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, Legend -Figure 1: MicroRNA Biogenesis and MicroRNA Modulating Strategies 641
, Canonical Biogenesis of miRNA: (adapted and modified with permission [113]) MiRNA 642 genes are transcribed by RNA polymerases in the nucleus forming large primary-miRNA, p.500
, bases) which harbours one or more stem-loop structures [5]. The RNA-binding protein (Di 644
, George syndrome Critical Region gene 8, DGCR8/Pasha) and a RNAse III endonuclease 645 (Drosha) recognizes the stemloop structures and releases the stem-loop Precursor miRNA, p.646
, ~70 nucleotides), vol.10
, Pre-miRNA is then transported out of the nucleus through 647 nuclear pore complexes (NPC) by binding to Exportin-5 (XPO5) and Ran-GTPase
, miRNA has two arms (5? and 3?) each might encode an active miRNA sequence named as miR-649
, Black strand) respectively. Once in the cytoplasm, the pre-650 miRNA loads into a pre-RISC (RNA induced silencing complex) consisting of Dicer, TAR RNA 651 binding protein (TRBP), one of the argonautes (AGO1 -AGO4) and chaperones, p.652
, Proteins, vol.8, p.9
, Dicer cleaves the stem-loop structure creating a, p.23
, Binding of seed sequence (2 -7 nucleotides at the 5?, p.655
, miRNA, highlighted in green) to the 3?-untranslated region (3?-UTR) of the target mRNA results 656 in the inhibition of protein synthesis either by messenger RNA (mRNA) destabilization (>75%) 657 and translational repression
, Other functions of miRNA and RISC: RISC complex also exerts nuclear functions by 659 shuttling between cytoplasm and nucleus via Exportin 1 and Importin 8 [18, 19, 114] and 660 possibly mitochondrial functions
, MicroRNA Modulation Strategies: Genome editing [47, vol.74
, Small Molecule miRNA 663
, , vol.66
, MicroRNA mimics and ss-siRNA, vol.82
, Antisense 664
, Oligonucleotides (ASO), vol.84
, MicroRNA Sponges (Gene Therapy), vol.81
, MicroRNA Masks 665, vol.115
, Can recombinant-AGO2 with targeting ligands acts as a RNAi delivery system?
, Legend -Figure 2: Role of miRNA in the Hallmarks of Cancer and Treatment Resistance 668
, Standard glioblastoma treatment involves radio and chemotherapy which damage DNA and 669 create double strand breaks (DSBs). Numbers 1 to 7 show various resistance mechanisms leading 670 to treatment failure. (1) Efflux pumps reduce intracellular drug concentration, vol.41, p.671
, DNA repair reverses lethal DSBs and DNA 672 damages; MGMT is a DNA repair enzyme targeted by miR-181d and miR-409, vol.24, p.673
, For detailed list of 674 resistance genes and mechanisms, see review [24]; (6) Tumour-microtubes and connexin 675 mediated invasion through exchange of resistance factors (e.g. miRNAs) helps cancer cells to 676 adapt to genotoxic treatments and surgery, vol.116, p.677
, Exosomal miRNAs (especially miR-21 and miR-29a) 681 can directly bind to Toll-like receptors (TLR7 and TLR8) in the endosomal compartment of 682 macrophages resulting in pro-metastatic inflammation, Small molecule drugs can be used to increase the expression of tumour-suppressor miRNAs and 678 block onco-miRs, vol.42, pp.39-43
, Functional significance of AGO2-miRNA complexes in GBM is yet unknown. (12) Legend 685 explaining the symbols
, Box 2: Argonaute as miRNA Delivery Systems 702
, Extracellular microRNAs are extremely stable and abundantly found in all body fluids, either 703 bound to argonaute proteins (especially AGO2) or encapsulated in extracellular vesicles (EV), p.704
, Ferreira et al showed 707 that AGO2 increased miRNA uptake in selective human endothelial cells (EC), derived from 708 cerebral arteriovenous malformation (AVM), normal and glioma endothelium, without the need 709 of any transfection agent [120]. AGO2 also protected miRNA from degradation after cellular 710 entry and they were functionally active under both in-vitro and in-vivo conditions [120]. In an 711 intracranial glioma model, intravenously injected AGO2-miR-18a complexes inhibited 712 angiogenesis by specific internalization by brain endothelium which is in direct contact with 713 blood stream [120]. The specificity of AGO2-miR uptake strongly indicates the involvement of 714 receptor mediated endocytosis, EV-associated 705 circulating miRNA and its role in cell-to-cell communication is increasingly becoming evident 706 while the functions of AGO2-associated miRNAs are explored very less
, GBM and other cancer cells which 717 makes them ideal targets for AGO2-mediated miRNA delivery and might explain the results 718 from Ferreira et al [118-120]. The AGO2-mediated RNAi delivery can be expanded to other 719 tissues by attaching ligands on its surface or producing recombinant AGO2-ligand fusion 720 proteins, see Figure 1. Also, AVM-EC can be used for AGO-miRNA delivery as it is known to 721 secrete AGO2. Unlike other delivery systems where the guide strand must be load into 722 endogenous AGO to be functional, AGO2-RNAi would be functional once reaching the 723 cytoplasm, NRP1 is highly expressed in vascular endothelial cells
, AGO:#Argonaute#is#a#core#element#of#RNA5induced#silencing#complex#(RISC)#which#comprises#of# a#single5stranded#guide#RNA,#argonaute#and#few#other#proteins.#This#complex#binds#to#the#target# mRNA,# interferes# protein# translation# or# mRNA# cleavage# resulting# in# gene# silencing.# Humans# express#four#different#AGOs#(AGO1#-#4)
, ASOs:#Anti5sense#oligonucleotides#are#synthetic#single#stranded#molecules#that#binds#to#mature# microRNA#by#complementary#base5paring.#They#act#as#microRNA#inhibitors#and#described#with# different#nomenclature#including,#antagomiRs,#anti5miR,#anti5microRNA#oligonucleotides#(AMO),# Locked#Nucleic#Acid#(LNA
, ceRNA:#Competitive#endogenous#RNAs#are#natural#miRNA5sponges,#which#includes#certain#long5 non5coding#RNAs#(lncRNA)#and#circular#RNAs#(circRNA),#that#attenuate#specific#miRNA#functions#
, Glioblastoma# are# aggressive,# grade# IV,# malignant# brain# tumours# with# most# frequent# occurrence#in#adults#and#has#poor#prognosis#(median#survival#less#than#2#years).#They#fall#under# diffuse# astrocytic# and# oligodendroglial# phenotype# with# occasional# mutation# in# IDH# gene#
, CRISPR5associated# protein)# is# a# bacterial# RNA5guided5endonuclease# complex# that# acts# as# an# immune# system# against# invading# plasmid# and# viral# DNA.# This# system# is# simplified# into# sgRNA# (single5guide# RNA)/Cas9# for# editing# genomes.# sgRNA# guides# Cas9# to# specific# locations# with# complementary# DNA# sequence.# Cas9# creates# site5specific# double5strand# breaks# which# is# subsequently# repaired# either# by# homologous# recombination# (accurate,# template5mediated)# or# nonhomologous#end5joining#(error5prone), Genome
, miRNA(mimics:#A#synthetic#double#stranded#molecule#with#identical#sequence#similarity#to#their# natural#microRNA#counterpart.#Used#for#microRNA5Replacement#therapy#and#functional#studies.# 8. TTF:(Tumour#Treating#Field#is#a#low#intensity#electric#field#of#intermediate#frequency#(~200kHz)# which# interferes# with# mitosis,# hinders# cell# division# and# consequently# induces# cell# death# in# dividing# cells.# TTF# is# produced# by# an# external,# non5invasive# transducer5array# (Optune®)# which# can#be#worn#by#patients#like#a#cap