

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human Dicer

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FUNCTION OF DICER

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Mechanism of RNAi

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Pathways of RNAi

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• A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses,

• which functions in RNA silencing and post-transcriptional regulation of gene expression

Introduction of miRNA

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Introduction of miRNA

• The first miRNA was discovered in 1993 by Victor Ambros, Rosalind Lee and Rhonda Feinbaum during a study of the lin-4 gene, which was known to control the timing of C. elegans larval development by repressing the lin-14 gene.

• The let-7 RNA was soon found to be conserved in many species, leading to the suggestion that let-7 RNA and additional "small temporal RNAs" might regulate the timing of development in diverse animals, including humans.

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Classification of miRNAS

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Overview of practice miRNA (or siRNA)

• miRNA suppresses translation of specific mRNA

• It seems that the RNAi agents involved in heterochromatin silencing.

• practice miRNA :1-mRNA degradation2-Inhibiting translation of mRNA3-Induced modifications of chromatin

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Applications of RNA Interference in Biology and Medicine

• control of gene expression In the post-transcriptional

• Evaluate the function of individual genes familial gene in different cells

• Phenotyping results of a gene or gene family

• Studied several unrelated genes and the effect of epistasis

• Study of human genetic diseases

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Applications of RNA Interference in Biology and Medicine

• treatment of Communicable disease

• Identification of enzymes and proteins involved in various physiological responses

• biology of stem cells

• treatment of infectious, fungal, parasitic and viral diseases

• Treatment of Fatal viral disease

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The biological role of miRNA

• miRNA , apoptosis and cancer:miRNA play a role in the regulation of apoptotic pathways.

• The first miRNA associated with apoptosis:miR-14 and bantam Which were discovered in Drosophila

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CLASSIFICATION OF APOPTOTICGENES

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GENES OF APOPTOSIS

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Therapeutic applications of RNAi:

• Cancer:

There are two general abnormalities in cancer cells: they exhibit dysregulation of the cell cycle resulting in uncontrolled growth and they are resistant to death as a result of abnormalities in one or more proteins that mediate apoptosis.

• The goals for RNAi approaches for cancer therapy are therefore to knock out the expression of a cell cycle gene and/or an anti-apoptotic gene in the cancer cells thereby stopping tumor growth and killing the cancer cells.

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Therapeutic applications of RNAi:

• Infectious Diseases:

• suppression of HIV-1 replication in human cells transfected with siRNA directed against tat and the rev gene.

• Transfection of human cells with siRNAs directed against different genes in the poliovirus genome resulted in resistance of the cells to infection with poliovirus.

• The ability of siRNAs targeting the gene encoding the death receptor Fas to protect liver failure and fibrosis in two models of autoimmune hepatitis was tested by Song and colleagues.

• Intravenous injection of Fas siRNA specifically reduced Fas protein levels in the livers of mice during a 10-day period

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Therapeutic applications of RNAi:

• Cardiovascular and Cerebrovascular Diseases:

Atherosclerosis involves damage to vascular endothelial cells, local production of inflammatory cytokines, and the recruitment of macrophages to the site forming foam cells; in addition, apoptosis of foam cells and vascular smooth muscle cells occurs. Although some of the cells die rapidly by necrosis, many other cells die more slowly by apoptosis.

• The production of cell adhesion molecules can be selectively suppressed in cultured cells.

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Therapeutic applications of RNAi:• Neurodegenerative Disorders:

Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis are examples of relatively common age-related neurodegenerative disorders that are increasing as average life expectancy increases.

• two different strategies for preventative and therapeutic interventions in neurodegenerative disorders:

One strategy is to block the disease-specific events that are believed to initiate the neurodegenerative process,

whereas the second strategy targets downstream events in the neurodegenerative cascade.

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miRNA as a marker

• Marker in cancer

• Fragile x mental retardation syndrome

• The role of miRNA in MS:miR326 ,miR26a

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Marker in cancer

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MICRORNA DYSREGULATION IN MULTIPLE SCLEROSIS

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The Future of RNA Interference in Biology and Medicine

• Even at this early stage of understanding the molecular mechanisms of RNAi and in the development of methods for the use of RNAi technology for selective gene silencing, it is clear that RNAi will be a widely used tool for establishing the functions of genes.

• The ability to selectively deplete a single protein of interest in cultured cells using siRNAs, and plasmids and viral vectors, is now established.

• many potential gene targets for therapeutic intervention using RNAi.

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Department of Genetics, Harvard University

Genetics is not perceived simply as a subject, but rather as a way of viewing and approaching biological

phenomena.

References1. Anghesom Ambesajira,Atul Kaushik,Jeevan J. Kaushik,Sham T. Petros.

(2012). RNA interference: A futuristic tool and its therapeutic applications . Saudi Journal of Biological Sciences, 395–403.

2. Fischer L. Tan and James Q. Yin . (2014). APPLICATION OF RNAi TO CANCER RESEARCH AND THERAPY.

3. Fischer L. Tan, and James Q. Yin . ( 2005). APPLICATION OF RNAi TO CANCER RESEARCH AND THERAPY . Frontiers in Bioscience , 1946-1960.

4. Hannon, G. J. (2002). RNA interference. Nature , 244-251.

5. Kyoung Min Kim and Sung-Kil Lim. (2014). Role of miRNAs in bone and their potential as therapeutic targets. Current Opinion in Pharmacology, 133-141.

6. L. Sudarsana Reddy, V. Sarojamma and V. Ramakrishna. (2007). Future of RNAi in Medicine: A Review. World Journal of Medical Sciences 2, 01-14.

7. Luqing Zhao,Xue Chen,and YaCao. (2011). New role of microRNA: carcinogenesis and clinical application in cancer (Vol. 43). china: Acta Biochim Biophys .

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References1. Michael Karbiener,Marcel Scheideler. (2014). MicroRNA functions in

brite/brown fat—Novel perspectives towards anti-obesitys trategies. Computational and Structural Biotechnology Journal, 5.

2. Ming Gao, Hao Yin, Zhe-Wei Fei. (2013). Clinical application of microRNA in gastric cancer in Eastern Asian area. World J Gastroenterol, 2019-2027.

3. Mohammad Amin Honardoost,Abbas Kiani-Esfahani,Kamran Ghaedi,Masoud Etemadifar,Mansoor Salehi. (2014). miR-326 and miR-26a,two potential markers for diagnosis of relapse and remission phases in patient with relapsing–remitting multiple sclerosis. Gene544, 128-133.

4. Nelson C Lau and Eric C Lai. (2005). Diverse roles for RNA in gene regulation. GenomeBiology.

5. René F. Ketting,Sylvia E.J. Fischer,. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes & Dev.

6. S.Patrick Nana-Sinkam,Carlo M.Croce. (2011). Non-coding RNAs in cancer initiation and progression and as novel biomarkers. MOLECULAR ONCOLOGY , 483-491. 41

References

شهر 1. مرادي حسين صادقي، مصطفي بابك، شهر مرادي محمد محمدي، حسين) . زمستان چكيده قديمى رضا حميد در RNAنقش). 89بابك، شونده كد غير هاى

. بيمارى و ژن بيان سوم، تنظيم هزاره در .ژنتيك

.2) . پاييز قائدى كامران رهگذر، سهيال محمدى، و). 90عصمت ساختار بر مرورىميكرو . RNAعملكرد سرطان در آن نقش سوم و هزاره در .ژنتيك

.3 ) . پاييز پور وندرجب فاطمه دلوئي، نوري ريز). 90محمدرضا در RNAنقش ها . سرطان درمان و تشخيص آپوپتوز، ژن، بيان دانشگاه تنظيم پزشكي علوم مجله

اسالمي .161-151, آزاد

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