Monday, February 07, 2011

siRNA knockdowns of ROR1 and FMOD

Small interfering RNAs (siRNAs) are a useful tool in finding out what genes do. Previously it was necessary to produce genetically engineered mice with particular genes 'knocked out' (knockout mice) in order to find out what genes did.

siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science.[Hamilton A, Baulcombe D (1999). "A species of small antisense RNA in posttranscriptional gene silencing in plants". Science 286 (5441): 950–2.] Shortly thereafter, in 2001, synthetic siRNAs were shown to be able to induce RNAi in mammalian cells by Thomas Tuschl, and colleagues in a paper published in Nature.[Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells". Nature 411 (6836): 494–988.] This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development. To me they sound like Nobel Prize candidates.

siRNAs have a well-defined structure: a short (usually 21-nucleotide) double strand RNA (dsRNA) with 2-nucleotide 3' overhangs on either end: Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. SiRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.

This technique has been used to look at two genes in CLL by Mellstedt's group in Sweden. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B-cells. It is located on chromosome 1p31.3 and codes for a 105 kDa protein. It is normally expressed in heart, lung and kidneys. It binds to Wnt5a in CLL cells.

Fibromodulin (FMOD) is a 59 kDA collegen binding protein coded for by a gene at 1q31.2 and is found in many types of connective tissue including cartilage, tendon, skin, sclera adn cornea. It is known to bind to TGF-beta.

Using siRNA knockdown the FMOD and ROR1 proteins were significantly reduced in CLL cells, and increased apoptosis occurred in CLL cells but not normal B cells.

Two more genes involved in apoptosis and possible targets in CLL.

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