in recent years , studies have shown that micrornas ( mirnas ) play a significant role in brain development .
for instance , mir-134 is localized to the synaptodendritic area in rat hippocampal neurons and is associated with synaptic development , maturation , and plasticity . in mammals , mir-9 is a neural - specific mirna and was not found to be expressed in any other tissues .
mir-9 is widely expressed in neural precursor cells and has lower expression in matured postmitotic neurons .
importantly , mir-9 regulates neurogenesis at the midbrain - hindbrain boundary in zebrafish brain models .
mir-124 is recognized as a brain - specific mirna and is the most abundant mirna in the mouse brain .
mir-124 is expressed in the mature neurons of adult mouse brain and is upregulated in differentiating neurons .
mirnas are also implicated in various neuropathologies , neurodegenerative diseases , and intellectual disabilities . 

mir-344 is a novel mirna that was first reported in 2004 as one of the many mirnas found in rat cortical neurons .
a study by royo et al . showed that mir-344 was one of the imprinted small rna genes at the prader - willi locus of the transgenic mouse model .
it was predicted to map between ndn and snrpn genes located at the prader - willi domain on mouse chromosome 7 .
however , mir-344 was not detected in homologous human prader - willi domain at 15q11q13 or any nonrodent genomes .
mir-344 family had nine known isoforms , mir-344a to mir-344i . however , limited studies were carried out on a few of these isoforms and implicated with roles in various pathological disorders .
mir-344h was one of the mirnas identified in a study that observed mirna expressional alteration of a mouse hippocampus after a traumatic brain injury .
another study also had showed that mir-344b , mir-344d , and mir-344h were downregulated in a neurotoxin - induced apoptosis in mouse mn9d cell line .
studies revealed that mir-344 is expressed during mouse brain development at e15.5 [ 10 , 15 ] and in the adult mouse brain .
recently , mir-344 - 3p was reported to be expressed in neural - specific regions during mouse embryonic development .
although evidence had shown that mir-344 , particularly mir-344b and mir-344c , was expressed in the developing mouse brain , the function of these mirnas had yet to be ascertained .
besides the developing brain , mir-344 had been implicated in mouse adipocyte differentiation [ 17 , 18 ] .
a high throughput microarray study revealed that mir-344 was one of the 29 mirnas identified which inhibits adipogenesis via wnt signally pathway .
moreover , mir-344 had been implicated in huntington disease and acute respiratory distress syndrome animal models .
mir-344 was found downregulated in the brain of huntington disease mouse models   while it was upregulated in the lungs of the rat model for acute respiratory distress syndrome . in this study , we profiled the expression of mir-344b and mir-344c in mouse brain development via in situ hybridization at both embryonic and postnatal stages .
a quantitative analysis was also carried out to determine the expression levels of these mirnas in the central nervous system and multiple organs .
bioinformatics analysis was employed to predict the potential downstream target genes of mir-344b and mir-344c .
a luciferase protein suppression assay was then performed as a downstream measurement of mirna efficiency on a target sequence .
mice were kept in a 12 h light/12 h dark cycle with access to unlimited food and water ; they were also not pharmacologically treated .
female mice were considered pregnant with the presence of vaginal plug and the gestation time was designated as embryonic day ( e ) 0.5 .
pregnant mice were culled , and embryos were harvested at e11.5 , e13.5 , e15.5 , e17.5 , and postnatal day ( p ) 1 .
adult mice were anaesthetized with isoflurane inhalation followed by cervical dislocation , and the brain and multiple organs were harvested . the number of mice ( n ) used in each group was seven , where n = 2 for in situ hybridization and n = 5 for real - time quantitative polymerase chain reaction ( rt - qpcr ) .
animals in this study were used in accordance with the animal care and use committee , universiti putra malaysia ( upm / fpsk / pads / br - uuh/00469 ) .
embryonic mouse brains at e11.5 , e13.5 , e15.5 , e17.5 , and p1 were harvested and fixed in 4% paraformaldehyde ( pfa ) for 24  h in a refrigerated shaker . as for adult mouse brains
, mice were first perfused with 4% pfa via transcardiac perfusion following anesthetization with 0.05  ml/10  g body weight of sodium pentobarbital ( sigma ) through intraperitoneal injection .
a 25-gauge needle was inserted at the apex of the heart , and the right atrium was lacerated with scissors .
the tissues were collected and fixed with 4% pfa with gentle agitation for 2  d at 4c .
the tissues were then subjected to standard tissue processing procedures ( incubation with 10% formalin , 80% ethanol , 95% ethanol ( twice ) , 100% ethanol ( thrice ) , and xylene ( thrice ) for 1  h each , and incubation with paraffin wax ( twice ) for 2  h each ) using a semiclosed bench top tissue processor ( leica tp1020 ) .
the tissues were then embedded in a paraffin block in a tissue embedding station ( leica eg1160 ) .
briefly , paraffin sections of the brain ( 8  m ) were deparaffinized , rehydrated , and fixed with pfa , followed by digestion with proteinase k ( 1.2  g/l in 0.05  m tris - hcl ( ph 7.5 ) and 0.05  m edta ) .
then , sections were refixed with pfa and acetylated with 0.1  m triethanolamine , 0.178%  ( v / v ) concentrated hcl , and 0.25%  ( v / v ) acetic anhydride .
prehybridization was performed in a humidified chamber of 50%  ( v / v ) formamide at 65c as previously described .
sections were covered with prehybridization buffer consisting of 50%  ( v / v ) deionized formamide , 3x saline sodium citrate buffer , 1x denhardt 's solution , 1x phosphate - buffered saline , 1  mg / ml yeast total rna , and 1  mg / ml herring sperm dna .
after 2  h of prehybridization , custom - made mir-344b , mir-344c , or mir - scrambled locked nucleic acid probes ( exiqon ) were added to the hybridization buffer to a final concentration of 0.020  pmol/l .
hybridization was carried out in an oven at 57c for 16  h as per the manufacturer 's recommendations .
after hybridization , sections were washed with serial concentrations of saline sodium citrate buffer ( 2x , 1x , 0.5x , and 0.1x ) for 15  min each at 48c .
then , they were rinsed with 0.1x saline sodium citrate and preblocking solution ( 0.1  m tris - hcl ( ph 7.5 ) , 0.15  m nacl , and 240  g / ml levamisole ( sigma ) ) for 5  min each at room temperature . in a humidified chamber , sections were blocked with 5%  ( v / v ) heat - inactivated fetal calf serum , 1%  ( v / v ) blocking powder in maleate buffer , and 0.1%  ( v / v ) tween-20 for 1  h at room temperature . after blocking ,
sections were incubated with 0.00015  u ( 1  :  1000 dilution ) of fab fragments anti - digoxigenin antibody conjugated with alkaline phosphatase ( roche diagnostics ) in blocking buffer for 1  h. the sections were later incubated with alkaline phosphatase buffer ( 0.1  m tris - hcl ( ph 9.5 ) , 0.1  m nacl , 0.05  m mgcl2 , 1% ( v / v ) tween-20 , and 240  g / ml levamisole ) for 10  min .
after blocking , 0.06x nitro - blue tetrazolium/5-bromo-4-chloro-3-indolyl - phosphate , toluidine salt ( nbt / bcip ) stock solution ( roche diagnostics ) in blocking buffer was added and incubated with sections for 58  d or until purple coloration sufficiently developed .
then , the sections were washed in tris - edta buffer , ph 8.0 ( 0.01  m tris - hcl ( ph 7.5 ) and 0.001  m edta ( ph 8.0 ) ) , for 10  min at room temperature and washed with 3 changes of fresh 1x pbs for 3 minutes at each step . for colocalization study
the sections were then dehydrated in a series of ethanol concentrations and xylene for 3  min each and subsequently mounted in dpx mounting medium and covered with a glass cover slip .
for immunofluorescence study , the sections were incubated in primary antibody , polyclonal rabbit anti - tuj1 ( sigma - aldrich ) , with dilution factor of 1  :  1000 in a humidified chamber at 4c for 16 hours . then
, the sections were washed thrice with 1x pbs and incubated with secondary antibody , anti - rabbit alexaflour 488 ( thermofisher scientific ) , with dilution factor of 1  :  1000 in a humidified chamber for four hours at room temperature .
the antibody was discarded and the sections were rinsed thrice in 1x pbs and subsequently mounted in prolong gold antifade mounting media with dapi ( thermofisher scientific ) and covered with a coverslip .
the slides were left to dry in the dark at room temperature before keeping in 4c for storage .
total rna was isolated from c57bl/6 whole mouse brain and multiple organs ( pancreas , thymus , skin , stomach , lung , spleen , liver , adipose tissue , ovary and fallopian tubes , testes , small intestine , heart , kidney , large intestine , and skeletal muscle ) using trizol ( invitrogen ) according to the manufacturer 's protocol .
a total of 2.0  g of total rna was reverse transcribed into cdna using 0.05  m of in - house designed stem - loop primer ( 5-gttggctctg gtaggatgcc gctctcaggg catcctacca gagccaaaca cwgtc-3 ) with 2.5  m oligo ( dt)20 ( invitrogen ) and a superscript iii reverse transcriptase kit ( invitrogen ) with modifications to the manufacturer 's protocol . the stem - loop primer was added after the initial denaturation step at 65c , and cdna was synthesized using stem - loop pulsed reverse transcription as previously described .
first strand cdna contained a target site for universal reverse primer ( 5-gtaggatgcc gctctcagg-3 ) and universal probelibrary ( upl ) probe # 21 ( roche diagnostics ) , which were used together with specific forward primers for mir-344b ( 5-ggaccattta gccaaagcct-3 ) and mir-344c ( 5-gcgtgatcta gtcaaagcct-3 ) , respectively .
pre - pcr steps , qpcr steps , and subsequent analysis were performed using lightcycler 480 software version 1.5 ( roche diagnostics ) .
four - data point standard curves for all analyses were constructed based on equally pooled pre - pcr products .
primers used to amplify u6 small nuclear rna were 5-cgcttcggca gcacatata-3 ( forward ) and 5-aaatatggaa cgcttcacgaat-3 ( reverse ) .
qpcr results for mir-344b and mir-344c were normalized against the u6 small nuclear rna used as endogenous controls .
one - way analysis of the variance was used to compare expression levels among the groups of samples .
four data mining tools , miranda ( august 2010 release ) , mirdb ( version 4.0 , january 2012 ) , targetscan mouse ( release 6.2 , june 2012 ) , and diana micro - t cds ( version 5.0 ) , were used to identify the candidate target genes of mir-344b and mir-344c . predicted downstream target genes were ranked according to the criteria set for each bioinformatics tool . both conserved and nonconserved sites were used to cover a wide range of target sites across various species .
threshold values were set to determine the specificity and sensitivity of the prediction to identify the target genes using the four data mining tools . using miranda bioinformatics , the predicted data with good mirsvr score and nonconserved mirna was downloaded .
however , with mirdb [ 23 , 24 ] , target genes with a target score between 60 and 100 were selected for further analysis . in targetscan mouse ,
a minimum total context+ score of 0.12 , irrespective of site conservation , was used in the analysis , and the top 40% of the selected genes were subjected to further analysis . on the other hand ,
target genes with a minimum mitg score threshold of 0.75 were selected for diana micro - t cds [ 26 , 27 ] .
the predicted genes from all data mining tools were then compiled into a venn diagram   to identify common target genes .
target genes that were predicted by at least three tools were subjected to expression analysis in allen brain atlas .
genes that were expressed in embryonic and adult stages were considered for the next downstream analysis .
this set of predicted target genes was then subjected to the protein analysis through evolutionary relationships classification system .
genes that played a role in transcription or gene regulation were further streamlined and selected for validation .
hek293 cells were cultured in dulbecco 's modified eagle 's medium ( sigma ) supplemented with 10% fetal bovine serum , 1% l - glutamine , 1% nonessential amino acids , 1% sodium pyruvate , and 1% penicillin - streptomycin ( all from gibco ) .
cells were plated in a 12-well plate and incubated at 37c with 5% co2 until they reached 9095% confluence .
transfection of plasmids with mir-344b , mir-344c , olig2 , and otx2 , purchased from genecopoeia , usa , was performed using lipofectamine3000 ( invitrogen ) as per the manufacturer 's protocol .
, hek293 cells were cotransfected with 0.8  g of the pezx - mt01 plasmid carrying the 3utr of the target gene and 0.8  g of the pezx - mr04 plasmid carrying pre - mirnas in six different transfection groups ( group 1 : negative control luciferase plasmid + pre - mirnas ; group 2 : target gene + mir - scramble ; group 3 : target gene ; group 4 : negative control luciferase plasmid ; group 5 : target gene + pre - mirnas ; group 6 : mock control ) .
negative control luciferase plasmid contained the firefly luciferase gene without 3utr , while ultrapure water was used as mock control .
firefly and renilla luciferase were measured at 24 , 36 , 48 , and 60  h after transfection using a luc - pair mir luciferase assay kit ( genecopoeia ) .
to investigate the expression profiles of mir-344b and mir-344c during mouse brain development , we performed in situ hybridization on the sagittal plane of mice at e11.5 , e13.5 , e15.5 , e17.5 , p1 , and p86 ( n = 2 ) .
mir-344b was expressed throughout the entire embryonic brain at e11.5 ( figure 1(a ) ) and e13.5 ( figure 1(b ) ) . at e15.5
, stronger expression was observed in derivatives of the telencephalon ( cerebral cortex and hippocampal formation ) compared to other parts of the developing brain ( figure 1(c ) ) .
subsequently , at e17.5 , expression of mir-344b decreased and was not detectable from postnatal stages ( figures 1(d)1(f ) ) onwards . on the other hand
, mir-344c showed strong expression throughout the brain from e11.5 to p1 ( figures 1(m)1(r ) ) . in the adult stage ( p86 ) , mir-344c was not expressed in the brain with exception of the olfactory bulb and granular cell layer of the cerebellum ( figure 1(r ) ) .
mir - scramble was used on age - matched mouse brains as a negative control ( figures 1(g)1(l ) and 1(s)1(x ) ) . to further profile the spatial expression of mir-344b during brain development , sagittal sections of three primary areas of the brain , namely , the telencephalon ( developing cerebral cortex ) , mesencephalon ( developing midbrain ) , and metencephalon ( developing cerebellum ) , were further evaluated ( figures 2 and 3 ) . at e11.5
, mir-344b expression was observed in the ventricular zone of the developing cerebral cortex ( figure 2(a ) ) . at e13.5 and e15.5 ,
its expression was found in the ventricular zone , intermediate zone , and cortical plate but not in the molecular zone of the cerebral cortex ( figures 2(d ) and 2(g ) ) . at e17.5
, mir-344b was expressed exclusively in the cortical plate ( figure 2(j ) ) . at p1 , mir-344b was not detectable in any cortical layer of the cerebrum ( figure 2(m ) ) and continued to show no expression in the adult cerebral cortex .
in the mesencephalon , mir-344b was expressed throughout the developing midbrain at e11.5 ( figure 2(b ) ) , e13.5 ( figure 2(e ) ) , and e15.5 ( figure 2(h ) ) .
expression of mir-344b was no longer detectable at late embryonic stages , e17.5 ( figure 2(k ) ) , and postnatal stages p1 ( figure 2(n ) ) and p86 . in the developing cerebellum
, mir-344b was expressed in the cerebellar neuroepithelium at e11.5 ( figure 2(c ) ) , e13.5 ( figure 2(f ) ) , and e15.5 ( figure 2(i ) ) . at e17.5 , mir-344b was lowly expressed in the purkinje and granular cell layer of the developing cerebellum ( figure 2(l ) ) . as development progressed to p1 , mir-344b was not expressed in any layer of the cerebellum ( figure 2(o ) ) , nor was it expressed in the adult cerebellum .
in contrast to mir-344b , mir-344c was globally expressed throughout brain development from e11.5 to p1 and decreased in adulthood ( figures 1(g)1(l ) ) . at e11.5
, mir-344c was expressed in both the ventricular zone and preplate of the developing cerebral cortex ( figure 3(a ) ) . at e13.5 and e15.5 , it continued to be expressed in the ventricular zone , intermediate zone , and cortical plate but not in the marginal zone of the developing cerebral cortex ( figures 3(d ) and 3(g ) ) . at e17.5 , mir-344c continued to be expressed in the cortical plate and intermediate zone but not the marginal zone ( figure 3(j ) ) . at p1
, mir-344c was expressed in layers i , ii , and iii of the cerebral cortex ( figure 3(m ) ) . 

mir-344c was expressed throughout the developing midbrain from e11.5 to p1 ( figures 3(b ) , 3(e ) , 3(h ) , 3(k ) , and 3(n ) ) but not in the adult stage at p86 . in the developing cerebellum , mir-344c was expressed in the cerebellar neuroepithelium at e11.5 ( figure 3(c ) ) , e13.5 ( figure 3(f ) ) , and e15.5 ( figure 3(i ) ) . at e17.5 and p1 ,
mir-344c was expressed in the molecular , purkinje , and granular cell layers of the developing cerebellum ( figures 3(l ) and 3(o ) ) . besides being expressed in adult ( p86 ) cerebral cortex ,
the mirna was expressed in the molecular , mitral , and granular cell layers ( figure 4(b ) ) . in the cerebellum ( figure 4(c ) ) , mir-344c was lowly expressed in the granular cell layer ( figure 4(d ) ) . in comparison
, mir-344b was not expressed in both the adult mouse olfactory bulb and cerebellum . to quantify the expression of mir-344b and mir-344c ( figure 5 ) , we performed stem - loop rt - qpcr in embryonic mouse whole brain and multiple organs of adult mice .
using whole brain samples ( n = 5 ) , a significant difference in mir-344b expression was found at e11.5 , e13.5 , e15.5 , e17.5 , p1 , and adult brain samples ( p < 0.0001 ; figure 5(a ) ) .
mir-344b then continued to express until the adult stage ( figure 5(a ) ) , which was in contrast with results from our in situ hybridization study .
we further compared expression of mir-344b among various adult mouse organs and found no significant difference in its expression among them ( p = 0.0609 ; figure 5(b ) ) .
the adult mouse pancreas had the highest expression of mir-344b , followed by the brain , skeletal muscle , skin , small intestine , large intestine , ovary and fallopian tubes , lung , thymus , kidney , heart , stomach , spleen , liver , adipose tissue , and testes ( figure 5(b ) ) .
the same analysis was performed on mir-344c using embryonic mouse whole brain and multiple adult mouse organs . using whole brain samples ( n = 5 )
, a significant difference was observed in mir-344c expression at e11.5 , e13.5 , e15.5 , e17.5 , or p1 , or in adult brain samples ( p < 0.0001 ; figure 5(c ) ) .
mir-344c was significantly increased from e11.5 to e13.5 and continued to express until the adult stage ( figure 5(c ) ) .
we further compared the expression of mir-344c in multiple adult organs and found significant differences in its expression among them ( p < 0.0001 ; figure 5(d ) ) .
the adult mouse brain had the highest mir-344c expression , followed by the pancreas , skin , kidney , liver , large intestine , stomach , lung , adipose tissue , thymus , heart , small intestine , ovary and fallopian tubes , spleen , testes , and skeletal muscle .
it was also found that the expression of mir-344c in thymus , heart , small intestine , ovary and fallopian tubes , spleen , testes , and skeletal muscle was significantly lower compared to the brain ( figure 5(d ) ) . to identify the genes targeted by mir-344b and mir-344c , we employed four online bioinformatics databases , namely , miranda , mirdb , targetscanmouse , and diana micro - t cds .
the venn diagram summarized the number of genes targeted by mir-344b ( figure 6(a ) ) and mir-344c ( figure 6(b ) ) using the different databases .
a total of 539 genes were predicted by miranda , while 162 genes were predicted by mirdb .
targetscanmouse predicted 885 genes , and diana micro - t cds predicted 302 genes . to increase the specificity of the predictions , we focused on genes predicted by three or four databases .
based on these criteria , a total of 63 target genes were predicted and seven genes were identified as transcription factors ( jmjd1c , med14 , olig2 , kbtbd7 , tox , st18 , and zranb2 ) . of these genes , only four were expressed in both embryonic and adult mouse brain according to allen brain atlas ( jmjd1c , olig2 , tox , and st18 ) . as for mir-344c ,
nine genes were commonly identified by using a similar set of criteria ( figure 6(b ) ) .
these genes were otx2 , pnpla8 , erich1 , fam118a , tmpo , olfr1426 , pou4f1 ( also known as brn3a ) , tmem131 , and stau1 .
of these nine genes , only otx2 and pou4f1 were transcription factors expressed in both embryonic and adult mouse brains .
we chose olig2 and otx2 genes as potential targets of mir-344b and mir-344c , respectively , for further validation .
olig2 has been established as a neuronal and glial fate determinant , and otx2 plays a role in formation and patterning of the rostral head .
we conducted a luciferase assay to determine whether olig2 and otx2 were targeted by mir-344b and mir-344c , respectively .
the luciferase assay was performed 24 , 36 , 48 , and 60  h ( n = 3 ) after transfection in six different cotransfected groups to investigate expression of the mirna and chimeric target gene over time .
transfection groups 1 and 2 served as negative controls that determined the specificity of the mirna to the target .
groups 3 and 4 were negative controls that determined the effect of host intrinsic factors on the chimeric targets .
group 5 was the assay test group , while group 6 was a mock control for background luminescence .
fluorescence micrographs were captured to determine the transfection efficacy ( figure 7(a ) ) . using green fluorescent protein as an indicator
stem - loop rt - qpcr was carried out to determine the overexpression of mir-344b and mir-344c ( figure 7(b ) ) .
it showed that both mirnas were present 24 , 36 , and 48  h after transfection after being normalized against hmbs .
normalized luciferase bioluminescence was not downregulated at 24 , 36 , 48 , or 60  h when the chimeric target gene olig2 was cotransfected with mir-344b ( figure 7(c ) ) .
similar to mir-344b , the normalized luciferase bioluminescence level was not affected by overexpression of mir-344c ( figure 7(d ) ) .
bioinformatics study had predicted olig2 and otx2 were target genes of mir-344b and mir-344c , respectively .
however , these target genes were not targeted by their respective mirnas as indicated in the luciferase assay .
therefore , we sought to determine the localization of mir-344b and mir-344c in the cells of the developing brain .
closer observations of ish brain sections revealed that both mir-344b and mir-344c were localized to the nuclei instead of the cytoplasm of the cell ( figures 8(a ) and 8(b ) ) .
fluorescence microscopy was performed using uv channel for dapi staining and fitc channel for tuj1 staining .
sagittal sections of e15.5 mouse cerebral cortex were observed with dapi ( figures 9(a ) , 9(d ) , 10(a ) , and 10(d ) ) , tuj1 ( figures 9(b ) , 9(e ) , 10(b ) , and 10(e ) ) , and brightfield microscopy ( figures 9(c ) , 9(f ) , 10(c ) , and 10(f ) ) .
dapi is known for staining the cell nucleus to distinguish individual cells . on the other hand ,
both expressions of mir-344b and mir-344c were found coexpressed with dapi ( figures 9(g ) , 9(j ) , 10(g ) , and 10(j ) ) as opposed to tuj1 staining where green fluorescence was only found at the periphery of the cell ( figures 9(h ) , 9(k ) , 10(h ) , and 10(k ) ) . merged images of three different channels confirmed the locality of mir-344b and mir-344c in the nucleus of a neuronal cell ( figures 9(i ) , 9(l ) , 10(i ) , and 10(l ) ) .
in this study , we demonstrated comprehensive spatiotemporal expression of mir-344b and mir-344c throughout mouse brain development via in situ hybridization .
the expression profiles for mir-344b and mir-344c were generally similar with slight differences at select brain regions or time points .
our findings concur with previous studies that showed mir-344 - 3p was expressed in embryonic and adult mouse brain .
liu et al . performed time point in situ hybridization using whole embryos mounted at earlier embryonic stages ( e9.5e11.5 ) and whole brain sections at e15.5 , e18.5 , and adult stages .
our analysis provides further insight into mir-344b and mir-344c at other time points ( e11.5 , e13.5 ) , as well as their expression in multiple adult mouse organs .
furthermore , a previous study by ling et al .   showed two mature isoforms of mir-344 - 3p ( mir-344b and mir-344c ) were expressed in the whole developing mouse brain ( e15.5 ) via northern blot .
let-7 , one of the first mirnas discovered , was found to be expressed in the neuroepithelium of e9.5 whole - mount mouse embryos , showing strong expression during neural differentiation processes .
mir-124 was shown to promote neurogenesis in the cerebral cortex   and regulate neurite growth during neuronal differentiation .
a different review also suggested that overexpression of mir-9 alters migration and proliferation processes of neural precursors .
the roles of spatiotemporally expressed mir-344b and mir-344c in brain development , however , are yet to be determined and warrant further characterization . at higher magnifications , mir-344c was found globally expressed across the developing mouse brain .
in addition , mir-344b and mir-344c were expressed throughout the brain all sections , suggesting a wide regulatory role for these mirnas during brain development , such as neuronal proliferation , migration , and differentiation .
this expression pattern may suggest a possible role as housekeeping mirna in maintaining basic cellular processes , as described elsewhere . in the adult mouse brain ( p86 ) , only mir-344c was expressed and found localized to the olfactory bulb and subgranular zone of the cerebellum .
interestingly , mir-344 - 3p was also found to be primarily expressed in the olfactory bulb and cerebellar cortex of the adult mouse brain . as mir-344c was expressed in the olfactory bulb , a brain region for odour recognition , mir-344c may be involved in transmission , integration , and processing of olfactory signals . in the cerebellum ,
mir-344c was found in the subgranular zone , which gives excitatory outputs to purkinje cells .
subsequent stem - loop rt - qpcr analysis of mir-344b and mir-344c was performed to validate our in situ hybridization findings .
both mir-344b and mir-344c had significantly increased from e11.5 to e13.5 and their expression remained in a steady state until adulthood .
stem - loop rt - qpcr is a specific and sensitive approach to quantify individual mirna present in the brain .
the expression of mir-344b and mir-344c may be widely diffused in the brain and it was not earlier detected via in situ hybridization , which was performed on a specific plane or section of the brain .
in addition to the whole mouse brain , we also performed similar analyses on mir-344b and mir-344c in various organs of the adult mouse .
comparison of multiple adult mouse organs showed the adult pancreas highly expressed both mir-344b and mir-344c .
mir-344b was lowly expressed in adult mouse testes while skeletal muscles have the lowest expression of mir-344c . besides the brain
, other studies have shown that mir-344 is expressed in the pancreas , lungs , and adipose tissue [ 18 , 19 ] , which concur with our findings in the current study .
in contrast , mirna array analysis suggested that mir-344 was expressed specifically in the brain when compared to liver and heart tissues of the adult mouse .
our study predicted olig2 and otx2 as the most probable targets of mir-344b and mir-344c , respectively .
the prediction model was limited to transcription factors involved in the regulation of dna transcription processes , which is the most common form of gene control .
it was used as a preliminary study to understand the functional role of mir-344b and mir-344c .
olig2 has been reported to regulate mammalian brain development as a neuronal and glial cell determinant .
furthermore , olig2 has antineurogenic properties and maintains multipotent neural progenitor cells . as the predicted target of mir-344c , otx2 has been shown to be localized in the nuclei of cells of the olfactory bulb .
otx2 is known to play a role in the formation and patterning of the developing brain .
our subsequent attempt to validate olig2 and otx2 as downstream targets of mir-344b and mir-344c unexpectedly did not concur with our earlier predictions , as chimeric olig2 and otx2 were not suppressed by their respective mirnas in the luciferase suppression assay .
both mir-344b and mir-344c were localized to the nucleus , suggesting that these mrnas were not direct targets of these mature mirnas .
although olig2 and otx2 were not validated as the targeted genes , other potential targets genes ( supplementary tables 1 and 2 , available online at http://dx.doi.org/10.1155/2016/1951250 ) predicted in the study warrant a more extensive validation in order to elucidate with the potential functional role of mir-344b and mir-344c . a closer look into the expression profiles revealed that both the mir-344b and mir-344c were localized in the nuclei of neuronal cells , suggesting that they may function in nuclei rather than cytosol in a noncanonical manner .
higher magnification on these neuronal cells showed that mir-344b and mir-344c expressions were unevenly distributed in the nucleus , with an average of 5  foci per nucleus .
paraspeckles are the rna - protein structures found in the interchromatin space of a mammalian cell .
they are also the vital subnuclear domain to control gene expression by retaining nuclear mrna .
however , more studies are required to validate the potential role and mechanisms of mir-344b and mir-344c in the cell nucleus .
the noncanonical role of mirnas has been described before , where mature mirnas were transported back into the nucleus via importin-8 . in the nucleus
this prevents further downstream mechanisms related to mirna biogenesis [ 47 , 48 ] . in the absence of nuclear targets
emerging evidence had suggested that nuclear mirnas play a role in a noncanonical manner to regulate the biogenesis and function of other noncoding rnas . a study by tang et al .
had shown that mir-709 is localised in the mouse nucleus and binds to mir-15a/16 - 1 recognition element and inhibits further processing of mir-15a/16 - 1 primary transcript ( pri - mir-15a/16 - 1 ) into mir-15a/16 - 1 precursor ( pre - mir-15a/16 - 1 ) . a different study by zisoulis et al .
revealed that mature let-7 was required in the association of protein argonaute - like gene 1 ( alg-1 ) to pri - let-7 transcripts in the nucleus of caenorhabditis elegans .
disrupted alg-1-pri - let-7 binding caused an increased pri - let-7 in the nucleus but decreased mature let-7 in cells .
these findings reinforced the hypotheses that mirnas regulate the biogenesis of other mirnas as well as its own .
in conclusion , our study shows that mir-344b and mir-344c are spatiotemporally expressed in the developing mouse brain . in multiple adult mouse organs
while mir-344b and mir-344c predicted downstream targets were olig2 and otx2 , respectively , these target genes were not validated via luciferase suppression assay .
further investigation to their expression pattern revealed that both mirnas are expressed in the nucleus when counterstained with eosin .
immunofluorescence staining confirmed that mir-344b and mir-344c were expressed in the nucleus of the neurons .
 micrornas ( mirnas ) are small noncoding rna known to regulate brain development . the expression of two novel mirnas , namely , mir-344b and mir-344c , was characterized during mouse brain developmental stages in this study . in situ hybridization analysis 
 showed that mir-344b and mir-344c were expressed in the germinal layer during embryonic brain developmental stages . 
 in contrast , mir-344b was not detectable in the adult brain while mir-344c was expressed exclusively in the adult olfactory bulb and cerebellar granular layer . 
 stem - loop rt - qpcr analysis of whole brain rnas showed that expression of the mir-344b and mir-344c was increased as brain developed throughout the embryonic stage and maintained at adulthood . 
 further investigation showed that these mirnas were expressed in adult organs , where mir-344b and mir-344c were highly expressed in pancreas and brain , respectively . 
 bioinformatics analysis suggested mir-344b and mir-344c targeted olig2 and otx2 mrnas , respectively . 
 however , luciferase experiments demonstrated that these mirnas did not target olig2 and otx2 mrnas . 
 further investigation on the locality of mir-344b and mir-344c showed that both mirnas were localized in nuclei of immature neurons . in conclusion , mir-344b and mir-344c were expressed spatiotemporally during mouse brain developmental stages . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion