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siRNA, miRNA, and shRNA: in vivo Applications
P.N. Pushparaj1,*,3,
J.J. Aarthi1,
J. Manikandan1 and
S.D. Kumar2,*
1 Department of Physiology, National University of Singapore, Singapore; and
2 Department of Anatomy, National University of Singapore, 2 Medical Drive, MD9 #01-05, Singapore 117597
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Figure 1. Mechanism of RNAi. (A) Synthetic siRNAs and shRNAs hypothetically represent perfect drugs for the specific blocking of unwanted or disease-causing gene products. These RNA sequences can be tailored to block just about any gene. They bind and then degrade the mRNA produced by the gene before that mRNA can start producing a harmful protein, which actually causes the illness. In contrast, miRNAs are being produced or coded by genes inherently within a cell and control the expression of various genes involved in key cellular processes. (B) The RNAi pathway involves the introduction of a long ‘triggering’ dsRNA with 2-nucleotide overhangs at the 3' terminal end that is subsequently processed into 21- to 25-bp small interfering RNAs (siRNA) by an RNaseIII-like enzyme called Dicer. The siRNAs are unwound by a helicase before entering into a multi-subunit RNA-induced silencing complex (RISC), and the strand complementary to the target mRNA is integrated into RISC. An endonuclease present within the RISC either degrades or inhibits translation of specific mRNA transcripts. In contrast, synthetic siRNAs (generally 21–25 bp long) skip the Dicer cleavage, directly unwind by helicase, and enter into the RISC for the specific blocking of mRNA transcripts.
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Figure 2. RNAi-mediated knockdown of splicing isoforms. (A) Bcl-xL-specific siRNA silences Bcl-xL protein and inhibits the proliferation of 5-fluorouracil and tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL)-resistant cells. (B) RNAi-mediated silencing of human growth hormone (hGH) splice variant without exon 3.
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Figure 3. Biogenesis of miRNA. (A) The biogenesis of miRNA involves several enzymatic steps. Following transcription by RNA polymerase II (Pol II), capped and polyadenylated primary miRNA transcripts (pri-miRNAs) are processed in the nucleus by the endonuclease Drosha into one or more pre-miRNAs (1). This pre-miRNA is exported from the nucleus to the cytoplasm and processed by another RNase enzyme called Dicer, which produces a transient 19- to 24-nucleotide duplex (2). The duplex is cleaved (3), and only one strand of the miRNA duplex (mature miRNA) is incorporated into the RISC (RNA-induced silencing complex), which retains only the single-stranded mature miRNA (4). This miRNA-programmed RISC negatively regulates the stability and/or translation of target mRNAs, depending on the degree of complementary sites between the miRNA and its target. (B) In the proposed model, amplification or overexpression of a miRNA that down-regulates a tumor suppressor or other important genes involved in differentiation might also contribute to tumor formation by stimulating proliferation, angiogenesis, and invasion. For example, amplifications of the oncogenic miRNAs, miR-17–92 cluster, miR-21, and miR-372 have been clearly associated with tumor initiation and progression, whereas miRNAs that normally down-regulate an oncogene can act as a tumor suppressor gene when lost in a tumor. Any abnormalities in miRNA biogenesis might result in an abnormal expression of the target oncogene, which subsequently contributes to tumor formation.
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Figure 4. Schematic representation of non-viral in vivo transfecting agent-mediated transfer of siRNA duplex for functional genomics and therapeutics. The transfecting agents are useful for both local and systemic delivery of siRNA, since most of the siRNA-transfecting agent complexes are stable in vivo. Subsequently, for the evaluation of the in vivo efficacy and stability of siRNAs, Western blot/RT-PCR/Q-PCR/flow-cytometry/non-invasive bio-imaging can be adopted. Based on the degree of knockdown, the specific siRNA sequence can be used to silence the specific disease-causing gene in various animal models of disease/disorder.
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Figure 5. The disease-causing genes in humans can be identified by molecular diagnosis, and the treatment testimonial could be formulated based on the expression analysis in an RNAi chip for specific genes using computational analysis in a typical clinical milieu.
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Journal of Dental Research, Vol. 87, No. 11,
992-1003 (2008)
DOI: 10.1177/154405910808701109
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