there are more than 20 regulator of g - protein signaling ( rgs ) proteins , and rgs5 is a member of the r4/b subfamily ( 1 - 4 ) .
rgs proteins are components of g - protein ( guanine nucleotide binding protein ) coupled receptor ( gpcr ) complexes , and they act to shorten the duration of signaling resulting from ligand binding to gpcrs by acting as activators of the gtpase activity of the  subunit of g proteins ( 5 ) .
gtpase activation of rgs5 is apparently specific for the gi and gq g protein subunits ( 6 , 7 ) .
gi and gq , respectively , can inhibit adenylyl cyclase and activate phospholipase c ( 8 , 9 ) .
in addition to g proteins , there is emerging evidence that rgs proteins also interact with other proteins downstream of gpcrs ( 10 ) .
there is abundant evidence for roles of rgs5 in vascular and cardiac remodeling and blood pressure homeostasis ( 11 - 13 ) .
it has been suggested that it is a stimulator of apoptosis of endothelial cells ( 14 ) .
a role for rgs5 in various cancers has also been reported ( 15 - 19 ) .
with respect to ocular functions , it was first found that rgs5 mrna is up - regulated in hypertensive monkey eyes ( 20 ) .
subsequently , high expression of rgs5 mrna was reported in human ciliary body and trabecular meshwork ( tm ) tissues and a novel alternative spliced variant of rgs5 mrna was identified in human ocular tissues ( 21 ) .
it is a major component of the conventional outflow pathway of aqueous humor and significantly modulates outflow of this fluid from the anterior chamber to venous blood via schlemm s canal ( 22 ) .
increased tm resistance causes decreased outflow and increased intraocular pressure , the latter being a major risk factor for glaucoma ( 23 ) .
our long standing interest in glaucoma prompted a study aimed at identification of non - housekeeping genes expressed in human tm cells ( 24 ) .
a meta - analysis on tm gene expression data derived from four microarray , two cdna library , and one sage ( serial analysis of gene expression ) experiment identified only ten non - housekeeping genes that were reported to be expressed in the human tm by all studies ( 24 - 30 ) .
their identification in all studies was taken to mean that these are highly and consistently expressed non - housekeeping genes in the human tm ( 24 ) .
micrornas ( mirnas ) , which are small ( ~22 nucleotides ) single stranded non - coding rnas , are now considered major components of the cellular machinery for gene expression regulation in multicellular eukaryotes ( 31 , 32 ) .
they have important roles in various biological processes , including development , cell growth , cell signaling , and apoptosis ( 33 ) .
mirnas have been implicated in the etiology of various diseases ( 34 - 36 ) .
they act at the post - transcriptional level , by promoting mrna degradation or by having inhibitory effects on translation , and they usually exert fine tuning regulatory effects ; on average , they affect a two - fold decrease in production of target proteins ( 37 - 39 )
. each mirna can have multiple mrna targets , and the expression of up to 60% of human genes is thought to be affected by mirnas ( 40 , 41 ) .
the most critical factor in mirna targeting is complementary pairing of sequences within the 3 untranslated ( 3-utr ) regions of mrnas with a seven nucleotide seed region within the mirnas ( 41 , 42 ) .
various bioinformatics tools use this feature and more subtle criteria to predict mirnas that may target specific genes and to predict genes that an mirna of interest may target ( 43 , 44 ) .
mirna expression and the genes and pathways they affect in ocular tissues have now been investigated in several studies ( 45 - 49 ) .
we recently reported the effect of mir-204 on the expression of foxc1 and other genes in human tm cells ( 50 ) . in a preliminary effort to gain a more complete understanding of aspects of rgs5 expression in ocular tissues , we here aimed to identify mirnas which may target rgs5 mrnas
candidate mirnas to be tested for targeting rgs5 mrna were selected in three steps . initially , mirwalk was used to identify mirnas with predicted binding sites within the mrna s 3-utr segment ( ( 51 ) ; http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/ ) .
this in silico bioinformatics tool relies on information accrued from predictions made by nine ( rna22 , miranda , mirdb , targetscan , rnahybrid , pita , pictar4 , pictar5 , and diana - microt ) established programs for binding of mirnas to 3-utrs of all known human genes .
mirwalk ultimately reports predictions made by ten algorithms , including its own algorithm and those of the nine programs specified above .
thus identified , those whose expression in the eye had been experimentally validated were chosen at the second level of selection .
expression in the eye was assessed on the basis of information available at mirwalk ( http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/ ) and review of the literature ( 47 ) .
finally , seven mirnas with high prediction scores were chosen to be tested for experimental verification of targeting the 3-utr segment of rgs5 mrna by the dual luciferase assay .
an eighth mirna that is a family member of the seven selected mirnas was also tested .
mirnas were purchased either as cloned sequences within pcmv - mir vectors ( origene , rockville , md , usa ) or as un - cloned mirs ( ambion , austin , tx , usa ) .
empty pcmv vector and a scrambled mimic mirna were used as negative controls for assessing the effects , respectively , of the cloned and un - cloned mirnas that were tested .
scrambled mimic mirna does not produce identifiable effects on known human mrna functions ( reference for snord ; http://www.invitrogen.com/1/1/11216-mir-vana-mirna-mimic-negative-control-1.html ) .
the 3-utr segment of rgs5 mrna consists of 5038 nucleotides ( nm_003617 ) . to facilitate cloning , genomic dna encoding the 3-utr segment
was amplified by pcr in three shorter amplicons , rgs5 - 1 ( nucleotides 687 - 2162 ) , rgs5 - 2 ( 2899 - 3992 ) , and rgs5 - 3 ( 3995 - 4967 ) . forward and reverse primers for all amplicons were designed to include , respectively , xho1 and not1 restriction enzyme recognition sites .
the three fragments collectively contained almost all recognition sites predicted by the mirwalk analysis ( figure 1a ) .
each fragment was cloned downstream of the renilla luciferase reporter gene , directly 3 to its amino acid coding region , in psicheck2 to create vectors psicheck2- rgs5 - 1 , psicheck2-rgs5 - 2 , and psicheck2-rgs5 - 3 ( promega corporation , madison , wi , usa ) .
the psicheck2 vector , in addition to the renilla gene which is expressed under the t7 promoter , contains the firefly luciferase gene adjacent to the hsv - tk promoter . for verification
the sequences of primers used for the cloning reactions are presented in table 1 . dual luciferase assay .
nucleotide positions are with reference to the translation termination codon , and the position of the first nucleotide after that codon is considered + 1 .
the thick lines represent rgs5 - 1 , rgs5 - 2 , and rgs5 - 3 that were cloned ; the nucleotide positions at the termini of the fragments are shown .
* indicates p<0.05 , * * indicates p<0.01 , and * * * indicates p<0.001 for comparison of renilla / firefly luciferase ratios between control transfections and transfections with mirnas .
rlu , relative light units sequences of primers used for pcr - amplification of rgs5 - 1 , rgs5 - 2 , and rgs5 - 3 * xhoi recognition sites on forward primers and noti recognition sites on reverse primers are shaded the effects of the mirnas were assessed in human embryonic kidney ( hek)-293 cells ( national institute of genetic engineering , tehran , iran ) .
empty or recombinant psicheck2 vectors ( 250 ng ) were co - transfected in 24-well plates ( 2  10 cells / well ) with cloned mirnas ( 250 ng pcmv - mir dna ( origene ; mir-7 : cat # sc400648 , mir-9 : z cat # sc400676 , mir-23a : cat # sc400294 , or mir-23b : cat # sc400295 ) ) or with un - cloned mirnas ( final concentration of 25 nm ( ambion ; mir-96 : pm10422 , mir-182 : pm12369 , mir-204 : pm11116 , or mir-211 : pm10168 ) ) using lipofectamine ltx reagent ( invitrogen , carlsbad , ca , usa ) .
forty eight hours after transfections , renilla and firefly luciferase activities were measured using dual luciferase assays ( promega corporation ) according to the manufacturer s instructions .
expression of cloned mirnas was verified by real time pcr using snord-47 mirna as control .
candidate mirnas to be tested for targeting rgs5 mrna were selected in three steps . initially , mirwalk was used to identify mirnas with predicted binding sites within the mrna s 3-utr segment ( ( 51 ) ; http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/ ) .
this in silico bioinformatics tool relies on information accrued from predictions made by nine ( rna22 , miranda , mirdb , targetscan , rnahybrid , pita , pictar4 , pictar5 , and diana - microt ) established programs for binding of mirnas to 3-utrs of all known human genes .
mirwalk ultimately reports predictions made by ten algorithms , including its own algorithm and those of the nine programs specified above .
thus identified , those whose expression in the eye had been experimentally validated were chosen at the second level of selection .
expression in the eye was assessed on the basis of information available at mirwalk ( http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/ ) and review of the literature ( 47 ) .
finally , seven mirnas with high prediction scores were chosen to be tested for experimental verification of targeting the 3-utr segment of rgs5 mrna by the dual luciferase assay .
an eighth mirna that is a family member of the seven selected mirnas was also tested .
mirnas were purchased either as cloned sequences within pcmv - mir vectors ( origene , rockville , md , usa ) or as un - cloned mirs ( ambion , austin , tx , usa ) .
empty pcmv vector and a scrambled mimic mirna were used as negative controls for assessing the effects , respectively , of the cloned and un - cloned mirnas that were tested .
scrambled mimic mirna does not produce identifiable effects on known human mrna functions ( reference for snord ; http://www.invitrogen.com/1/1/11216-mir-vana-mirna-mimic-negative-control-1.html ) .
the 3-utr segment of rgs5 mrna consists of 5038 nucleotides ( nm_003617 ) . to facilitate cloning , genomic dna encoding the 3-utr segment
was amplified by pcr in three shorter amplicons , rgs5 - 1 ( nucleotides 687 - 2162 ) , rgs5 - 2 ( 2899 - 3992 ) , and rgs5 - 3 ( 3995 - 4967 ) . forward and reverse primers for all amplicons were designed to include , respectively , xho1 and not1 restriction enzyme recognition sites .
the three fragments collectively contained almost all recognition sites predicted by the mirwalk analysis ( figure 1a ) .
each fragment was cloned downstream of the renilla luciferase reporter gene , directly 3 to its amino acid coding region , in psicheck2 to create vectors psicheck2- rgs5 - 1 , psicheck2-rgs5 - 2 , and psicheck2-rgs5 - 3 ( promega corporation , madison , wi , usa ) .
the psicheck2 vector , in addition to the renilla gene which is expressed under the t7 promoter , contains the firefly luciferase gene adjacent to the hsv - tk promoter . for verification
nucleotide positions are with reference to the translation termination codon , and the position of the first nucleotide after that codon is considered + 1 .
the thick lines represent rgs5 - 1 , rgs5 - 2 , and rgs5 - 3 that were cloned ; the nucleotide positions at the termini of the fragments are shown .
* indicates p<0.05 , * * indicates p<0.01 , and * * * indicates p<0.001 for comparison of renilla / firefly luciferase ratios between control transfections and transfections with mirnas .
rlu , relative light units sequences of primers used for pcr - amplification of rgs5 - 1 , rgs5 - 2 , and rgs5 - 3 * xhoi recognition sites on forward primers and noti recognition sites on reverse primers are shaded
the effects of the mirnas were assessed in human embryonic kidney ( hek)-293 cells ( national institute of genetic engineering , tehran , iran ) . empty or recombinant
psicheck2 vectors ( 250 ng ) were co - transfected in 24-well plates ( 2  10 cells / well ) with cloned mirnas ( 250 ng pcmv - mir dna ( origene ; mir-7 : cat # sc400648 , mir-9 : z cat # sc400676 , mir-23a : cat # sc400294 , or mir-23b : cat # sc400295 ) ) or with un - cloned mirnas ( final concentration of 25 nm ( ambion ; mir-96 : pm10422 , mir-182 : pm12369 , mir-204 : pm11116 , or mir-211 : pm10168 ) ) using lipofectamine ltx reagent ( invitrogen , carlsbad , ca , usa ) .
forty eight hours after transfections , renilla and firefly luciferase activities were measured using dual luciferase assays ( promega corporation ) according to the manufacturer s instructions .
expression of cloned mirnas was verified by real time pcr using snord-47 mirna as control .
based on mirwalk and review of the literature , targeting of the 3-utr of rgs5 mrna has not yet been experimentally validated for any mirna .
seven hundred seventy six mirnas were computationally predicted to target this 3-utr by the algorithm used by mirwalk or at least one of the mirna prediction programs included therein .
four hundred thirty five of these met our threshold selection criterion of being predicted by at least four programs .
of these , the expression of at least 55 has been experiment- tally evidenced in the eye ( http://www.umm .
as em - pirical evaluation of all these was beyond the scope of the present study , seven of the mirnas were chosen on the basis of predictive scores to be tested by the dual luciferase assay .
the seven mirnas were mir-7 , mir-9 , mir-23a , mir-23b , mir-96 , mir-182 , and mir-204 .
an eighth mirna ( mir-211 ) was also selected because it belongs to the same family as mir-204 which is known to be an important mirna with respect to various ocular functions , and because mir-211 has been shown to be relevant to glaucoma related functions of the tm ( 45 , 46 , 49 , 52 ) . the distribution of the predicted recognition sites of these mirnas on the 3-utr of rgs5 mrna and on these cloned fragments is shown in figure 1a .
the sequences of the 3-utr fragments cloned in psicheck2- rgs5 - 1 , psicheck2-rgs5 - 2 , and psicheck2-rgs5 - 3 were shown to be correct .
the number of predicted target sites for each mirna in each 3-utr fragment tested was either one or two .
expression of the cloned mirnas after transfection into hek293 cells was confirmed by real time pcr ( not shown ) .
the results of effects of the mirnas as assessed by dual luciferase assays are presented in figure 1b and table 2 .
effects on fragment rgs5 - 2 were comprehensively tested , but effects of mir-182 on rgs5 - 1 and mir-23a , mir-23b , and mir-182 for rgs5 - 3 were not assessed .
results of replicate assays were impressively similar as evidenced by small standard deviations in the renilla / firefly luciferase ratios ( figure 1b ) .
renilla / firefly luciferase ratios were not significantly different in cells transfected with only psicheck2 backbone , scrambled mirna , or any of the mirnas being tested ( not shown ) .
this means that the differences observed in the presence of psicheck2 vectors containing 3-utr sequences are due to mirna effects that were elicited only in the presence of those sequences . among the thirteen assays performed with psicheck2 vectors containing 3-utrs , the tested mirnas decreased target gene expression at a statistically significant level in seven of the reactions
this was evidenced by decreased renilla / firefly luciferase ratio in presence of the tested mirna as compared to control reactions ( figure 1b ) .
unexpectedly , mir-182 caused increased renilla / firefly luciferase ratio in the presence of psicheck2-rgs5 - 2 .
the effect of mir-204 in presence of psicheck2-rgs5 - 2 ( 1.4-fold ) was notable because it reached statistical significance at the level of p<0.0002 .
mir-23a and mir-23b , that share common seed sequences , are each predicted to have four recognition sites in the 3-utr of rgs5 .
effects of these mirnas on the single site present in rgs5 - 3 were not tested ; however they caused decreased renilla luciferase expression in the presence of rgs5 - 1 and rgs5 - 2 that contain the remaining recognition sites ( figure 1b , table 2 ) .
effects of mirn-23a and mir-23b on rgs5 - 2 , respectively , were 2.0- and 1.8-fold .
having two as compared to one recognition site did not necessarily result in increased mirna effect ( table 2 ) . among the five assays in which statistically significant effects of mirnas were not observed , four had two recognition sites for the tested mirna ; among the seven reactions in which the mirnas reduced renilla luciferase expression , four had one recognition site and three had two .
we have shown by the luciferase assay that all mirnas tested except mir-182 had a highly reproducible effect on down regulation of the renilla luciferase gene when the gene was associated with a partial 3-utr segment of the rgs5 gene .
the up - regulatory effect of mir-182 in the presence of rgs5 - 2 may have been indirect and caused by effects of this mirna on other regulatory components active in hek-293 cells .
the observed effects of mir-7 , mir-9 , mir-23a , mir-23b , mir-96 , mir-204 , and mir-211 on rgs5 - 1 , rgs5 - 2 , and/or rgs5 - 3 were modest . although we did not observe that having two rather than one binding site was correlated with increased down regulation ; it is generally believed that the presence of multiple target sites does increase effects of mirnas . in this light , it is possible that the presence of the entire 3-utr segment ( 5038 200 nucleotides ) downstream of the renilla luciferase gene would have a more pronounced effect .
mirnas mir-7 , mir-9 , mir-23a , and mir-23b are each predicted to have four or five target sites within the entire 3-utr segment , whereas there was a maximum of two target sites in the segments tested ( figure 1a ) .
known ocular related functions of the mirnas here tested , were reviewed with the consideration that the functions may shed light on their putative effects on rgs5 expression .
mir-7 has been reported to be among the mirnas whose levels where changed in tm cells upon treatment with mechanical stress which is a factor that potentially contributes to the pathogenesis of glaucoma ( 53 ) .
mir-9 is necessary for proper eye development , and has a role in inflammatory responses of retinal microglia cells ( 54 ) .
mir-23a and/or mir-23b are thought to be essential for proper eye development , to protect retinal epithelial cells from oxidative damage , and to be involved in choroidal neovascularization ( 54 )
. both are present in human aqueous humor and may have roles in the etiology of cataract ( 47 , 48 ) . among the mirnas tested ,
mir-204 is likely to be the most important with respect to ocular development and ocular functions .
some of its functions are mediated through the important eye transcription factors meis2 and foxc1 ( 46 , 50 ) .
mir-204 is known to be expressed in murine eye tissues from early developmental stages through adulthood , and has been shown to regulate multiple aspects of eye development in the medaka fish ( 45 , 46 , 55 ) ; it is expressed in human tm cells ( 49 , 52 ) .
the mirna may have roles in the etiology of cataract and is involved in the regulation of apoptosis , endoplasmic reticulum stress response , and inflammation in human tm cells ( 48 , 49 ) .
the above findings do not provide an exact signal for the biological significance of the effects of the mirnas on rgs5 expression .
nevertheless , given that rgs5 is definitively expressed in the human tm ( 23 , 25 - 27 ) , and that it is up - regulated in hypertensive eyes ( 20 ) , the results presented here justify further investigations on the regulatory functions of the mirnas on rgs5 expression and on genes downstream of g - protein coupled receptors that associate with gi and gq .
this having been said , we emphasize that our results are preliminary , and that the effects of the mirnas need to be studied at the protein level , and most importantly , in ocular relevant cells such as tm cells .
 objective(s):an earlier meta - analysis on gene expression data derived from four microarray , two cdna library , and one sage experiment had identified rgs5 as one of only ten non - housekeeping genes that were reported to be expressed in human trabecular meshwork ( tm ) cells by all studies . 
 rgs5 encodes regulator of g - protein signaling-5 . 
 the tm tissue is the route of aqueous fluid outflow , and is relevant to the pathology of glaucoma . 
 micrornas constitute the most recently identified components of the cellular machinery for gene regulation in eukaryotic cells . given our long standing interest in glaucoma , we aimed to identify mirnas that may target rgs5.materials and methods : eight mirnas were selected for study using bioinformatics tools and available data on mirnas expressed in the eye . 
 their effects were assessed using the dual luciferase assay . 
 3-utr segments of rgs5 mrna were cloned downstream of a luciferase coding gene in psicheck2 vectors , and these were co - transfected with each of the mirnas into hek293 cells.results:the outcomes evidenced that one or more of the segments are in fact targeted by mir-7 , mir-9 , mir-96 , mir-23a , mir-23b , mir-204 , and mir-211 . 
 down regulations by the mirnas were statistically significant . 
 the effect of mir-204 is considered particularly important as this mirna is well known to regulate eye development and to affect multiple ocular functions.conclusion:our results justify further studies on regulation of rgs5 expression and rgs5 downstream functions by these mirnas . 
Introduction
Materials and Methods
In silico analysis for miRNA selection
Preparation of psiCHECK2 constructs containing 3-UTR sequences
Plasmid transfections and dual luciferase assays
Results
Discussion
Conclusion