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The ole o siRNA and A gonau e 2 in Cance The apy: Molecula mechanisms and
d ug design pe spec i es
Md Sho i Uddin *, Md Roknuzzaman Faisal, A i Abyad Hossain, Adib Azwad Hossain, Nus a Jahan B is y,
Ms Umma Fa ama, Md A a a Rahman Koko and Tauhedul Islam
Depa men o Pha macy, School o Pha maceu ical Sciences, Zhengzhou Uni e si y, Henan, China.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 24(02), 125-148
Publica ion his o y: Recei ed on 26 Sep embe 2025; e ised on 02 No embe 2025; accep ed on 04 No embe 2025
A icle DOI: h ps://doi.o g/10.30574/wjbphs.2025.24.2.0971
Abs ac
RNA in e e ence (RNAi) has eme ged as one o he key mechanisms o pos - ansc ip ional gene egula ion and one o
he mos p omising he apeu ic app oaches o p ecision cance he apy. These include small in e e ing RNAs (siRNAs)
and he ca aly ic A gonau e 2 (Ago2) p o ein ac ing alone o join ly o silence a ge mRNAs in a sequence-speci ic way
by an i e e sible mechanism eminiscen o enzyma ic deg ada ion. This s udy e iews he s uc u al and unc ional
ace s o siRNAs and Ago2, along wi h hei syne gis ic ole in a ge ed cance gene silencing. We poin ou he
he apeu ic use o siRNA-di ec ed oncogene (KRAS, MYC, BCL-2) silencing and explain how Ago2 pa icipa es in
oncogenic signaling pa hways such as MAPK/ERK, PI3K/AKT, and FAK. The chemical modi ica ion and nanoca ie -
based deli e y sys ems ha e go siRNA much mo e s able, speci ic, and bioa ailable, hus i ually sol ing he majo
p oblems o ansla ional esea ch such as nuclease deg ada ion and immune ac i a ion. The coupling o a i icial
in elligence, bioin o ma ics, and CRISPR echnologies has p o oundly changed he concep ion and accu acy o RNAi-
based he apeu ics. Deli e y obs acles, immunogenici y, and la ge-scale manu ac u ing p oblems s ill exis , bu ecen
in en ions like sel -deli e ing siRNAs, ci cula siRNAs, and hyb id CRISPR-RNAi sys ems a e opening up he way o
siRNA-Ago2 clinical applica ions a a as pace. Al oge he , hese b eak h oughs make RNAi a e olu iona y ou e in
molecula ly a ge ed cance he apy.
Keywo ds: siRNA; A gonau e 2 (Ago2); RNA In e e ence (RNAI); Gene Silencing; Oncogene Ta ge ing; Nanoca ie
Deli e y; Chemical Modi ica ion; KRAS; MYC; BCL-2; PI3K/AKT Pa hway; Cance The apeu ics; CRISPR–RNAI Hyb id
1. In oduc ion
RNA in e e ence (RNAi) o ms a cen al pa o he in insic homeos a ic mechanism o all cells in na u e and has been
e olu iona ily conse ed [1]. Disco e ed o iginally in Caeno habdi is elegans as a mechanism o gene-speci ic silencing
induced by double s anded RNA (dsRNA) [2], he phenomenon has become i mly es ablished as a gene al mechanism
o egula ion ac oss euka yo ic o ganisms [3]. The p ocess in ol es he gene a ion o small RNAs—mainly small
in e e ing RNAs (siRNAs) and mic oRNAs (miRNAs)— ha guide RISC o he a ge messenge RNA (mRNA) molecules
which a e hen deg aded o ansla ion is inhibi ed [4].
RNAi has eme ged as a po en ool in unc ional genomics and is an e icien and lexible means o unco e gene unc ion,
alida e a ge s, and o he apeu ic pu poses [5,6]. Due o i s excep ional sequence speci ici y, disease- ela ed genes
conside ed p e iously as “und uggable” can be speci ically supp essed by small molecule inhibi o o monoclonal
an ibody [7].
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The p incipal e ec o s o RNAi a e sho in e e ing RNAs (siRNA) ypically 21–23 nucleo ide molecules o igina ing
om long dsRNA p ecu so s ha upon enzyma ic p ocessing by he ibonuclease Dice [2,6]. This enables inco po a ion
o he esul an siRNAs in o RISC, wi h a guide s and ha emains bound and i s pa ne (passenge ) s and being
deg aded [8,9]. A gonau e 2 (Ago2, a p incipal ep esen a i e o he a gonau e p o ein amily) is he ca aly ically ac i e
co e o RISC ha media es endo-nucleoly ic clea age o a ge mRNAs [8,10]. Suppo ed by he siRNA sequence, Ago2
clea es i s a ge exac ly be ween nucleo ides 10 and 11 o he guide s and, leading o a e y apid deg ada ion o hese
agmen s [7].
Human Ago2 can be di ided in o ou s uc u al domains, N- e minal, PAZ, MID and PIWI ha se e o media e RNA
binding and ca alysis [10]. The PIWI domain con ains he DEDH ca aly ic e ad ha is equi ed o i s slice ac i i y. A
de ailed knowledge o hese s uc u al and mechanis ic cha ac e is ics has been c ucial in he a ional design o siRNAs
wi h p e e ed s and selec ion, he modynamic s abili y and minimal o - a ge e ec s [6].
Al e ed gene exp ession is a well-es ablished cha ac e is ic o cance e iology and p og ession, esul ing om mu a ions
in co e signaling machine y as well as dis up ion o ansc ip ional and pos - ansc ip ional con ol [3,11]. siRNA-
di ec ed app oaches o e an e ec i e way o speci ically down- egula e oncogenes, umo -p omo ing ac o s o d ug-
esis ance media o s wi h g ea speci ici y [12,13]. In con as o classical chemo he apy o a ge ed inhibi o s, siRNA
he apeu ics ac a mRNA le el and, he e o e, p o ide po en ial supp ession o pa hological p o eins independen o
hei s uc u al d aggabili y [4].
The e ec i eness o siRNA-media ed knockdown o oncogenes such as KRAS, MYC and BCL-2 has also been es ablished
in mul iple s udies wi h induc ion o cell g ow h inhibi ion, apop o ic cell dea h and ab oga ed me as a ic po en ial in
p eclinical models [14]. Mo eo e , siRNA-media ed he apy in combina ion wi h con en ional chemo he apeu ics
sensi izes umo s and ab oga es mul id ug esis ance, subs an ia ing he po en ial o RNAi as a new gene a ion gene-
a ge ed cance he apeu ic [12].
Chemical modi ica ion and nanoca ie -media ed deli e y ha e ecen ly ad anced siRNAs o enhance s abili y and
po ency. Modi ica ions including 2′-O-me hyl (2′-OMe), 2′- luo o (2′-F) and phospho o hioa e bonds inc ease nuclease
esis ance, educe immune ac i a ion, whe eas lipid and polyme ic nanopa icles enables e icien packaging, speci ic
a ge ing deli e y and endosomal escape [4,15].
FDA-app o ed siRNA d ugs, such as Pa isi an and Inclisi an ha e demons a ed clinical success suppo ing he
he apeu ic po en ial o RNAi, and es ablishing a pla o m o b oade implemen a ion in oncology [16,17]. Inno a i e
deli e y sys ems, o ins ance sel - anspo ing siRNAs (sd-siRNAs), ci cula siRNA, and s imuli-sensi i e nanoca ie s
a e pushing he esea ch owa d enhanced p ecision o ac ion, s onge he apeu ic e ec [15].
Ne e heless, siRNA-based he apeu ics s ill emain o add ess majo issues such as nuclease deg ada ion, immune
s imula ion, apid sys emic clea ance and limi ed in acellula deli e y [4]. O e coming hese limi a ions will
necessi a e he combina ion o chemical op imiza ion wi h a ge ed deli e y ha enables imp o emen in s abili y,
bioa ailabili y and umo a ge ing [7].
No el c oss-disciplina y me hods a e also in luencing he de elopmen o RNAi-based he apeu ics. The combina ion
o AI wi h bioin o ma ics and s uc u al biology is acili a ing he design o mo e po en and speci ic siRNAs beyond
chemical enhancemen , as well as enginee ed mu an Ago2. Fu he mo e, no el hyb id app oaches o RNAi wi h
CRISPR-based genome edi ing a e being de eloped o enable long- e m con ol o e p og ammable and speci ic gene
egula ion [1,18]. Collec i ely, hese de elopmen s place RNAi as a b eak h ough echnology o p ecision oncology and
molecula he apy [12,17].
2. siRNA: S uc u e, Biogenesis, and Mechanism o Ac ion
Small in e e ing RNAs (siRNAs) a e a class o small, double-s anded non-coding RNA molecules ha play a signi ican
pa in he gene silencing p ocess RNA in e e ence [19–21]. Usually 21–23 nucleo ides (n ) long [19,21], hese en i ies
a e p oduced by he RNase III enzyme Dice du ing he p ocesses o splicing and modi ica ion o RNA [20]. Each siRNA
is composed o wo s ands —a guide (an isense) s and, which engages he a ge mRNA speci ically o silencing, and
a passenge (sense) s and ha is emo ed du ing RISC ac i a ion [22]. Dice ac i i y cu s long dsRNA subs a es in o
21-n siRNAs wi h ypical s uc u al p ope ies o 2-n (nucleo ide) 3′ o e hangs on each s and. E e y s and has a 5′
e minus monophospha e and a 3′ e minus hyd oxyl [23,24] his speci ic chemical p ope y e lec s genuine Dice
p ocessing and is necessa y o e icien silencing by he RNA-induced silencing complex (RISC) [23,24].
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The modynamic asymme y de e mines which o he s ands will be assigned as guide: he less s ably pai ed 5′ end o
he s and, usually AU- ich a he han GC- ich, is p e e en ially inco po a ed in o a gonau e (Ago) p o eins in RISC.
The ounda ional p inciple o which is well-es ablished in seminal wo k done by Kh o o a e al. (2003) and Schwa z e
al. (2003) [23], gua an ees di ec ional a ge ing. S and selec ion is also in luenced by o he ac o s, such as he na u e
o he 5′ nucleo ide (p e e en ially U/A) and he p esence o loading co ac o s o RISC (like TRBP and PACT) [23].
Chemical modi ica ions—such as 2′-O-me hyl, locked nucleic acids (LNAs), o phospho o hioa e linkages—ha e he
capaci y o enhance esis ance o nucleases [25,26], educe immune-s imula ion, and a ec s and bias, hus making
hem c i ical componen s in he de elopmen o he apeu ic siRNAs.
2.1. Biogenesis o siRNA
2.1.1. O igins o dsRNA P ecu so s
In plan s, p edominan ly in e eb a es, siRNAs a e p oduc s o RNA-dependen RNA pols (RdRPs) ha syn hesize long
dsRNAs om RNA empla es [27,28]. Dice p ocesses hese dsRNAs in he cy oplasm and hen loads i in o RISC o
media e sequence-speci ic gene silencing [28,29]. In plan cells, his is o en ini ia ed by en i onmen al s ess,
ansposon ac i i y o i al in ec ion. Unlike in mammals ha do no ha e RdRPs and i was p esumed ha hey only
p oduce exogenous siRNAs [27]. App ecia ion o his iewpoin was al e ed by he disco e y o a e endogenous
sou ces o dsRNA as ollows [28]: Re o ansposons[30]. Fo example, LINE-1 elemen s in humans p oduce
bidi ec ional polyadenyla ed RNA ansc ip s om a double p omo e sys em exp essed in bo h di ec ions o ien ed
wi hin genes and which a e he e o e copied in o complemen a y RNAs ha hyb idize o o m dsRNA [31]. Pseudogenes
con aining in e ed epea s [32].These s uc u es pe mi he o ma ion o long in amolecula hai pins [32],
eminiscen in s uc u e o shRNA p ecu so s. T ansc ip ional uni s o e lapped: An isense ansc ip ion om p o ein-
coding loci o noncoding loci may p oduce wo complemen a y RNAs, sui able o dsRNA syn hesis [33].
Endogenous sou ces o such dsRNA ha e been ound in a numbe o o ganisms. AGO2-bound small RNAs in D osophila
con ain a unique 21 n siRNA class ha includes pseudogene and in e ed epea de i ed species [30]. SiRNAs in oocy es
ha e been desc ibed in mice, a e Dice -dependen , and occasionally a ge p o ein-coding genes [33,34].
2.1.2. Dice P ocessing
In he cy oplasm, Dice diges s long double-s anded RNAs (dsRNAs) in o small in e e ing RNA (siRNA) duplexes wi h
speci ic 2-nucleo ide 3′ o e hangs and a phospho yla ed 5′ e mini in he cy oplasm en i onmen [25,26]. An siRNA
duplex consis s o a guide s and ha ac i ely is inco po a ed in o he RNA-induced silencing complex (RISC) and a
passenge s and, which will be deg aded [35].
RISC Loading and S and Selec ion
The siRNA duplex binds o AGO2, he ca aly ically ac i e mammalian A gonau e p o ein, and o ms he p e-RISC
complex [36,37]. The he modynamic asymme y be ween he duplex e mini, which d i es s and selec ion [38],
gene ally he less s able 5' end is designa ed as he guide s and. The passenge s and is subsequen ly excised and
deg aded, esul ing in he o ma ion o he ma u e RISC [39].
Ta ge Recogni ion and Gene Silencing
RISC is a ge ed o mRNAs wi h high sequence complemen a i y by he guide s and. Ta ge RNA clea age by AGO2 a a
single si e be ween nucleo ides 10 and 11 ela i e o he guide s and igge s ansc ip deg ada ion. Whils mic oRNAs
equen ly ha e misma ches and media e ansla ional ep ession, siRNAs gene ally lead o e icien mRNA clea age
wi h nea -pe ec base pai ing [40].
2.1.3. Endogenous s. Exogenous siRNAs
In i o, mos siRNA expe imen s in mammals ha e employed exogenous deli e y ei he using syn he ic siRNA duplexes
o using sho hai pin RNAs (shRNAs) exp essed om plasmid o i al ec o s [41]. shRNAs, d i en by Pol II o Pol III
p omo e called an RNAi ec o [42], a e ansc ibed in nucleus a ge ed o be p ocessed in o p e-shRNA by he
Mic op ocesso (D osha–DGCR8), hen expo in-5 ans e ed in o cy oplasmic space o dicing in o siRNA duplexes o
RISC complex.
Endogenous mammalian siRNAs a e spa sely exp essed and less es ablished as egula o y molecules ela i e o miRNAs
and piRNAs [43]. They p obably e ol ed om e olu iona y ancien an i i al de ense sys ems and he loci ha gi e ise
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 24(02), 125-148
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o hem migh s ill be unde selec i e p essu e [43,44]. The unc ional oles o mammalian endo-siRNAs, pa icula ly
hose ha a ge p o ein-coding genes, s ill need o be elucida ed in he cou se o u he esea ch [45].
2.2. Mechanism o mRNA silencing
siRNAs igge gene silencing by becoming pa o he RNA-induced silencing complex (RISC) [19,21]. A i icially
syn hesized siRNAs, gene ally 21–23 nucleo ides long wi h 2-n -long 3′ o e hangs [46–48], a e deli e ed o cy oplasm
h ough chemically modi ied deli e y agen s such as lipid nanopa icles, conjuga es, o i al ec o s [19,27,49]. One
impo an s ep is he endosomal escape as cy oplasmic localiza ion is necessa y o RISC o ma ion [49].
Figu e 1 S uc u al ea u es o exogenous siRNA. Exogenous siRNAs a e ypically designed as 19-20 bp RNA duplexes
wi h wo-nucleo ide 3' o e hangs composed o DNA nucleo ides ( ed #E07F80). The canonical 5' monophospha e and
3' hyd oxyl e mini a e p ese ed o ensu e p ope a gonau e loading and gene-silencing ac i i y
In RISC, he double-s anded siRNA is subjec o s and selec ion, wi h he passenge s and elimina ed, p ima ily ia
Ago2-ca alysed clea age o by dissocia ion wi h he help o accesso y ac o s [50]. The passenge s and is
he modynamically locked on o Ago2, he ca aly ic engine o he complex [51]. The guide s and guides RISC o mRNA
sequences ha a e complemen a y ia Wa son–C ick base pai ing [52]. Recogni ion is di ec ed by he seed egion
(posi ions 2–8 om he 5′ end o he guide s and), and silencing e iciency co ela es wi h he size o base pai ing [4].
Figu e 2 Schema ic illus a ion o he small in e e ing RNA (siRNA) media ed gene silencing
In cases o nea ly pe ec base pai ing, ypically in a coding egion, Ago2 ca alyzes endonucleoly ic clea age o he a ge
mRNA be ween he nucleo ides opposi e he guide posi ions 10 and 11. These esul ing RNA agmen s a e hen
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deg aded apidly by cellula exonucleases, including XRN1 and he exosome complex [53], he eby inducing as ,
i e e sible, and po en silencing o gene exp ession. This clea age-based pa hway is he majo o m o ac ion o siRNA
he apeu ics and designed o be ully homologous o hei a ge s. In con as , in he case o only pa ial
complemen a i y, usually in he 3′UTR, slicing ac i i y o Ago2 is no in ol ed. Ins ead, RISC ac s o ec ui e ec o
p o eins, which block ansla ion ini ia ion, induce ibosome disassembly, and p omo e mRNA decay a he le el o
deadenyla ion and decapping, equen ly wi hin p ocessing bodies (P-bodies) [25,26,54]. E en hough his mode o
ac ion seems o be mo e ypical o miRNA-media ed egula ion [55], o - a ge e ec s in he o m o non-clea age can
be elici ed by siRNAs unde limi ing complemen a i y. The e o e, siRNA-induced gene silencing is mainly ini ia ed ia
Ago2-clea age o he mRNA [54,56], leading o he subsequen seconda y e en s such as ansla ion ep ession and
a ge mRNA deg ada ion [48]. The siRNA he apeu ic oppo uni y emana es om hei abili y o manipula e his
endogenous sys em wi h speci ici y and po ency [57], depending on he success ul add essing o deli e y, s abili y and
endosome escape hu dles.
3. A gonau e 2: S uc u e, Func ion, and Role in RNAi
3.1. The a gonau e P o ein Family
The A gonau e (Ago) p o eins a e a g oup o specialized small-RNA-binding p o eins ha ac as he main e ec o s in
RNA-silencing pa hways [58]. Small RNAs mus be inco po a ed in o A gonau e-associa ed ibonucleop o ein
complexes, collec i ely known as RNA-induced silencing complexes (RISCs) [59], in o de o ca y ou hei egula o y
unc ions. A gonau es a e p ocessed in o complexes ha use small RNA guides o iden i y complemen a y nucleic acid
sequences, allowing o pos - ansc ip ional gene egula ion, ansc ip ional silencing o genomic de ense wi hin hese
complexes [59].
The name o his amily o igina es om a pheno ype desc ibed in A abidopsis haliana o a mu an wi h knockou o
AGO1 ha had abno mal lea shape simila o en acles o he oc opus A gonau a A go [60]. A gonau es a e di ided in o
wo majo subclasses, he Ago sub amily, which is simila o A abidopsis AGO1, and he Piwi sub amily, which is
homologous o D osophila PIWI p o eins [8]. Ago sub amily is widely exp essed in soma ic cell ypes, and mainly
unc ions by associa ing wi h miRNAs and siRNAs. The Ago sub amily con ains AGO1, AGO2, AGO3, and AGO4 in he
human genome [61]. O hese, AGO2 is unique in ha i e ains endonucleoly ic "slice " ac i i y o clea e a ge RNAs
while he o he pa alogs mainly exe ansla ional ep ession and mRNA des abiliza ion ac i i y [62]. Despi e a wide
conse a ion o Ago p o eins be ween species, he copy numbe is highly a iable: om one in Schizosaccha omyces
pombe [60], i e in D osophila, eigh in humans, en in A abidopsis haliana, and as many as wen y-se en in
Caeno habdi is elegans. In e es ingly, al hough se e al eme gen yeas s, including Saccha omyces Cas ellii, ha e
unc ional a gonau e genes and gene a e siRNAs by means o a di e gen Dice enzyme, he widely used model o ganism
Saccha omyces ce e isiae lacks A gonau es al oge he , along wi h canonical RNAi pa hways. On he o he hand, he
Piwi sub amily has a much mo e limi ed exp ession pa e n, mainly exp essed in ge mline cells, whe e Piwi p o eins
bound o Piwi-in e ac ing RNAs (piRNAs) silence ansposable elemen s in he ge mline o main ain genomic in eg i y
[63]. Al hough ou piwi p o eins (HIWI (PIWIL1), HILI (PIWIL2), HIWI3 (PIWIL3) and HIWI2 (PIWIL4)) a e encoded
in he human genome [64], mos s udies in mammals ha e ocused on HIWI and HILI [63]. Th ee Piwi p o eins, MIWI,
MILI, and MIWI2, a e exp essed du ing spe ma ogenesis o mice and play a key ole in ansposon egula ion. Likewise,
a gonau e p o eins ha e been de ec ed in a ious lineages o bac e ia and a chaea bu , he e, he dis ibu ion seems
a he e a ic. Comp ising a highly conse ed ou -domain a chi ec u e (N, PAZ, MID, and PIWI) [65,66], hese
p oka yo ic A gonau es (pAgos) equen ly employ DNA as a guide a he han RNA [67], using a a ie y o o eign DNA
and RNA subs a es as pa o hei de ense sys ems agains in ading elemen s (phage and plasmids) [68], al hough
Pagos ha e so a ecei ed much less a en ion ela i e o euka yo ic A gonau es [69].
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Table 1 Classi ica ion: Ago s. Piwi clades. [8,70,71]
Fea u e
AGO Clade
PIWI Clade
Small RNA
Guides
Binds siRNAs (∼21 n ) and miRNAs
(∼22 n ) o egula e gene exp ession
ia RISC.
Binds piRNAs (∼24–31 n ) o silence ansposons and
p ese e genome in eg i y, especially in he ge mline.
Exp ession
Pa e n
Ubiqui ously exp essed ac oss soma ic
cells in mos o ganisms.
P ima ily es ic ed o ge mline cells in animals
Func ion
Facili a es pos - ansc ip ional gene
silencing ia mRNA clea age o
ansla ional ep ession.
Media es ansposon silencing h ough cy oplasmic
clea age and/o nuclea silencing, o en in ol ing
he e och oma in o ma ion.
Ta ge
Recogni ion
Rules
Requi es s ic seed pai ing (guide
nucleo ides g2–g8) o a ge
ecogni ion and silencing.
Displays elaxed complemen a i y equi emen s,
ole a ing misma ches e en nea he clea age si e—
ad an ageous o comba ing apidly di e ging
ansposon.
Slice Ac i i y
Many AGO p o eins ha e
endonucleoly ic (“slice ”) ac i i y, e.g.,
human AGO2 clea es a ge mRNAs
di ec ly.
Some PIWIs (like Aub and Ago3 in D osophila) a e
slice s in he cy oplasm, bu o he s (like Piwi) lack
slice ac i i y and ac as a sca old o co-
ansc ip ional silencing.
E olu iona y
Conse a ion
AGO and PIWI a e wo phylogene ically
dis inc sub amilies wi hin he
A gonau e supe amily
PIWI p o eins a e animal-speci ic, e ol ing unique
unc ions in ge mline de ense agains ansposons
3.2. S uc u al domains o Ago2
Recen s uc u al obse a ions suppo a bi-lobe a chi ec u e o human AGO2 as de ined by he N–PAZ lobe connec ed
h ough wo lexible linke s (L1 and L2) o he MID–PIWI lobe (in wha is e e ed o as mid-ke nel exchange) [72].
Collec i ely, hese lobes c ea e a cen al cle ha binds he guide RNA, as well as i s complemen a y a ge s and,
allowing AGO2 o se e as he ca aly ic engine o RNA-induced silencing complexes (RISCs) [72].
Figu e 3 Linea ep esen a ion o human AGO2, illus a ing i s ou majo domains along wi h hei co e unc ions.
Domain
Func ion
N
Unwinds RNA duplex
PAZ
Binds 3′ o e hang o guide RNA
MID
Ancho s 5′ phospha e o guide s and
PIWI
Ca aly ic ac i i y (endonuclease/slice )
C- e minal
S uc u al ex ension (no de ined domain)
3.2.1. N- e minal Domain
The N- e minal domain plays an essen ial ole in RISC complex assembly, pa icula ly in acili a ing he unwinding o
he small RNA duplex and passenge s and elimina ion. This componen ep esen s a a e-limi ing s ep in RISC
ac i a ion. The N-domain, al hough no essen ial o RNA loading and slicing, is equi ed o assembly and unc ional
ma u a ion o RISC [73]. In addi ion, he N-domain makes a second con ibu ion o guide- a ge pai ing, by limi ing
pai ing ou side o he 16 h nucleo ide o he guide RNA, hus ensu ing ha only a speci ic loca ion is clea ed [72,74],
al hough his unc ion is no ye de ini i ely shown o euka yo ic AGO2.
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3.2.2. PAZ Domain
The PAZ domain sha ed be ween AGO2 and Dice has he oligonucleo ide/oligosaccha ide-binding (OB) old s uc u e
ha speci ically binds o he 3′ wo-base o e hang o guide RNAs. This allows o inc eased s abili y o he guide RNA
wi hin he assembly and ensu es ha hey a e o ien a ed co ec ly [72,75].S udies using mu agenesis show ha
mu a ions in he PAZ domain o AGO2 p o eins allow binding o small RNAs, bu loss he duplex unwinding abili y and
he abili y o o m e ec o cogna e RISC, indica ing an impo an ole o he PAZ domain in RISC assembly [76,77].
Figu e 4 S uc u e and schema ic o human a gonau e 2 (Ago2, PDB ID: 4Z4F), highligh ing he N, PAZ, MID, and PIWI
domains ha media e small RNA binding and a ge RNA clea age
3.2.3. MID Domain
The MID domain has a Rossmann-like e ia y s uc u e and c ea es a highly conse ed pocke ha binds he 5′
phospha e o guide RNAs, hus en o cing pola i y and s and selec ion [72,78]. S uc u al s udies ha e de ined
addi ional binding si es nea he co e ac i e si e s abilized by sul a e ions and conse ed lysine esidues (e.g., Lys599,
Lys638) and could play a ole in binding he m7GpppG cap o a ge mRNAs. This sugges s ha he MID domain p o ides
s uc u al and egula o y unc ions o AGO2 ac i i y [79].
3.2.4. PIWI Domain
The PIWI domain ac s as he ca aly ic cen e o AGO2, displays s uc u al esemblance o RNase H, and con ains a
conse ed DEDH ca aly ic e ad ha media es endo-nucleoly ic "slice " ac i i y. I is known ha his domain clea es
a ge RNAs 10 and 11 nucleo ides om he guide s and [80]. In addi ion o i s ca aly ic unc ion, he PIWI domain
con ains yp ophan-binding pocke s ha a e equi ed o he ec ui men o GW182 and o he yp ophan- ich
co ac o s essen ial o ansla ional ep ession and mRNA decay. In addi ion, he PIWI domain s eng hens he con ac s
be ween he MID–PIWI lobe and DNA o RNA subs a es, and some speci ic esidues such as Asp603 a e c ucial o
s uc u al in eg i y [72,81] .
3.3. Mechanism o AGO2-Media ed Slicing
Recen biochemical and s uc u al s udies ha e p o ided an in eg a i e model o how human AGO2 media es RNA
clea age (slicing). This can be sys ema ically classi ied in o sequen ial s ages in ol ing guide- a ge ecogni ion,
con o ma ional i ing, and ca alysis [82].Ta ge mRNAs a e i s ecognized by base-pai ing in e ac ions be ween he
seed egion (guide nucleo ides g2–g8) o he small RNA bound o AGO2. In he ini ial phase, base-pai ing in e ac ions
occu wi h he 3′ supplemen a y egion (g13–g16) a e which addi ional in e ac ions wi h he ac ion cen e o he guide
(g9–g12) ake place while he cen al egion emains unpai ed. A he ime pe ec duplexes o m be ween he seed and
3′ supplemen a y egions and he a ge , AGO2 d i es a o a ional mo emen o he 3′ supplemen a y duplex egion
[83,84]. This change enables he guide cen e (g9–g12) o base-pai wi h he a ge sequence o be de e mined i i is
complemen a y. Howe e , he lack o es ic ion on he cen al and 3′ supplemen a y segmen s allows o his
con o ma ion despi e AGO2 s abilizing he phospha e backbone o he seed egion [83]. When base-pai ing is ully
ex ended o he cen al egion o he a ge RNA, he a ge is p ecisely posi ioned wi hin he PIWI domain o AGO2
con aining he conse ed D–E–D–H ca aly ic e ad [83]. These agged esidues wo k alongside he di alen me al ions
o s imula e a wa e molecule o a nucleophilic a ack o he phosphodies e backbone. Ta ge Clea age AGO2 clea es
he a ge RNA endo-nucleoly ically be ween 10 ( = 1) and 11 (P = 5′ end o he guide RNA). Du ing his p ocess a ge
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 24(02), 125-148
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RNA is clea ed in o wo unique agmen s, which a e hen eleased and deg aded esul ing in e icien pos -
ansc ip ional gene slicing [84].
Figu e 5 Ago2-media ed RNA clea age. “Figu e adap ed om Nucleic Acids Res, Volume 50, Issue 12, 8 July 2022,
Pages 6618-6638, h ps://doi.o g/10.1093/na /gkac519
4. siRNA-Media ed Silencing o Oncogenes and Downs eam Pa hways
Speci ic supp ession o oncogenes wi h an essen ial ole in umo ini ia ion and p og ession can be achie ed using
siRNA echnology [85]. They ha e he abili y o di ec he deg ada ion o i s complemen a y oncogenic mRNAs h ough
RNA-induced silencing complex (RISC) media ion [86], leading o no p o ein ou pu . C ucially, silencing an oncogene
also dis up s he downs eam signaling pa hways i con ols, ampli ying he he apeu ic e ec [85]. This module
combines oncogenic a ge s KRAS, MYC, and BCL2 wi h hei ele an pa hways [14], emphasizing how siRNA-media ed
knockdown e-moni o s cance signals [87].
4.1. KRAS and MAPK/ERK, PI3K/AKT Pa hways
Mu a ions o KRAS a e one o he mos common oncogenic d i e s in lung, panc ea ic and colo ec al cance [88]. KRAS
s imula es p ominen signaling pa hways, speci ically MAPK/ERK and PI3K/AKT cascades ha go e n cell g ow h,
su i al and me as asis [89]. siRNA-induced deple ion o KRAS diminishes bo h ERK and AKT phospho yla ion and
subsequen umo cell g ow h and apop osis [85]. KRAS siRNAs deli e ed by nanopa icles led o subs an ial inhibi ion
o umo g ow h and me as asis in p eclinical lung cance models [90]. Fu he mo e, simul aneous KRAS ep ession
and MYC ab oga ion has esul ed in sus ained umo eg ession aising pe spec i es ha such a dual assaul may ha e
e en mo e p onounced he apeu ic e ec s [91].
4.2. MYC and PI3K/AKT Pa hway
The ansc ip ion ac o MYC ha con ols cell cycle, me abolism and apop osis ele an genes has been ound
o e exp essed in many hema ological and malignancies and solid umo s [92]. MYC exp ession is also inhibi ed by
siRNA, and esul s in ma kedly dec eased cell p oli e a ion media ed by KB-3-1 and K562 cells [93]. Biologically,
supp ession o MYC also in e e es wi h he PI3K/AKT pa hway ha is pi o al in he me abolic adap a ion and su i al
o cance cells [94]. Inhibi ion o his pa hway causes supp ession o glycolysis, dec eased biosyn hesis and inc eased
sensi i i y o apop o ic signals [95]. These da a indica e ha MYC is an impo an hub h ough which siRNA he apy can
in e sec wi h PI3K/AKT-s imula ed umo igenic signaling mechanism.
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4.3. BCL2 and Apop o ic Signaling
BCL2 is a classical oncogene whose an i-apop o ic ac i i y a e o e -exp essed in a ound 50% o all human cance s wi h
app oxima ely 50–70% o pa ien s wi h b eas cance s displaying de egula ed exp ession [96]. O e exp ession inhibi s
p og ammed cell dea h, p omo ing chemo esis ance and adio esis ance [97]. The nanoliposomal o mula ions (NL-
Bcl-2 siRNA) o siRNA-based silencing o BCL2 exe po en an i umo e ec s on es ogen ecep o -posi i e (MCF7) and
es ogen ecep o -nega i e (MDA-MB-231) b eas cance xenog a s [87]. Mechanis ically, he BCL2 inhibi ion included
in silencing would e-enable apop o ic machine y by eeing p o-apop o ic p o eins (e.g., BAX, BAK) [98], which u he
yields a adia ion- esis an s a us o umo cells upon con en ional he apies [87]. This shows how siRNA can di ec ly
e-engage in insic dea h pa hways ha cance cells e ade.
Table 2 siRNA-Media ed Silencing o Oncogenes and Downs eam Pa hways in Cance
Ta ge
(oncogene and
pa hway)
Cance model
siRNA Deli e y
Me hod
Obse ed E ec
Re .
KRAS / MAPK–
ERK, PI3K–AKT
Lung Cance
(xenog a models)
Nanopa icle-
deli e ed KRAS siRNA
Reduced KRAS exp ession, inhibi ed
ERK and AKT signaling, supp essed
umo g ow h and me as asis
[99]
BCL2 / Apop o ic
Pa hway
B eas Cance (ER⁺
MCF7, ER⁻ MDA-MB-
231 xenog a s)
Nanoliposomal BCL2
siRNA (NL-Bcl-2
siRNA)
Supp essed umo g ow h, enhanced
chemosensi i i y, es o ed apop o ic
signaling
[87]
MYC / PI3K–AKT
Leukemia (K562
cells), Ce ical
ca cinoma (KB-3-1
cells)
Syn he ic siRNA
agains c-myc
Dec eased MYC mRNA, inhibi ed
p oli e a ion, induced apop osis,
dis up ed PI3K–AKT su i al
signaling
[100]
KRAS + MYC /
MAPK–ERK and
PI3K–AKT
Lung Cance models
Combined siRNA
silencing
Mo e equen and du able umo
eg ession compa ed o single gene
a ge ing
[101]
5. Role o A gonau e 2 (Ago2) in Cance
Among he a gonau e amily, he only ca aly ically ac i e membe Ago2 has been iden i ied as a co e componen o he
RNA-induced silencing complex (RISC) and a pi o al playe in mic oRNA (miRNA)-guided gene silencing [10,72]. Ago2
has i al oles in media ing pos - ansc ip ional egula ion ha a ec no mal cellula homeos asis and cance [10,72].
Ago2 has been widely s udied in he ield o cance o i s abe an exp ession and dys egula ion in nume ous ypes o
cance s, highligh ing i s ole in umo igenesis and p og ession [102,103].
5.1. Exp ession Pa e ns o Ago2 in Tumo s Ve sus No mal Tissues
Mul iple s udies ha e shown ha Ago2 is o en up egula ed in umo issue compa ed o adjacen no mal issues.
Inc eased exp ession in bladde u o helial ca cinoma, hepa ocellula ca cinoma, glioma, hypopha yngeal ca cinoma,
ad enoco ical ca cinoma, gas ic ca cinoma, o a ian ca cinoma, and colon cance . In hese cance s, inc eased Ago2
le els co ela e wi h highe cellula p oli e a ion, mig a ion, me as asis, and o e all agg essi eness o umo s. Howe e ,
dec eased o a iable exp ession has been obse ed in melanoma wi h low Ago2 p o ein ye s able mRNA exp ession
and in ER posi i e b eas cance s whe e ac i e es ogen signaling supp essed Ago2 exp ession. Ye hese he e ogeneous
pa e ns e eal Ago2 's cance - ype-specific unc ions.
Table 3 Exp ession Pa e ns o Ago2 in Tumo s Ve sus No mal Tissues
Cance Type
Exp ession Pa e n
Compa ison wi h No mal
Re .
U o helial Ca cinoma
Up egula ed
Tumo > no mal bladde issue
[104]
Hepa ocellula Ca cinoma
(HCC)
Up egula ed
Tumo > adjacen no mal li e
issue
[105,106]
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 24(02), 125-148
140
S a emen o e hical app o al
Md Sho i Uddin*, Md Roknuzzaman Faisal, A i Abyad Hossain, Adib Azwad Hossain and Nus a Jahan B is y played
pi o al oles in shaping his e iew pape . Thei con ibu ions encompassed he comp ehensi e li e a u e e iew,
concep ual amewo k de elopmen , da a collec ion, o iginal d a p epa a ion, igu e illus a ion, and he o e all
gene a ion o ideas ha guided he di ec ion o he manusc ip .
Ms Umma Fa ama, Md A a a Rahman Koko, Tauhedul Islam con ibu ed signi ican ly h ough o mal da a analysis,
me iculous da a cu a ion, igu e illus a ion, able cons uc ion, g amma ical e inemen , so wa e- ela ed adjus men s,
and ho ough p oo eading o enhance he cla i y and quali y o he inal manusc ip .
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