The role of siRNA and Argonaute 2 in Cancer Therapy: Molecular mechanisms and drug design perspectives

 Corresponding author: Md Shorif Uddin Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. The role of siRNA and Argonaute 2 in Cancer Therapy: Molecular mechanisms and drug design perspectives Md Shorif Uddin *, Md Roknuzzaman Faisal, Afif Abyad Hossain, Adib Azwad Hossain, Nusrat Jahan Bristy, Mst Umma Fatama, Md Arafat Rahman Koko and Tauhedul Islam Department of Pharmacy, School of Pharmaceutical Sciences, Zhengzhou University, Henan, China. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 Publication history: Received on 26 September 2025; revised on 02 November 2025; accepted on 04 November 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.2.0971 Abstract RNA interference (RNAi) has emerged as one of the key mechanisms of post-transcriptional gene regulation and one of the most promising therapeutic approaches for precision cancer therapy. These include small interfering RNAs (siRNAs) and the catalytic Argonaute 2 (Ago2) protein acting alone or jointly to silence target mRNAs in a sequence-specific way by an irreversible mechanism reminiscent of enzymatic degradation. This study reviews the structural and functional facets of siRNAs and Ago2, along with their synergistic role in targeted cancer gene silencing. We point out the therapeutic use of siRNA-directed oncogene (KRAS, MYC, BCL-2) silencing and explain how Ago2 participates in oncogenic signaling pathways such as MAPK/ERK, PI3K/AKT, and FAK. The chemical modification and nanocarrierbased delivery systems have got siRNA much more stable, specific, and bioavailable, thus virtually solving the major problems of translational research such as nuclease degradation and immune activation. The coupling of artificial intelligence, bioinformatics, and CRISPR technologies has profoundly changed the conception and accuracy of RNAibased therapeutics. Delivery obstacles, immunogenicity, and large-scale manufacturing problems still exist, but recent inventions like self-delivering siRNAs, circular siRNAs, and hybrid CRISPR-RNAi systems are opening up the way to siRNA-Ago2 clinical applications at a fast pace. Altogether, these breakthroughs make RNAi a revolutionary route in molecularly targeted cancer therapy. Keywords: siRNA; Argonaute 2 (Ago2); RNA Interference (RNAI); Gene Silencing; Oncogene Targeting; Nanocarrier Delivery; Chemical Modification; KRAS; MYC; BCL-2; PI3K/AKT Pathway; Cancer Therapeutics; CRISPR–RNAI Hybrid 1. Introduction RNA interference (RNAi) forms a central part of the intrinsic homeostatic mechanism of all cells in nature and has been evolutionarily conserved [1]. Discovered originally in Caenorhabditis elegans as a mechanism for gene-specific silencing induced by double stranded RNA (dsRNA) [2], the phenomenon has become firmly established as a general mechanism of regulation across eukaryotic organisms [3]. The process involves the generation of small RNAs—mainly small interfering RNAs (siRNAs) and microRNAs (miRNAs)—that guide RISC to the target messenger RNA (mRNA) molecules which are then degraded or translation is inhibited [4]. RNAi has emerged as a potent tool in functional genomics and is an efficient and flexible means to uncover gene function, validate targets, and for therapeutic purposes [5,6]. Due to its exceptional sequence specificity, disease-related genes considered previously as “undruggable” can be specifically suppressed by small molecule inhibitor or monoclonal antibody [7]. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 126 The principal effectors of RNAi are short interfering RNAs (siRNA) typically 21–23 nucleotide molecules originating from long dsRNA precursors that upon enzymatic processing by the ribonuclease Dicer [2,6]. This enables incorporation of the resultant siRNAs into RISC, with a guide strand that remains bound and its partner (passenger) strand being degraded [8,9]. Argonaute 2 (Ago2, a principal representative of the argonaute protein family) is the catalytically active core of RISC that mediates endo-nucleolytic cleavage of target mRNAs [8,10]. Supported by the siRNA sequence, Ago2 cleaves its target exactly between nucleotides 10 and 11 of the guide strand, leading to a very rapid degradation of these fragments [7]. Human Ago2 can be divided into four structural domains, N-terminal, PAZ, MID and PIWI that serve to mediate RNA binding and catalysis [10]. The PIWI domain contains the DEDH catalytic tetrad that is required for its slicer activity. A detailed knowledge of these structural and mechanistic characteristics has been crucial in the rational design of siRNAs with preferred strand selection, thermodynamic stability and minimal off-target effects [6]. Altered gene expression is a well-established characteristic of cancer etiology and progression, resulting from mutations in core signaling machinery as well as disruption of transcriptional and post-transcriptional control [3,11]. siRNAdirected approaches offer an effective way to specifically down-regulate oncogenes, tumor-promoting factors or drugresistance mediators with great specificity [12,13]. In contrast to classical chemotherapy or targeted inhibitors, siRNA therapeutics act at mRNA level and, therefore, provide potential suppression of pathological proteins independent of their structural draggability [4]. The effectiveness of siRNA-mediated knockdown of oncogenes such as KRAS, MYC and BCL-2 has also been established in multiple studies with induction of cell growth inhibition, apoptotic cell death and abrogated metastatic potential in preclinical models [14]. Moreover, siRNA-mediated therapy in combination with conventional chemotherapeutics sensitizes tumors and abrogates multidrug resistance, substantiating the potential of RNAi as a new generation genetargeted cancer therapeutic [12]. Chemical modification and nanocarrier-mediated delivery have recently advanced siRNAs to enhance stability and potency. Modifications including 2′-O-methyl (2′-OMe), 2′-fluoro (2′-F) and phosphorothioate bonds increase nuclease resistance, reduce immune activation, whereas lipid and polymeric nanoparticles enables efficient packaging, specific targeting delivery and endosomal escape [4,15]. FDA-approved siRNA drugs, such as Patisiran and Inclisiran have demonstrated clinical success supporting the therapeutic potential of RNAi, and establishing a platform for broader implementation in oncology [16,17]. Innovative delivery systems, for instance self-transporting siRNAs (sd-siRNAs), circular siRNA, and stimuli-sensitive nanocarriers are pushing the research toward enhanced precision of action, stronger therapeutic effect [15]. Nevertheless, siRNA-based therapeutics still remain to address major issues such as nuclease degradation, immune stimulation, rapid systemic clearance and limited intracellular delivery [4]. Overcoming these limitations will necessitate the combination of chemical optimization with targeted delivery that enables improvement in stability, bioavailability and tumor targeting [7]. Novel cross-disciplinary methods are also influencing the development of RNAi-based therapeutics. The combination of AI with bioinformatics and structural biology is facilitating the design of more potent and specific siRNAs beyond chemical enhancement, as well as engineered mutant Ago2. Furthermore, novel hybrid approaches of RNAi with CRISPR-based genome editing are being developed to enable long-term control over programmable and specific gene regulation [1,18]. Collectively, these developments place RNAi as a breakthrough technology for precision oncology and molecular therapy [12,17]. 2. siRNA: Structure, Biogenesis, and Mechanism of Action Small interfering RNAs (siRNAs) are a class of small, double-stranded non-coding RNA molecules that play a significant part in the gene silencing process RNA interference [19–21]. Usually 21–23 nucleotides (nt) long [19,21], these entities are produced by the RNase III enzyme Dicer during the processes of splicing and modification of RNA [20]. Each siRNA is composed of two strands —a guide (antisense) strand, which engages the target mRNA specifically for silencing, and a passenger (sense) strand that is removed during RISC activation [22]. Dicer activity cuts long dsRNA substrates into 21-nt siRNAs with typical structural properties of 2-nt (nucleotide) 3′ overhangs on each strand. Every strand has a 5′ terminus monophosphate and a 3′ terminus hydroxyl [23,24] this specific chemical property reflects genuine Dicer processing and is necessary for efficient silencing by the RNA-induced silencing complex (RISC) [23,24]. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 127 Thermodynamic asymmetry determines which of the strands will be assigned as guide: the less stably paired 5′ end of the strand, usually AU-rich rather than GC-rich, is preferentially incorporated into argonaute (Ago) proteins in RISC. The foundational principle of which is well-established in seminal work done by Khvorova et al. (2003) and Schwarz et al. (2003) [23], guarantees directional targeting. Strand selection is also influenced by other factors, such as the nature of the 5′ nucleotide (preferentially U/A) and the presence of loading cofactors for RISC (like TRBP and PACT) [23]. Chemical modifications—such as 2′-O-methyl, locked nucleic acids (LNAs), or phosphorothioate linkages—have the capacity to enhance resistance to nucleases [25,26], reduce immune-stimulation, and affect strand bias, thus making them critical components in the development of therapeutic siRNAs. 2.1. Biogenesis of siRNA 2.1.1. Origins of dsRNA Precursors In plants, predominantly invertebrates, siRNAs are products of RNA-dependent RNA pols (RdRPs) that synthesize long dsRNAs from RNA templates [27,28]. Dicer processes these dsRNAs in the cytoplasm and then loads it into RISC to mediate sequence-specific gene silencing [28,29]. In plant cells, this is often initiated by environmental stress, transposon activity or viral infection. Unlike in mammals that do not have RdRPs and it was presumed that they only produce exogenous siRNAs [27]. Appreciation of this viewpoint was altered by the discovery of rare endogenous sources of dsRNA as follows [28]: Retrotransposons[30]. For example, LINE-1 elements in humans produce bidirectional polyadenylated RNA transcripts from a double promoter system expressed in both directions oriented within genes and which are therefore copied into complementary RNAs that hybridize to form dsRNA [31]. Pseudogenes containing inverted repeats [32].These structures permit the formation of long intramolecular hairpins [32], reminiscent in structure to shRNA precursors. Transcriptional units overlapped: Antisense transcription from proteincoding loci or noncoding loci may produce two complementary RNAs, suitable for dsRNA synthesis [33]. Endogenous sources of such dsRNA have been found in a number of organisms. AGO2-bound small RNAs in Drosophila contain a unique 21 nt siRNA class that includes pseudogene and inverted repeat derived species [30]. SiRNAs in oocytes have been described in mice, are Dicer-dependent, and occasionally target protein-coding genes [33,34]. 2.1.2. Dicer Processing In the cytoplasm, Dicer digests long double-stranded RNAs (dsRNAs) into small interfering RNA (siRNA) duplexes with specific 2-nucleotide 3′ overhangs and a phosphorylated 5′ termini in the cytoplasm environment [25,26]. An siRNA duplex consists of a guide strand that actively is incorporated into the RNA-induced silencing complex (RISC) and a passenger strand, which will be degraded [35]. RISC Loading and Strand Selection The siRNA duplex binds to AGO2, the catalytically active mammalian Argonaute protein, and forms the pre-RISC complex [36,37]. The thermodynamic asymmetry between the duplex termini, which drives strand selection [38], generally the less stable 5' end is designated as the guide strand. The passenger strand is subsequently excised and degraded, resulting in the formation of the mature RISC [39]. Target Recognition and Gene Silencing RISC is targeted to mRNAs with high sequence complementarity by the guide strand. Target RNA cleavage by AGO2 at a single site between nucleotides 10 and 11 relative to the guide strand triggers transcript degradation. Whilst microRNAs frequently have mismatches and mediate translational repression, siRNAs generally lead to efficient mRNA cleavage with near-perfect base pairing [40]. 2.1.3. Endogenous vs. Exogenous siRNAs In vivo, most siRNA experiments in mammals have employed exogenous delivery either using synthetic siRNA duplexes or using short hairpin RNAs (shRNAs) expressed from plasmid or viral vectors [41]. shRNAs, driven by Pol II or Pol III promoter called an RNAi vector [42], are transcribed in nucleus targeted to be processed into pre-shRNA by the Microprocessor (Drosha–DGCR8), then exportin-5 transferred into cytoplasmic space for dicing into siRNA duplexes for RISC complex. Endogenous mammalian siRNAs are sparsely expressed and less established as regulatory molecules relative to miRNAs and piRNAs [43]. They probably evolved from evolutionary ancient antiviral defense systems and the loci that give rise World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 128 to them might still be under selective pressure [43,44]. The functional roles of mammalian endo-siRNAs, particularly those that target protein-coding genes, still need to be elucidated in the course of further research [45]. 2.2. Mechanism of mRNA silencing siRNAs trigger gene silencing by becoming part of the RNA-induced silencing complex (RISC) [19,21]. Artificially synthesized siRNAs, generally 21–23 nucleotides long with 2-nt-long 3′ overhangs [46–48], are delivered to cytoplasm through chemically modified delivery agents such as lipid nanoparticles, conjugates, or viral vectors [19,27,49]. One important step is the endosomal escape as cytoplasmic localization is necessary for RISC formation [49]. Figure 1 Structural features of exogenous siRNA. Exogenous siRNAs are typically designed as 19-20 bp RNA duplexes with two-nucleotide 3' overhangs composed of DNA nucleotides (red #E07F80). The canonical 5' monophosphate and 3' hydroxyl termini are preserved to ensure proper argonaute loading and gene-silencing activity In RISC, the double-stranded siRNA is subject to strand selection, with the passenger strand eliminated, primarily via Ago2-catalysed cleavage or by dissociation with the help of accessory factors [50]. The passenger strand is thermodynamically locked onto Ago2, the catalytic engine of the complex [51]. The guide strand guides RISC to mRNA sequences that are complementary via Watson–Crick base pairing [52]. Recognition is directed by the seed region (positions 2–8 from the 5′ end of the guide strand), and silencing efficiency correlates with the size of base pairing [4]. Figure 2 Schematic illustration of the small interfering RNA (siRNA) mediated gene silencing In cases of nearly perfect base pairing, typically in a coding region, Ago2 catalyzes endonucleolytic cleavage of the target mRNA between the nucleotides opposite the guide positions 10 and 11. These resulting RNA fragments are then World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 129 degraded rapidly by cellular exonucleases, including XRN1 and the exosome complex [53], thereby inducing fast, irreversible, and potent silencing of gene expression. This cleavage-based pathway is the major form of action for siRNA therapeutics and designed to be fully homologous to their targets. In contrast, in the case of only partial complementarity, usually in the 3′UTR, slicing activity of Ago2 is not involved. Instead, RISC acts to recruit effector proteins, which block translation initiation, induce ribosome disassembly, and promote mRNA decay at the level of deadenylation and decapping, frequently within processing bodies (P-bodies) [25,26,54]. Even though this mode of action seems to be more typical of miRNA-mediated regulation [55], off-target effects in the form of non-cleavage can be elicited by siRNAs under limiting complementarity. Therefore, siRNA-induced gene silencing is mainly initiated via Ago2-cleavage of the mRNA [54,56], leading to the subsequent secondary events such as translation repression and target mRNA degradation [48]. The siRNA therapeutic opportunity emanates from their ability to manipulate this endogenous system with specificity and potency [57], depending on the successful addressing of delivery, stability and endosome escape hurdles. 3. Argonaute 2: Structure, Function, and Role in RNAi 3.1. The argonaute Protein Family The Argonaute (Ago) proteins are a group of specialized small-RNA-binding proteins that act as the main effectors in RNA-silencing pathways [58]. Small RNAs must be incorporated into Argonaute-associated ribonucleoprotein complexes, collectively known as RNA-induced silencing complexes (RISCs) [59], in order to carry out their regulatory functions. Argonautes are processed into complexes that use small RNA guides to identify complementary nucleic acid sequences, allowing for post-transcriptional gene regulation, transcriptional silencing or genomic defense within these complexes [59]. The name of this family originates from a phenotype described in Arabidopsis thaliana of a mutant with knockout of AGO1 that had abnormal leaf shape similar to tentacles of the octopus Argonauta Argo [60]. Argonautes are divided into two major subclasses, the Ago subfamily, which is similar to Arabidopsis AGO1, and the Piwi subfamily, which is homologous to Drosophila PIWI proteins [8]. Ago subfamily is widely expressed in somatic cell types, and mainly functions by associating with miRNAs and siRNAs. The Ago subfamily contains AGO1, AGO2, AGO3, and AGO4 in the human genome [61]. Of these, AGO2 is unique in that it retains endonucleolytic "slicer" activity to cleave target RNAs while the other paralogs mainly exert translational repression and mRNA destabilization activity [62]. Despite a wide conservation of Ago proteins between species, the copy number is highly variable: from one in Schizosaccharomyces pombe [60], five in Drosophila, eight in humans, ten in Arabidopsis thaliana, and as many as twenty-seven in Caenorhabditis elegans. Interestingly, although several emergent yeasts, including Saccharomyces Castellii, have functional argonaute genes and generate siRNAs by means of a divergent Dicer enzyme, the widely used model organism Saccharomyces cerevisiae lacks Argonautes altogether, along with canonical RNAi pathways. On the other hand, the Piwi subfamily has a much more limited expression pattern, mainly expressed in germline cells, where Piwi proteins bound to Piwi-interacting RNAs (piRNAs) silence transposable elements in the germline to maintain genomic integrity [63]. Although four piwi proteins (HIWI (PIWIL1), HILI (PIWIL2), HIWI3 (PIWIL3) and HIWI2 (PIWIL4)) are encoded in the human genome [64], most studies in mammals have focused on HIWI and HILI [63]. Three Piwi proteins, MIWI, MILI, and MIWI2, are expressed during spermatogenesis of mice and play a key role in transposon regulation. Likewise, argonaute proteins have been detected in various lineages of bacteria and archaea but, here, the distribution seems rather erratic. Comprising a highly conserved four-domain architecture (N, PAZ, MID, and PIWI) [65,66], these prokaryotic Argonautes (pAgos) frequently employ DNA as a guide rather than RNA [67], using a variety of foreign DNA and RNA substrates as part of their defense systems against invading elements (phage and plasmids) [68], although Pagos have so far received much less attention relative to eukaryotic Argonautes [69]. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 130 Table 1 Classification: Ago vs. Piwi clades. [8,70,71] Feature AGO Clade PIWI Clade Small RNA Guides Binds siRNAs (∼21 nt) and miRNAs (∼22 nt) to regulate gene expression via RISC. Binds piRNAs (∼24–31 nt) to silence transposons and preserve genome integrity, especially in the germline. Expression Pattern Ubiquitously expressed across somatic cells in most organisms. Primarily restricted to germline cells in animals Function Facilitates post-transcriptional gene silencing via mRNA cleavage or translational repression. Mediates transposon silencing through cytoplasmic cleavage and/or nuclear silencing, often involving heterochromatin formation. Target Recognition Rules Requires strict seed pairing (guide nucleotides g2–g8) for target recognition and silencing. Displays relaxed complementarity requirements, tolerating mismatches even near the cleavage site— advantageous for combating rapidly diverging transposon. Slicer Activity Many AGO proteins have endonucleolytic (“slicer”) activity, e.g., human AGO2 cleaves target mRNAs directly. Some PIWIs (like Aub and Ago3 in Drosophila) are slicers in the cytoplasm, but others (like Piwi) lack slicer activity and act as a scaffold for cotranscriptional silencing. Evolutionary Conservation AGO and PIWI are two phylogenetically distinct subfamilies within the Argonaute superfamily PIWI proteins are animal-specific, evolving unique functions in germline defense against transposons 3.2. Structural domains of Ago2 Recent structural observations support a bi-lobe architecture of human AGO2 as defined by the N–PAZ lobe connected through two flexible linkers (L1 and L2) to the MID–PIWI lobe (in what is referred to as mid-kernel exchange) [72]. Collectively, these lobes create a central cleft that binds the guide RNA, as well as its complementary target strand, allowing AGO2 to serve as the catalytic engine of RNA-induced silencing complexes (RISCs) [72]. Figure 3 Linear representation of human AGO2, illustrating its four major domains along with their core functions. Domain Function N Unwinds RNA duplex PAZ Binds 3′ overhang of guide RNA MID Anchors 5′ phosphate of guide strand PIWI Catalytic activity (endonuclease/slicer) C-terminal Structural extension (no defined domain) 3.2.1. N-terminal Domain The N-terminal domain plays an essential role in RISC complex assembly, particularly in facilitating the unwinding of the small RNA duplex and passenger strand elimination. This component represents a rate-limiting step in RISC activation. The N-domain, although not essential for RNA loading and slicing, is required for assembly and functional maturation of RISC [73]. In addition, the N-domain makes a second contribution to guide-target pairing, by limiting pairing outside of the 16th nucleotide of the guide RNA, thus ensuring that only a specific location is cleaved [72,74], although this function is not yet definitively shown for eukaryotic AGO2. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 131 3.2.2. PAZ Domain The PAZ domain shared between AGO2 and Dicer has the oligonucleotide/oligosaccharide-binding (OB) fold structure that specifically binds to the 3′ two-base overhang of guide RNAs. This allows for increased stability of the guide RNA within the assembly and ensures that they are orientated correctly [72,75].Studies using mutagenesis show that mutations in the PAZ domain of AGO2 proteins allow binding of small RNAs, but loss the duplex unwinding ability and the ability to form effector cognate RISC, indicating an important role for the PAZ domain in RISC assembly [76,77]. Figure 4 Structure and schematic of human argonaute 2 (Ago2, PDB ID: 4Z4F), highlighting the N, PAZ, MID, and PIWI domains that mediate small RNA binding and target RNA cleavage 3.2.3. MID Domain The MID domain has a Rossmann-like tertiary structure and creates a highly conserved pocket that binds the 5′ phosphate of guide RNAs, thus enforcing polarity and strand selection [72,78]. Structural studies have defined additional binding sites near the core active site stabilized by sulfate ions and conserved lysine residues (e.g., Lys599, Lys638) and could play a role in binding the m7GpppG cap of target mRNAs. This suggests that the MID domain provides structural and regulatory functions to AGO2 activity [79]. 3.2.4. PIWI Domain The PIWI domain acts as the catalytic center of AGO2, displays structural resemblance to RNase H, and contains a conserved DEDH catalytic tetrad that mediates endo-nucleolytic "slicer" activity. It is known that this domain cleaves target RNAs 10 and 11 nucleotides from the guide strand [80]. In addition to its catalytic function, the PIWI domain contains tryptophan-binding pockets that are required for the recruitment of GW182 and other tryptophan-rich cofactors essential for translational repression and mRNA decay. In addition, the PIWI domain strengthens the contacts between the MID–PIWI lobe and DNA or RNA substrates, and some specific residues such as Asp603 are crucial for structural integrity [72,81] . 3.3. Mechanism of AGO2-Mediated Slicing Recent biochemical and structural studies have provided an integrative model for how human AGO2 mediates RNA cleavage (slicing). This can be systematically classified into sequential stages involving guide-target recognition, conformational fitting, and catalysis [82].Target mRNAs are first recognized by base-pairing interactions between the seed region (guide nucleotides g2–g8) of the small RNA bound to AGO2. In the initial phase, base-pairing interactions occur with the 3′ supplementary region (g13–g16) after which additional interactions with the action center of the guide (g9–g12) take place while the central region remains unpaired. At the time perfect duplexes form between the seed and 3′ supplementary regions and the target, AGO2 drives a rotational movement of the 3′ supplementary duplex region [83,84]. This change enables the guide center (g9–g12) to base-pair with the target sequence to be determined if it is complementary. However, the lack of restriction on the central and 3′ supplementary segments allows for this conformation despite AGO2 stabilizing the phosphate backbone of the seed region [83]. When base-pairing is fully extended to the central region of the target RNA, the target is precisely positioned within the PIWI domain of AGO2 containing the conserved D–E–D–H catalytic tetrad [83]. These tagged residues work alongside the divalent metal ions to stimulate a water molecule for a nucleophilic attack of the phosphodiester backbone. Target Cleavage AGO2 cleaves the target RNA endo-nucleolytically between t10 (t = 1) and t11 (P = 5′ end of the guide RNA). During this process target World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 132 RNA is cleaved into two unique fragments, which are then released and degraded resulting in efficient posttranscriptional gene slicing [84]. Figure 5 Ago2-mediated RNA cleavage. “Figure adapted from Nucleic Acids Res, Volume 50, Issue 12, 8 July 2022, Pages 6618-6638, https://doi.org/10.1093/nar/gkac519 4. siRNA-Mediated Silencing of Oncogenes and Downstream Pathways Specific suppression of oncogenes with an essential role in tumor initiation and progression can be achieved using siRNA technology [85]. They have the ability to direct the degradation of its complementary oncogenic mRNAs through RNA-induced silencing complex (RISC) mediation [86], leading to no protein output. Crucially, silencing an oncogene also disrupts the downstream signaling pathways it controls, amplifying the therapeutic effect [85]. This module combines oncogenic targets KRAS, MYC, and BCL2 with their relevant pathways [14], emphasizing how siRNA-mediated knockdown re-monitors cancer signals [87]. 4.1. KRAS and MAPK/ERK, PI3K/AKT Pathways Mutations of KRAS are one of the most common oncogenic drivers in lung, pancreatic and colorectal cancer [88]. KRAS stimulates prominent signaling pathways, specifically MAPK/ERK and PI3K/AKT cascades that govern cell growth, survival and metastasis [89]. siRNA-induced depletion of KRAS diminishes both ERK and AKT phosphorylation and subsequent tumor cell growth and apoptosis [85]. KRAS siRNAs delivered by nanoparticles led to substantial inhibition of tumor growth and metastasis in preclinical lung cancer models [90]. Furthermore, simultaneous KRAS repression and MYC abrogation has resulted in sustained tumor regression raising perspectives that such a dual assault may have even more pronounced therapeutic effects [91]. 4.2. MYC and PI3K/AKT Pathway The transcription factor MYC that controls cell cycle, metabolism and apoptosis relevant genes has been found overexpressed in many hematological and malignancies and solid tumors [92]. MYC expression is also inhibited by siRNA, and results in markedly decreased cell proliferation mediated by KB-3-1 and K562 cells [93]. Biologically, suppression of MYC also interferes with the PI3K/AKT pathway that is pivotal in the metabolic adaptation and survival of cancer cells [94]. Inhibition of this pathway causes suppression of glycolysis, decreased biosynthesis and increased sensitivity to apoptotic signals [95]. These data indicate that MYC is an important hub through which siRNA therapy can intersect with PI3K/AKT-stimulated tumorigenic signaling mechanism. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 133 4.3. BCL2 and Apoptotic Signaling BCL2 is a classical oncogene whose anti-apoptotic activity are over-expressed in around 50% of all human cancers with approximately 50–70% of patients with breast cancers displaying deregulated expression [96]. Overexpression inhibits programmed cell death, promoting chemoresistance and radioresistance [97]. The nanoliposomal formulations (NLBcl-2 siRNA) of siRNA-based silencing of BCL2 exert potent antitumor effects on estrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-231) breast cancer xenografts [87]. Mechanistically, the BCL2 inhibition included in silencing would re-enable apoptotic machinery by freeing pro-apoptotic proteins (e.g., BAX, BAK) [98], which further yields a radiation-resistant status to tumor cells upon conventional therapies [87]. This shows how siRNA can directly re-engage intrinsic death pathways that cancer cells evade. Table 2 siRNA-Mediated Silencing of Oncogenes and Downstream Pathways in Cancer Target (oncogene and pathway) Cancer model siRNA Delivery Method Observed Effect Ref. KRAS / MAPK– ERK, PI3K–AKT Lung Cancer (xenograft models) Nanoparticledelivered KRAS siRNA Reduced KRAS expression, inhibited ERK and AKT signaling, suppressed tumor growth and metastasis [99] BCL2 / Apoptotic Pathway Breast Cancer (ER⁺ MCF7, ER⁻ MDA-MB231 xenografts) Nanoliposomal BCL2 siRNA (NL-Bcl-2 siRNA) Suppressed tumor growth, enhanced chemosensitivity, restored apoptotic signaling [87] MYC / PI3K–AKT Leukemia (K562 cells), Cervical carcinoma (KB-3-1 cells) Synthetic siRNA against c-myc Decreased MYC mRNA, inhibited proliferation, induced apoptosis, disrupted PI3K–AKT survival signaling [100] KRAS + MYC / MAPK–ERK and PI3K–AKT Lung Cancer models Combined siRNA silencing More frequent and durable tumor regression compared to single gene targeting [101] 5. Role of Argonaute 2 (Ago2) in Cancer Among the argonaute family, the only catalytically active member Ago2 has been identified as a core component of the RNA-induced silencing complex (RISC) and a pivotal player in microRNA (miRNA)-guided gene silencing [10,72]. Ago2 has vital roles in mediating post-transcriptional regulation that affect normal cellular homeostasis and cancer [10,72]. Ago2 has been widely studied in the field of cancer for its aberrant expression and dysregulation in numerous types of cancers, highlighting its role in tumorigenesis and progression [102,103]. 5.1. Expression Patterns of Ago2 in Tumors Versus Normal Tissues Multiple studies have shown that Ago2 is often upregulated in tumor tissue compared to adjacent normal tissues. Increased expression in bladder urothelial carcinoma, hepatocellular carcinoma, glioma, hypopharyngeal carcinoma, adrenocortical carcinoma, gastric carcinoma, ovarian carcinoma, and colon cancer. In these cancers, increased Ago2 levels correlate with higher cellular proliferation, migration, metastasis, and overall aggressiveness of tumors. However, decreased or variable expression has been observed in melanoma with low Ago2 protein yet stable mRNA expression and in ER positive breast cancers where active estrogen signaling suppressed Ago2 expression. Yet these heterogeneous patterns reveal Ago2 's cancer-type-specific functions. Table 3 Expression Patterns of Ago2 in Tumors Versus Normal Tissues Cancer Type Expression Pattern Comparison with Normal Ref. Urothelial Carcinoma Upregulated Tumor > normal bladder tissue [104] Hepatocellular Carcinoma (HCC) Upregulated Tumor > adjacent normal liver tissue [105,106] World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 125-148 140 Statement of ethical approval Md Shorif Uddin*, Md Roknuzzaman Faisal, Afif Abyad Hossain, Adib Azwad Hossain and Nusrat Jahan Bristy played pivotal roles in shaping this review paper. 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