**2.1 Mechanisms of siRNA**

SiRNA is a class of dsRNA molecules with 20-25 nucleotides in length. When dsRNA enters cells, it can be lysed into siRNA by Dicer enzyme. On the other hand, dsRNA proliferates in the presence of RNA-directed RNA polymerase (RdRP) and is then lysed in the presence of Dicer enzyme. Double-stranded siRNA is processed by Dicer into single-stranded siRNA, which can form a complex via binding with some related proteins. This complex can bind simultaneously with complementary mRNA, thereby inducing mRNA lysis.

SiRNA-induced RNAi is initiated from a cleavage program. Long dsRNA is first cleaved by Dicer into siRNAs in cytoplasm. The Dicer contains an N-terminal helicase domain, a PAZ(Piwi/Argonaute/Zwille)RNA domain, two RNAase III domains, and a dsRNA binding domain (dsRBD) (Bernstein *et al,* 2001). The RNA helicase domain sequence is highly stable and N-terminal and C-terminal are core components. The N-terminal has an ATP-binding and hydrolysis subunit, and the C-terminal contains a RNA-binding subunit. With these subunits, the RNA helicase domain can play a helicase function by making use of the energy provided by ATP (Ma *et al,* 2004). The PAZ domain of Dicer can specifically recognize and bind with the 5' phosphate bond with two nucleotide protrusions in the dsRNA3' end (Cordin *et al,* 2006). There is a long α-helix between the PAZ domain and the RNase III domain, and this helix is encircled by a conserved N-terminal protein, forming a platform domain consisting of an anti-parallel β-sheet and three α-helixes, and at both ends of the platform domain are hinge1 and hinge 2 as a polypeptide chain to hinge with the PAZ domain and the RNase III domain respectively. Of them, hinge1 contains a Pro-266 kink, which enables the PAZ domain to move a 5Å distance. Unlike hinge 1, hinge 2 does not have the link, but it can enable the RNase III domain to make a 5° rotation along the long axis of the Dicer molecule. Therefore, Dicer is able to adapt to substrates of different shapes by regulating the platform, hinge 1 and hinge 2 for the purpose of cleaving different substrates (Macrae *et al,* 2006). Each RNase III domain possesses two active centers, and each center has the activity of cleaving the phosphodiester bond, and can cleave dsRNA into small RNA with two nucleotide protrusions in the dsRNA3' end, which is the typical characteristic of the RNase III product (Collins&Cheng,2005). Although Dicer contains two RNase III domains and four active cleavage centers, each RNase III domain provides only one active center for cleavage. The distance between the active centers of the two RNase III domains is 65Å apart, equaling 25 bp RNA in length. This structural characteristic ensures Dicer to specifically produce small RNA of 21~25 bp in length, and enables it to change the length of the cleaved product by mirco-regulating the distance between the two RNase III domains (Blaszczyk *et al,* 2001). In the C-terminal of Dicer is the dsRBD that contains about 70 amino acids existing in the form of αβββα, of which the α-helix is close to one side of βsheet in a reverse and parallel form (Hallegger *et al,* 2006). dsRBD and dsRNA interact with each other across a width of 16 bp, which is just the distance between the two small adjacent grooves and the large groove on one side of α-dsRNA(Wu *et al,* 2004). This interacting region can be further divided into three small regions. Region 1: the four 2'-OH at the first

of dsRNA can induce RNAi, but only few types of dsRNA can lead to potent interference effect (Fire *et al,* 1998). The molecules that can trigger RNAi include small interference RNA (siRNA), microRNA (miRNA) and small hairpin RNA (shRNA). The mechanisms of RNAi

SiRNA is a class of dsRNA molecules with 20-25 nucleotides in length. When dsRNA enters cells, it can be lysed into siRNA by Dicer enzyme. On the other hand, dsRNA proliferates in the presence of RNA-directed RNA polymerase (RdRP) and is then lysed in the presence of Dicer enzyme. Double-stranded siRNA is processed by Dicer into single-stranded siRNA, which can form a complex via binding with some related proteins. This complex can bind

SiRNA-induced RNAi is initiated from a cleavage program. Long dsRNA is first cleaved by Dicer into siRNAs in cytoplasm. The Dicer contains an N-terminal helicase domain, a PAZ(Piwi/Argonaute/Zwille)RNA domain, two RNAase III domains, and a dsRNA binding domain (dsRBD) (Bernstein *et al,* 2001). The RNA helicase domain sequence is highly stable and N-terminal and C-terminal are core components. The N-terminal has an ATP-binding and hydrolysis subunit, and the C-terminal contains a RNA-binding subunit. With these subunits, the RNA helicase domain can play a helicase function by making use of the energy provided by ATP (Ma *et al,* 2004). The PAZ domain of Dicer can specifically recognize and bind with the 5' phosphate bond with two nucleotide protrusions in the dsRNA3' end (Cordin *et al,* 2006). There is a long α-helix between the PAZ domain and the RNase III domain, and this helix is encircled by a conserved N-terminal protein, forming a platform domain consisting of an anti-parallel β-sheet and three α-helixes, and at both ends of the platform domain are hinge1 and hinge 2 as a polypeptide chain to hinge with the PAZ domain and the RNase III domain respectively. Of them, hinge1 contains a Pro-266 kink, which enables the PAZ domain to move a 5Å distance. Unlike hinge 1, hinge 2 does not have the link, but it can enable the RNase III domain to make a 5° rotation along the long axis of the Dicer molecule. Therefore, Dicer is able to adapt to substrates of different shapes by regulating the platform, hinge 1 and hinge 2 for the purpose of cleaving different substrates (Macrae *et al,* 2006). Each RNase III domain possesses two active centers, and each center has the activity of cleaving the phosphodiester bond, and can cleave dsRNA into small RNA with two nucleotide protrusions in the dsRNA3' end, which is the typical characteristic of the RNase III product (Collins&Cheng,2005). Although Dicer contains two RNase III domains and four active cleavage centers, each RNase III domain provides only one active center for cleavage. The distance between the active centers of the two RNase III domains is 65Å apart, equaling 25 bp RNA in length. This structural characteristic ensures Dicer to specifically produce small RNA of 21~25 bp in length, and enables it to change the length of the cleaved product by mirco-regulating the distance between the two RNase III domains (Blaszczyk *et al,* 2001). In the C-terminal of Dicer is the dsRBD that contains about 70 amino acids existing in the form of αβββα, of which the α-helix is close to one side of βsheet in a reverse and parallel form (Hallegger *et al,* 2006). dsRBD and dsRNA interact with each other across a width of 16 bp, which is just the distance between the two small adjacent grooves and the large groove on one side of α-dsRNA(Wu *et al,* 2004). This interacting region can be further divided into three small regions. Region 1: the four 2'-OH at the first

simultaneously with complementary mRNA, thereby inducing mRNA lysis.

triggered by these molecules are not exactly the same.

**2.1 Mechanisms of siRNA** 

small groove of dsRBD N-terminal α-helix connect with five nucleotides of RNA. Region 2: a stem loop between β1and β2 of dsRBD connects with 2-5 nucleotides of RNA in the second small groove through 2'-OH. Region 3: the α-helix at the C-terminal of dsRBD travels across the large groove of RNA and connects with the phosphodiester bond in RNA (Ryter & Schultz, 1998).

The long dsRNA that enters cells is processed by Dicer into double-stranded siRNA, and then bind with Dicer, trans-activitor response region RNA-binding protein (TRBP), and protein kinase R (PKR)-activating protein (PACT) to form a RNA-induced silencing complex (RISC) (Hammond *et al,* 2000).

RNAi induced by completely processed single-stranded siRNA (mostly exogenous) can be directly installed into RISC without the participation of TRBP/PACT-Dicer complex. However, in other RNAi processes, RNAi cannot be installed into RISC without the participation of TRBP/PACT-Dicer complex. Through TRBP/PACT, siRNA-TRBP/PACT-Dicer complex binds with Argonaute 2 (Ago2/EIF2C2) to form RISC loading complex (RLC) (Robb&Rana,2007). RLC initiates disentanglement of siRNA and distinguishs the guide strand and the passenger strand. The guide strand 5'-end has the property of a low thermodynamic stability and can be bound into RISC by priority(Tomari *et al,* 2004). The passenger strand is cleaved by Ago 2 and separated from RLC (Matranga *et al,* 2005). The specific sequence for binding between the RISC-mediated RNA guide strand and target mRNA is mainly located in the "seed" or "core" region of 2-8 nucleotides of siRNA 5'-end (Ma *et al,* 2005). mRNA oligonucleotide is cleaved from the middle in the presence of Ago2 and loses the expression function (Schwarz *et al,* 2004; Hammond,2005).

Ago 2 is the only protein that has the endonuclease function in RLC, mainly consisting of an N-terminal domain, a PAZ domain, a middle domain and a PIWI domain, of which the Nterminal domain, middle domain and PIWI domain form a crescent basin structure, with one "arm" supporting the PAZ domain above the "basin" (Song *et al,* 2004;Yuan *et al,* 2005). This structure forms a groove carrying positive charge. The N-terminal domain and PIWI domain form the small groove, which can bind with nucleic acid. The PAZ domain and the crescent "basin" form the large groove, which can contain double strands and locate the easily broken phosphate bond directly to the catalytic site of Rnase H sheet on the PIWI domain. Ago 2 can melt siRNA, forming single-stranded antisense siRNA complementary to target mRNA. The Rnase H PIWI domain of Ago 2 is located in the 5'-end, and the PAZ domain can recognize siRNA3'-end. When siRNA binds with complementary target mRNA, Ago 2 begins digesting mRNA, forming RNA fragments with 5'-carboxylate and 3' phosphate to degrade mRNA of the endogenous target gene, thus preventing gene expression. During the process of degradation, mRNA comes in between the N-terminal and the PAZ domain and goes out between the PAZ domain and the middle domain. When mRNA moves between the PAZ domain and the "crescent basin", it is cleaved by the active site of the PIWI domain.

RISC can be recycled. When a target mRNA is degraded, RISC can continue to degrade other target mRNA, thus amplifying the PTGS effect of the target gene (Gregory *et al,* 2005) and this amplification can maintain for few days in dividing cells. Obviously, it is advantageous over other therapeutic methods, and is a potent method for specific inhibition of gene expression. In addition, a few studies(Meister *et al,* 2005) also found that RISC also

RNA Interference for Tumor Therapy 65

siRNA in vivo is to clone the siRNA sequence into the plasmid as a "short hairpin". When the hairpin is delivered to the animal body, its sequence is expressed, forming "double-

The first-generation shRNA is decorated and cloned to the viral carrier on the pre-miRNA platform, and transcribed into 50-70bp single-stranded molecules with the unique "stemloop" structure in the presence of RNA Pol III in the nucleus. When it enters the cytoplasm, its stem-loop structure is cut off by Dicer, forming siRNAs. Finally, it participates in the production of the RISC complex and induces RNAi (Paddison *et al,* 2004; Brummelkamp *et al,* 2003; Berns *et al,* 2004). With further research on miRNA, researchers have re-modeled the well-known structure of human miRNA-mir-30 and successfully developed a carriermediated second-generation RNAi trigger shRNAmir using pri-miRNA as the platform. Unlike pri-mir-30, the "stem" of this carrier-mediated shRNAmir has been replaced by other double helixes specific to different target genes. This structural re-design and transformation do not affect normal maturity of mir-30. Compared with the first-generation shRNA, the design of the second-generation shRNAmir makes use of the endogenous Drosha digestion process, thus increasing the specificity of Dicer in identifying Pre-miRNA. In addition, shRNAmir enters the intracellular RNAi pathway than shRNA or siRNA and can be treated

Bifunctional shRNA can make use of cleavage-dependent and non cleavage-dependent RNAi (Rao *et al,* 2010). Such shRNA contains two stem loops: one with complete-matching passenger and guide strands, which can combine with cleavage-dependent RISC, and the other with non-matching strands, which can combine with non cleavage-dependent RISC. This shRNA is more efficient than shRNA RNAi with one RISC complex. In experiments directly comparing the same siRNA target RNAi, it was found that bifunctional shRNA was more efficient (the same dosage of shRNA could produce more powerful gene knockout

RNAi has a standard procedure in intracellular gene knockout and can be used to identify gene functions by inhibiting specific genes and analyzing the change of gene appearance. As such, by analyzing gene functions, the signal pathway of tumor development could be identified. This further provides the feasibility to assess the medical or RNAi therapeutic target(Devi,2006). In addition, systemic treatment approaches with cancer-related miRNA or miRNA offer opportunities for tumor therapy. There have been a few overexpressed target

Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor specific to vascular endothelial cells, and can induce angiogenesis in vivo. VEGF and anti-VEGF coexist in normal tissues and remain in a relatively balanced state. In the presence of tumor growth, multiple carcinogenic factors trigger dramatic increase in VEGF, the amount and function of which are by far greater than those of anti-VEGF, thus causing massive growth

function) and its pharmacokinetics also underwent change(Rao *et al,* 2009,2010).

genes for RNAi study *in vivo*. The section is a retrospective review of these targets.

**3. The application of RNAi for silencing of tumor-related genes** 

stranded RNA" (shRNA), which is treated by the RNAi pathway.

by Drosha and Dicer, thus producing more siRNA(Silva *et al,* 2005).

**2.4 Mechanisms of bifunctional shRNA** 

**3.1 Vascular endothelial growth factor** 

has some other protein components, including Vasa intronic gene (VIG) protein, DmFXR, Tudor-SN, potential RNA helicase Dmp68 and Gemin3. These proteins are not necessary for the activity of RISC nuclease, but may provide additional functions such as RISC turnover, RISC subcellular location, and degradation after mRNA cleavage.

### **2.2 Mechanisms of miRNA**

miRNAs are a class of endogenous non-coding RNA that serves a specific function in eukaryotic organisms, with about 20~25 nucleotides. Mature miRNAs are processed from relatively long primary transcripts through a series of nuclease cutting, and then assembled into the RNA-induced silencing complex to identify target mRNA through base pairing, and instruct the silencing complex to degrade or block the translation of target mRNA depending on the degree of complementation.

miRNA genes are usually transcribed by RNA polymerase II (pol II) in the nucleus, and the primary product is pre-miRNA with a large cap-structure (7MGpppG) and a poly-A tail (AAAAA). Pre-miRNA is treated in stem loop-structured pre-mRNA containing 60-110 nucleotides in the presence of Rnase III enzyme Drosha (RNASEN) and its cofactor DGCR8 (a double-strand RNA domain-binding protein)(Tang,2005), and this pre-miRNA is transported from the nucleus to the cytoplasm by RAN-GTP and exportin 5 (EXP 5), and then cleaved by another nuclease Dicer to produce miRNA:miRNA double strands about 22 nucleotides in length, which are quickly introduced into RISC containing Ago2. One of the single-stranded mature miRNA is retained in the complex and expressed in response to regulatory genes in a manner similar to siRNA at the compensatory site of mRNA via base pairing (Meister&Tuschl,2004). Apart from Ago2, Ago1, Ago3 and Ago4 in the argonaute family that have no endonuclease activity also participate in the RNAi process of RISC through regulation on RISC rather than through cleavage (Preall&Sontheimer,2005).

Identification of target mRNA by miRNA requires precise pairing of nucleotides with miRNA seed sequence (Sethupathy *et al,* 2006). If miRNA and mRNA compensate with each other precisely, Ago 2 can cleave mRNA in the same way as it cleaves siRNA (Meister *et al,*  2004). However, there is a convex sequence in the middle of the A-type helical structure from non-precise combination of compensatory miRNA and the target mRNA, which makes mRNA cleavage impossible but can inhibit the expression of target mRNA at the protein translation level. This cleavage-independent mechanism plays the main role in miRNAnduced RNAi (Pillai *et al,* 2007). The miRNA-binding site using this mechanism is usually in 3'-end non-translation region of mRNA. Each miRNA can have multiple target genes, while several miRNAs can also regulate the same gene. This complex regulatory network can regulate expression of multiple genes through one miRNA, or precisely regulate the expression of a particular gene through combination of several miRNAs (Sethupathy *et al,*  2006). About one-third of encoding protein mRNA can be regulated by miRNA(Krek *et al,*  2005).

#### **2.3 Mechanisms of shRNA**

shRNA contains two short inverted repeat sequences separated by a stem loop in the middle forming a hairpin structure, which is controlled by Pol III promoter and then connects with 5-6 T as the transcription terminator of RNA Pol III. One of the methods of transporting

has some other protein components, including Vasa intronic gene (VIG) protein, DmFXR, Tudor-SN, potential RNA helicase Dmp68 and Gemin3. These proteins are not necessary for the activity of RISC nuclease, but may provide additional functions such as RISC turnover,

miRNAs are a class of endogenous non-coding RNA that serves a specific function in eukaryotic organisms, with about 20~25 nucleotides. Mature miRNAs are processed from relatively long primary transcripts through a series of nuclease cutting, and then assembled into the RNA-induced silencing complex to identify target mRNA through base pairing, and instruct the silencing complex to degrade or block the translation of target mRNA

miRNA genes are usually transcribed by RNA polymerase II (pol II) in the nucleus, and the primary product is pre-miRNA with a large cap-structure (7MGpppG) and a poly-A tail (AAAAA). Pre-miRNA is treated in stem loop-structured pre-mRNA containing 60-110 nucleotides in the presence of Rnase III enzyme Drosha (RNASEN) and its cofactor DGCR8 (a double-strand RNA domain-binding protein)(Tang,2005), and this pre-miRNA is transported from the nucleus to the cytoplasm by RAN-GTP and exportin 5 (EXP 5), and then cleaved by another nuclease Dicer to produce miRNA:miRNA double strands about 22 nucleotides in length, which are quickly introduced into RISC containing Ago2. One of the single-stranded mature miRNA is retained in the complex and expressed in response to regulatory genes in a manner similar to siRNA at the compensatory site of mRNA via base pairing (Meister&Tuschl,2004). Apart from Ago2, Ago1, Ago3 and Ago4 in the argonaute family that have no endonuclease activity also participate in the RNAi process of RISC

through regulation on RISC rather than through cleavage (Preall&Sontheimer,2005).

Identification of target mRNA by miRNA requires precise pairing of nucleotides with miRNA seed sequence (Sethupathy *et al,* 2006). If miRNA and mRNA compensate with each other precisely, Ago 2 can cleave mRNA in the same way as it cleaves siRNA (Meister *et al,*  2004). However, there is a convex sequence in the middle of the A-type helical structure from non-precise combination of compensatory miRNA and the target mRNA, which makes mRNA cleavage impossible but can inhibit the expression of target mRNA at the protein translation level. This cleavage-independent mechanism plays the main role in miRNAnduced RNAi (Pillai *et al,* 2007). The miRNA-binding site using this mechanism is usually in 3'-end non-translation region of mRNA. Each miRNA can have multiple target genes, while several miRNAs can also regulate the same gene. This complex regulatory network can regulate expression of multiple genes through one miRNA, or precisely regulate the expression of a particular gene through combination of several miRNAs (Sethupathy *et al,*  2006). About one-third of encoding protein mRNA can be regulated by miRNA(Krek *et al,* 

shRNA contains two short inverted repeat sequences separated by a stem loop in the middle forming a hairpin structure, which is controlled by Pol III promoter and then connects with 5-6 T as the transcription terminator of RNA Pol III. One of the methods of transporting

RISC subcellular location, and degradation after mRNA cleavage.

**2.2 Mechanisms of miRNA** 

2005).

**2.3 Mechanisms of shRNA** 

depending on the degree of complementation.

siRNA in vivo is to clone the siRNA sequence into the plasmid as a "short hairpin". When the hairpin is delivered to the animal body, its sequence is expressed, forming "doublestranded RNA" (shRNA), which is treated by the RNAi pathway.

The first-generation shRNA is decorated and cloned to the viral carrier on the pre-miRNA platform, and transcribed into 50-70bp single-stranded molecules with the unique "stemloop" structure in the presence of RNA Pol III in the nucleus. When it enters the cytoplasm, its stem-loop structure is cut off by Dicer, forming siRNAs. Finally, it participates in the production of the RISC complex and induces RNAi (Paddison *et al,* 2004; Brummelkamp *et al,* 2003; Berns *et al,* 2004). With further research on miRNA, researchers have re-modeled the well-known structure of human miRNA-mir-30 and successfully developed a carriermediated second-generation RNAi trigger shRNAmir using pri-miRNA as the platform. Unlike pri-mir-30, the "stem" of this carrier-mediated shRNAmir has been replaced by other double helixes specific to different target genes. This structural re-design and transformation do not affect normal maturity of mir-30. Compared with the first-generation shRNA, the design of the second-generation shRNAmir makes use of the endogenous Drosha digestion process, thus increasing the specificity of Dicer in identifying Pre-miRNA. In addition, shRNAmir enters the intracellular RNAi pathway than shRNA or siRNA and can be treated by Drosha and Dicer, thus producing more siRNA(Silva *et al,* 2005).
