mirnas ( micrornas ) are endogenous non - coding rna ( ncrna ) molecules of 22 nt , which inhibit target gene expression by either inducing mrna degradation or by blocking protein translation . some mirnas have important cellular functions , including cellular differentiation , proliferation , and apoptosis.2 , 3 the seed region is present from bases two to seven of the mature mirna , and it is especially important for recognition of target mrna and proper regulation of protein expression .
the most abundantly expressed mirna in the cornea and lens is mir-184.4 , 5 mir-184 effects its biological function through the competitive inhibition of mir-205 when binding to the mrna of  inppl1 ( inositol polyphosphate - like 1 ) and itgb4 ( integrin , beta  4 ) .
reported that decreased levels of mir-184 are responsible for the aberrant activation of wnt signaling in ischemia - induced retinal neovascularization .
in addition , an earlier study reported that mir-184 is implicated in ocular neovascularization in the oir ( oxygen - induced retinopathy ) model and the non - ischemia retinopathy ( laser - induced choroidal nv [ neovascularization ] ) model .
further , it is reported that mir-184 is involved in a competitive rna network in control of mouse secondary cataract disease .
an mir-184 inhibitor can attenuate secondary cataract expansion , migration of lens epithelial cells , and signs of epithelial - to - mesenchymal transition , such as -smooth muscle actin expression .
recently , studies have reported a heterozygous c - to - u transition ( r.57c > u ) in the seed region of mir-184 in a three - generation northern ireland family with keratoconus and cataracts , a family with edict ( endothelial dystrophy , iris hypoplasia , congenital cataract , and stromal thinning ) syndrome , and a five - generation family with cataracts and varying corneal abnormalities , including severe keratoconus and non - ectatic corneal thinning , from galicia , spain.6 , 10 , 11 , 12 however , no mutation was detected within the genomic region of mir-184 in keratoconus patients from saudi arabia .
the biological function of the m - mir-184 ( mutant mir-184 , r.57c > u ) is still not clear in ocular disease . with the wide - scale adoption of high - throughput sequencing techniques , circrnas ( circular rnas ) were recently described as a novel type of endogenous ncrna , and they represent a potential research hotspot in the field of rna .
circrnas derived from the exon and intron of genes have been identified in all domains of life , including eukaryotes , archaea , bacteria , and viruses.14 , 15 functional studies have found that circrnas can act as a sponge for certain mirnas ; an example of this is cirs-7 , which harbors more than 70 conventional mir-7 binding sites .
this new circrna / mirna regulatory interaction has already generated an interesting new field of study .
however , no study has addressed whether mature mirna or its mutant counterparts can influence the expression of circrna . in an effort to enrich our understanding of the m - mir-184-mediated progression of ocular disease , we explored a variety of biological entities produced only in the presence of m - mir-184 .
furthermore , since circrnas have been shown to have a sponge role in interaction with mirna in cells , we also sequenced libraries of circrna obtained from the same biological samples . in this study
, we attempted to describe the expression of previously identified genes and circrnas and identify those that may be differentially regulated by m - mir-184 in ocular disease .
thus , the mir-184 , m - mir-184 , or a nc ( negative control , a random sequence mirna mimic molecule that has been extensively tested in human cell lines and tissues and validated not to produce identifiable effects on known mirna function ) was transfected into hle ( human lens epithelial ) cells , and the effects on circrna and the cellular transcriptome were investigated .
a total of six samples ( n  = 2 mir-184/m - mir-184/nc ) was sequenced after rrna depletion . in total from all 6 samples , 214.6 million raw reads
were produced , in pair end , with an average of 35 million reads per sample .
after quality trimming , 211.6 million high - quality reads remained ( data s1 ) .
the proportion of total reads in the six mirna treatment transcriptome libraries that mapped to the genome ranged from 90.51% to 91.81% .
the distribution of the numbers of degs ( differentially expressed genes ) across the six mirna treatment cells is shown in figure  1a .
we found 1,063 degs between hle - mir-184 and hle - m - mir-184 cells .
moreover , there were 674 degs identified when comparing hle - mir-184 and hle - nc cells .
a go - based enrichment analysis of these genes indicated that protein phosphorylation ( go : 0006468 ) was the most significantly enriched term ( p  = 2.75e06 ) .
furthermore , 973 degs were identified when  comparing hle - m - mir-184 and hle - nc cells , and the biological process macromolecule modification ( go : 0043412 ) was  shown to be the most significantly enriched go term ( p  =  3.79e06 ) .
only 33 genes showed common differential expressions in comparisons of mir-184 versus nc , m - mir-184 versus nc , and mir-184 versus m - mir-184 , indicating that hle cells have a different response to mir-184 , m - mir-184 , and nc treatment ( data s2 ) .
we aimed to perform a global analysis of the degs between the hle - mir-184 and hle - m - mir-184 and the hle - nc cells .
as shown in figure  1b , there were 496 downregulated and 477 upregulated genes in hle - m - mir-184 cells .
however , there were only 246 downregulated and 428 upregulated genes in hle - mir-184 cells .
the number of overlapping downregulated and upregulated genes between hle - mir-184 and hle - m - mir-184 were 112 and 198 , respectively , leaving 384 unique downregulated degs in hle - m - mir-184 cells and 134 unique downregulated degs in hle - mir-184 cells .
further , a minimum count after normalization of ten reads , absolute fold change 1.5 , and a corrected p value  0.05 was set as the threshold for significant differential expression .
in addition to the 26 overlapping downregulated genes , the numbers of the unique downregulated genes in hle - m - mir-184 and hle - mir-184 are 20 and 7 separately .
the much higher proportion of downregulated genes in the hle - m - mir-184 ( figure  1c ) implies that m - mir-184 has a broader spectrum of targets when compared to mir-184 .
moreover , the most significantly downregulated gene , plbd2 ( phospholipase b domain containing 2 , log2 fold change 2.3769 , q value 5.65e53 ) , has one perfect seed match with m - mir-184 and the most upregulated gene was ina , associated with morphogenesis of neurons ( internexin neuronal intermediate filament protein , alpha , log2 fold change 2.1782 , q value 0.019968 ) .
furthermore , these were different from the most downregulated gene , epb41l5 ( erythrocyte membrane protein band 4.1 like 5 , log2 fold change 1.9299 , q  value 1.11e16 ) and the most upregulated gene foxp4 ( forkhead box p4 , log2 fold change 1.7735 , q value 5.61e26 ) in the hle - mir-184 cells .
there were 16 significantly degs between mir-184- and m - mir-184-treated cells . among these , nine were membrane and calcium ion association genes , including epb41l5 ( erythrocyte membrane protein band 4.1 like 5 ) , cacng7 ( calcium channel , voltage - dependent , gamma subunit 7 ) , s100a16 ( s100 calcium - binding protein a16 ) , coro2b ( coronin , actin - binding protein , 2b ) , zdhhc18 ( zinc finger , dhhc - type containing 18 ) , cdh24 ( cadherin 24 , type 2 ) , sv2a ( synaptic vesicle glycoprotein 2a ) , wrb ( tryptophan - rich basic protein ) , and mpp2 ( membrane protein , palmitoylated 2 ) ; three are enzymatic genes , including aldh4a1 ( aldehyde dehydrogenase 4 family , member a1 ) , aldh5a1 ( aldehyde dehydrogenase 5 family , member a1 ) , and pip4k2c ( phosphatidylinositol-5-phosphate 4-kinase , type ii , gamma ) kinase .
the expression level of aldh4a1 was 1.74-fold higher and aldh5a1 was 1.74-fold lower in m - mir-184- than mir-184-treated cells , and a kegg ( kyoto encyclopedia of genes and genomes)-based pathway analysis of aldh4a1 and aldh5a1 returned to the same alanine , aspartate , and glutamate metabolism pathway , implying an interesting relationship between these two genes .
one was an ubiquitin protein gene klhl8 ( kelch - like family member 8) , two were growth factor genes igfbp3 ( insulin - like growth factor - binding protein 3 ) and egr3 ( early growth response 3 ) , and one was lincrna ( large intergenic ncrna ) rp11 - 482m8.1 ( figure  1d ; data s3 ) . among the 16 genes ,
56% were membrane- and calcium ion - relevant genes ( figure  1e ) , indicating that the m - mir-184 mainly disorders membrane and calcium ion functions .
next , we computed all possible interactions of these 16 degs with mir-184 and m - mir-184 seed sequences using miranda .
all genes that were downregulated by m - mir-184 presented a perfect seed match with m - mir-184 seed sequence ( 7-mer - m8 , ccatcc ) .
moreover , the genes that were downregulated by mir-184 also contained a perfect seed match with the mir-184 seed sequence ( 7-mer - m8 , ccgtcc ) .
various stress conditions can induce apoptosis of the lens epithelial cells , leading to eventual cataract formation.17 , 18 , 19 we investigated the changes in apoptosis genes across the six mirna treatment groups .
the distribution of the expression of different apoptosis genes are shown in data s4 . among the 96 genes known to be associated with apoptosis ( taqman low density array configuration , life technologies )
, there were no different apoptosis - associated genes across mir-184 , m - mir-184 , and nc treatment cells .
further , we found one apoptosis gene , casp7 , upregulated in hle - mir-184 and hle - m - mir-184 when compared to hle - nc cells ( figure  1f ) .
it appears that the m - mir-184 does not affect the fate of hle cells by disordering the apoptotic pathway .
further , the apoptosis rate of hle cells transfected with mir-184 , m - mir-184 , or nc was determined by flow cytometry .
results indicated that there was no difference in the rate of apoptosis across these three samples ( figure  2f ) . to identify putative mirna - inducible circrnas , we isolated rna from hle cells treated for 24  hr with mir-184 , m - mir-184 , or nc , and we performed two rounds of rna sequencing using previously described procedures .
we identified a total of 20,522 circrna candidates in the six mirna - treated cells using rna sequencing .
as shown in figure  2a , apart from a few antisense , lincrna candidates and others , most putative circrnas overlapped known ncrna , protein - coding , and antisense transcripts . in addition , of the 20,522 candidates , only 579 are listed in the circbase database ( figure  2b ) .
based on the circrnas identified in both biological replicates , there  were 2,000 distinct circrna candidates in hle - mir-184 cells , 1,879 circrna candidates in hle - m - mir-184 cells , and 1,916 circrna candidates in hle - nc cells .
overall , a total of 2,367 circrnas was identified after removing the duplicate circrnas between samples ( data s5 ) .
the detailed distribution and the length of the identified circrnas are shown in figures 2c and 2d .
further , no significant difference in the numbers of circrnas originating from each chromosome was found ( figure  2e ) .
the quantitation of circrna raw counts was normalized using tpm  ( transcripts per million ) normalization .
several circrnas appeared differentially expressed but none of them displayed a corrected p value  0.05 .
taken together , the individual circrna expression seems to be highly  random across the mir-184 , m - mir-184 , and nc treatment cells .
next , we quantified the number of canonical mirna seed sequences in each circrna as follows : 7-mer - perfect match to the 6-nt mirna seed augmented ; 7-mer - a1-perfect match to the 6-nt mirna seed augmented by an a at target position 1 ; and 7-mer - m8-perfect match to the 6-nt mirna seed with an additional match to nucleotide 8 of the mirna .
we counted a total of three seed pairings for mir-184 or m - mir-184 against all circrna sequences identified .
of 2,367 circrnas , there were 149 circrnas that had a mir-184 binding site , and the distribution of these circrnas in the three different treatment groups was random .
but the numbers of binding sites in circrna that matched with m - mir-184 were less than those for mir-184 .
this indicated that the mir-184 and m - mir-184 binding site can exist in the same circrna . in summary ,
the interaction between mir-184/m - mir-184 and circrna is compatible and not exclusive , which indicates that the mechanism of interaction between mirna and circrna may be complex .
as alus are the most successful active tes ( transposable elements ) in the human genome , in terms of copy number , and they occupy two - thirds of the sines ( short interspersed nuclear elements ) , we tested whether alus contributed to the sequence of circrnas . to investigate this , we divided sines into two subgroups , alus and mirs ( miniature inverted repeats ) , and we investigated them separately .
after running the sequences in the repeatmasker program , no repeat sequences were found in 587 circrnas , but the vast majority ( 1,780 circrnas ) contained at least one repeat sequence .
the four major types ( line [ long interspersed nuclear element ] , sine , ltr element , and dna element ) of tes are shown in data s6 .
results indicated that sines accounted for 87.33% of tes in circrnas and 80.41% of all of the sines contained at least one alu sequence .
figure  3 shows the kernel density distribution of these four different te types in circrnas derived from data s6 .
the percentage was converted to a scale from zero to one and plotted with the kernel density function using r statistical software .
figures 3a and 3 g suggest that masked bases were mainly composed of sines and alus .
the relationships of different tes were then used in a pearson correlation coefficient analysis . as shown in figure  4 ,
the masked bases of  circrnas were correlated with sines and lines ( r  = 0.512 and r  = 0.564 , respectively ) .
there was low correlation between masked bases and ltr elements ( r  = 0.164 , p  < 0.05 ) , but there was no correlation between masked bases and dna elements ( r  = 0.036 , p  > 0.05 ) . since m - mir-184 ( r.57c >
u ) appeared in familial hereditary ocular diseases , including keratoconus and cataracts , we decided to focus our data integration analysis on this mutant mirna .
of the 19 significantly downregulated degs in hle - m - mir-184 when compared to hle - nc cells , 12 possessed at least one perfect seed match in their 3  utr with m - mir-184 .
a go - based enrichment analysis of these genes returned the molecular function binding ( go : 0005488 ) to be the most significantly enriched ( p  = 0.032 ) .
thus , the genes plbd2 , sv2a , rab11fip4 ( rab11 family - interacting protein 4 [ class ii ] ) , aldh5a1 , klhl8 , pip4k2c , egr3 , dusp2 ( dual - specificity phosphatase 2 ) , mpp2 , ephb3 ( eph receptor b3 ) , fkbp4 ( fk506-binding protein 4 , 59  kda ) , and prdm1 ( pr domain - containing 1 , with znf domain ) all demonstrated a perfect seed match with m - mir-184 in their 3 utr , indicating that their expression may be inhibited by m - mir-184 directly ( data s7 ) .
although some of these genes are not really informative , we noticed that aldh5a1 , a mitochondrial nad ( + ) -dependent succinic semi - aldehyde dehydrogenase , and gabra3 , a gaba - a receptor , were downregulated in hle - m - mir-184 cells .
both aldh5a1 and gaba ( -aminobutyric acid ) associate with the alanine , aspartate , and glutamate metabolism pathway .
it is reported that the ssadh ( succinic semi - aldehyde dehydrogenase ) deficiency is a rare autosomal recessive disorder , and it affects the degradation of gaba , leading to intractable seizures and psychomotor retardation .
studies have also shown that inherited disorders of gaba metabolism , including ssadh and gaba - t ( gaba transaminase ) deficiencies , are associated with developmental delay , hypotonia , and epilepsy . furthermore , because the 3 utr of aldh5a1 has five perfect putative seed matches with m - mir-184 , the downregulation of aldh5a1 may be directly caused by m - mir-184 .
as there are no putative seed matches on the gabra3 3 utr , the downregulation of gabra3 may be regulated by m - mir-184 indirectly .
furthermore , we analyzed the nine upregulated genes in hle - m - mir-184 ( data s7 ) .
l - glutamate is also an important intermediate in the alanine , aspartate , and glutamate metabolism pathway .
it is notable that grik5 , aldh5a1 , and gabra3 all affect this alanine , aspartate , and glutamate metabolism pathway .
the specific expression changes of the grik5 , aldh5a1 , and gabra3 were also investigated by real - time pcr .
the results indicated that mrna levels of aldh5a1 decreased 0.619   0.068-fold ( p  < 0.05 ) , gabra3 decreased 0.883   0.017-fold ( p  < 0.05 ) , and girk5 increased 1.616   0.054-fold ( p 
< 0.01 ) in hle - m - mir-184 cells ( figure  5a ) .
the girk5 gene expression increased 2.056   0.165-fold ( p  < 0.05 ) , but aldh5a1 and gabra3 were unaffected in the hle - mir-184 ( figure  5b ) .
moreover , the mrna expression of girk5 was greater in hle - mir-184 compared with hle - m - mir-184 ( figure  5c ) .
western blot analysis indicated that aldh5a1 and gabra3 expression was reduced and girk5 was unaffected when hle was transfected with m - mir-184 ( figure  5d ) . taken together , we are now able to formulate the following hypothesis : the overexpression of m - mir-184 in hle cells directly inhibits aldh5a1 expression and downregulates gabra3 , disordering the alanine , aspartate , and glutamate metabolism pathway ( figure  5e ) . in the absence of aldh5a1 ,
the insufficiency of succinate may cause disorder of the citrate cycle and ultimately lead to ocular disease .
the mirnas are small ncrnas binding to complementary sequences in the 3 utr of target mrna transcripts , inhibiting mrna translation or promoting mrna degradation .
mir-184 located on the q  arm of chromosome 15 is known to play a key role in neurological development and apoptosis and is highly expressed in mouse brain ,  mouse corneal epithelium , lens , and retinal pigment epithelium.24 , 25 , 26 also , mir-184 can be oxidatively modified by ros ( reactive oxygen species ) and mismatch with the 3 utrs of bcl - xl and bcl - w .
the mismatch of oxidized mir-184 with bcl - xl and bcl - w is involved in the apoptosis of rat heart cell line h9c2 and mouse models .
furthermore , m - mir-184 is found in families with edict syndrome or cataracts and varying corneal abnormalities . how the m - mir-184 influences ocular disease is not clear . recently , evidence has suggested that circrna can act as a sponge for certain mirnas .
so we focused on describing the differential expression of mrnas and circrnas in cells expressing m - mir-184 in an effort to elucidate a possible mechanism for ocular disease .
our analysis reports 16 significant degs between m - mir-184 and mir-184 treatments , nine of which are membrane and calcium ion genes .
moreover , we report 47 significant degs between hle - mir-184 and hle - nc and 63 significant degs between hle - m - mir-184 and hle - nc cells .
we found that the number of downregulated degs in hle - m - mir-184 cells is almost three times higher than in hle - mir-184 cells .
results indicated that m - mir-184 downregulates a broader spectrum of genes when compared to the wild - type mir-184 .
also , the upregulation of grik5 and downregulation of aldh5a1 and gabra3 in hle - m - mir-184 cells suggest that there is some differential regulation of the alanine , aspartate , and glutamate metabolism pathway , which may be linked to ocular disease .
the apoptosis of hle cells is a common cause of non - congenital cataract development in humans and animals .
our data investigated a global change in the apoptosis genes across the six mirna treatment cells .
there are no significant differences in the expression of these genes in any of the groups .
we only found casp7 to be upregulated in hle - mir-184 and hle - m - mir-184 when compared to the control .
further , we have shown that there was no difference in apoptosis rate across the three groups .
it appears that the m - mir-184 is not associated with the apoptotic pathway when compared to mir-184 .
although the sequencing data and real - time pcr results showed that grik5 was highly expressed in hle - m - mir-184 cells , the protein expression was not confirmed by western blot .
both mrna and protein levels of aldh5a1 and gabra3 were downregulated in hle - m - mir-184 cells , but there was a greater degree of aldh5a1 downregulation than that of gabra3 .
integration of these different data types leads us to the hypothesis that the overexpression of m - mir-184 in hle cells directly inhibits aldh5a1 expression , indirectly downregulating gabra3 .
a kegg enrichment analysis shows that these two genes are related to the alanine , aspartate , and glutamate metabolism pathway .
fonnum and henke reported that there is a marked reduction of glutamate and aspartate in the optic nerve and the superficial tectal layers after retinal ablation .
also , there is a good correlation between the topographical distribution of glutamate carboxylase activity and the concentration of gaba in the tectum .
there is evidence indicating that the dysregulation of aldh5a1 , gaba , and grik5 is associated with epilepsy , psychomotor retardation , developmental delay , and hypotonia.22 , 23 obviously , this hypothesis provides a new perspective for the association of m - mir-184 with ocular disease .
moreover , our study reports the first prediction of circrna expression patterns in the presence of m - mir-184 and mir-184 .
our prediction reveals that , within a single cell type , circrna expression patterns appear globally variable .
total circrna expression and the number of chromosomal origins for the circrna also appear to be random under our conditions .
we found that the mir-184 and m - mir-184 can bind the same circrna , although the numbers of binding sites for the mutant form seem lower than for the wild - type mirna .
our analysis suggests that the interaction between these mirnas and the circrna is compatible and not exclusive .
further , of the total 2,376 circrnas , 1,780 are observed within tes , indicating that this is a common phenomenon .
most of the tes were composed of sines ( 87.33% ) , and alus accounted for 80.41% of these sines .
these results suggest a tendency for masked bases in circrna to be composed of alus .
further , sines show a high correlation with alus ( r  = 0.965 ) and masked bases show correlation with sines ( r  = 0.512 ) and lines ( r  = 0.564 ) after pearson correlation coefficient analysis . taken together
, our study shows that circrnas are abundant in alu sequences and have mir-184 and m - mir-184 binding sites ; but , we have not identified a specific biological function for these ncrnas .
our study presents an innovative data integration analysis in which mrna , mir-184/m - mir-184 , and circrnas are studied .
we provide a catalog of predicted circrnas in hle cell lines , which may be of relevance for other research groups .
the identification of the alanine , aspartate , and glutamate metabolic pathway as a target for m - mir-184 offers a new avenue for understanding ocular disease progression ; but , further complete proteomic and metabolomic profiling is needed to elucidate how m - mir-184 influences ocular disease through disorder of aldh5a1 and gabra3 expression and the long - term effects of m - mir-184 on cells , especially regarding potential clinical application .
culture reagents and molecular biology products including opti - mem reduced serum medium were purchased from gibco ( life technologies ) .
the synthesized mirna mimics ( mir-184 and m - mir-184 ) and the negative control were from genepharma .
hle cells were maintained as a monolayer in a humidified incubator containing 5% co2 at 37c .
the cells were cultured in growth medium comprising dmem / f-12 50/50 ( dmem / han s f-12 50/50 mix ; corning ) with l - glutamine , 15  mm hepes , and 10% fbs ( fetal bovine serum ) . transfections were done using 30  nm mir-184 , m - mir-184 , and the nc in hle cells at a cell density of 1   10 cells .
after the cells had been transfected with mirnas for 24  hr , total rna was extracted using trizol reagent according to the manufacturer s instructions ( ambion , life technologies ) . rna degradation and contamination
rna concentration was measured using qubit rna assay kit in qubit 2.0 flurometer ( life technologies ) .
rna integrity was assessed by rna nano 6000 assay kit of the bioanalyzer 2100 system ( agilent technologies ) .
a total amount of 3  g rna per sample was used as input material for the rna sample preparations .
sequencing libraries were generated using next ultra rna libraryprep kit for illumina ( new england biolabs ) , following the manufacturer s recommendations .
the clustering of the index - coded samples was performed on a cbot cluster generation system using truseq pe cluster kit v3-cbot - hs ( illumina ) , according to the manufacturer s instructions .
after cluster generation , the library preparations were sequenced on an illumina hiseq2500 platform and 125-bp paired - end reads were generated .
the raw data from this paper have been submitted to the genebank  database https://www.ncbi.nlm.nih.gov/sra/ under accession numbers : srp077606 , srp077680 , and srp077681 .
rna - seq ( rna sequencing ) reads were trimmed to the first 80  bp and then aligned to the ensembl human genome using bowtie v2.2.3 using the default parameters .
the paired - end clean reads were then aligned to the reference genome using tophat v2.0.12 .
a total of 5  g rna was treated with epicenter ribo - zero rrna removal kit and rnase r ( epicenter ) .
sequencing libraries were generated using the next ultra directional rna libraryprep kit for illumina ( neb ) , following the manufacturer s recommendations .
the clustering of the index - coded samples was performed on a cbot cluster generation system using hiseq pe cluster kit ( illumina ) .
after cluster generation , the library preparations were sequenced on an illumina hiseq 4000 platform , and 150-bp paired - end reads were generated .
briefly , the rna - seq reads mapping to the human genome were discarded , retaining only the spliced reads .
next , we extracted 20mers from both ends and aligned them independently to find unique anchor positions within spliced exons . finally , the anchor alignments were extended to the original read sequence alignments , and the inferred breakpoint was flanked by the required gu / ag splice signals .
genome references used for all mapping and subsequent analyses were from the ensembl human genome , release_77 .
all circrnas identified from sequencing data were annotated in the circbase database . following the primary analysis , each biological entity ( mrna and circrna )
the mrna raw counts were normalized using fpkm ( fragments per kilobase of transcript sequence per million base pairs sequenced ) normalization .
differential expression analysis of the two groups was performed using the deseqr package ( 1.18.0 ) , and p values were adjusted using the benjamini and hochberg approach for controlling the false discovery rate .
further , we estimated significant differentially expressed entities between the two treatment groups using the following criteria : ( 1 ) a minimum count after normalization of ten reads , ( 2 ) absolute fold change  1.5 , and ( 3 ) a corrected p  value  0.05 .
since we worked with two biological replicates per condition , we also removed the biological entities for which the two biological replicates were discordant ( e.g. , opposite fold change direction ) or for which a single sample represented > 75% of all the read counts .
the quantitation of circrna raw counts was normalized as tpm ( transcript per million tags ) .
putative interactions between the mir-184/m - mir-184 seed sequence and the predicted mrna / circrnas were evaluated using miranda , investigating only perfect seed matching without gap or wobble pairing . to this end ,
the 3 utr of all mrnas expressed in our samples was downloaded using ensembl biomart , and the circrna bed output file was converted into fasta format dna sequences .
accumulating evidence demonstrates that widespread alu elements are distributed throughout the human genome in a non - random manner and play an important role in genome and gene evolution and epigenetic and gene regulation.36 , 37 as one of the most important and abundant tes that is still active in the human genome , alu , a sine family te , has demonstrated its indispensable regulatory function .
there are three major subfamilies as follows : the oldest aluj , intermediately aged alus , and the youngest aluy .
alus regulate gene expression and the function of rnas , including mirnas , circrnas , and lncrnas ( long ncrnas ) .
moreover , recent findings indicate that inverted alu repeats appear to promote the formation of circrnas by facilitating back - splicing of pre - mrnas .
we analyzed circrna sequence on repeatmasker program and assigned all the tes to four major types ( line , sine , ltr , and dna ) .
the kernel density of the four te types was calculated , and the relationship of different tes was then used in a pearson correlation coefficient analysis .
differentially expressed aldh5a1 , gabra3 , and girk5 were validated by real - time pcr and western blot according to the previously published experimental methods used in our laboratory .
 m - mir-184 ( mutant mir-184 , r.57c > u ) appears in familial hereditary ocular diseases , including keratoconus , cataracts , edict ( endothelial dystrophy , iris hypoplasia , congenital cataract , and stromal thinning ) syndrome , severe keratoconus , and non - ectatic corneal thinning . 
 the biological function of m - mir-184 in these ocular diseases remains unclear . with the emergence of high - throughput sequencing 
 , it is now possible to discover many different biological components simultaneously . using two different rna libraries , we sequenced the complete transcriptome of hle cells treated with mir-184 , m - mir-184 , and a negative control . 
 data were integrated in an effort to identify any novel gene affected by m - mir-184 . 
 notably , we concluded that aldh5a1 and gabra3 were disordered by m - mir-184 , which might lead to ocular disease . 
 moreover , circrna ( circular rna ) expression was highy random across mir-184 , m - mir-184 , and negative control treatment groups . 
 the sequences of the circrnas did reveal a particularly high level of alu sequences . in summary 
 , we provide a new avenue for understanding the role of m - mir-184 in ocular diseases . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest