BACKGROUND
lr is of great clinical significance in various liver-associated diseases  <cit> .
as one of the vital organs in body, liver not only has the ability of detoxification, metabolism, biliation and immune defense, but has a high capacity to regenerate.
following 2/ <dig> partial hepatectomy , hepatocytes can be activated, and then enter cell cycle and proliferate to restore the original mass and function, which is called lr  <cit> .
lr is a complex process regulated by growth factors, cytokines and non-coding rnas  <cit> .
circrnas, a novel type of non-coding rnas, compose a class of rna developing covalently closed loop structures without 5’–3’ polarities  <cit>  but with widespread, abundant and tissue-specific expression across animals  <cit> .
circrnas are generally formed by ‘backsplicing’ in which an upstream splice acceptor is joined to a downstream splice donor .
although there isn’t consistent conclusion as to the function of circrnas, a number of studies have revealed that circrnas are classified into three types: circular exonic rnas , circular intronic rnas  and exon-intron circrnas   <cit> .
in recent years, the roles of circrnas in the organism have been increasingly illuminated.
circrnas can function as mirna sponges, as templates for viroid and viral replication, as intermediates in rna processing reactions, as regulators of transcription in cis, as snornas   <cit> , and so on.
different types of circrnas may have distinct properties.
cirnas and eicirnas might tend to be enriched in the nucleus and associate with the polymerase ii elongation machinery to enhance expression of their corresponding parent genes  <cit> .
however, ecircrnas have been identified to act as mirna decoys to regulate the mirna effects on their coding rna targets by competing for mirna binding, which seems like playing a regulation for competing endogenous rna activities  <cit> .
hansen et al.
<cit>  attested that circrnas could act as mirna sponges to regulate the expression of corresponding genes at the post-transcription level.
for example, cdr1as  with circularization into circrna harboured  <dig> conserved binding sites for targeting mir- <dig>  so it interacted with mir- <dig> and strongly suppressed mir- <dig> activity with resistant to mirna-mediated target destabilization, resulting in increased levels of mir- <dig> targets  <cit> .
meanwhile, cdr1as was highly complementary to mature mir- <dig> and could be cleavaged by mir- <dig>  <cit> .
similarly, the transcript of sex-determining region y  mainly in the form of circrna in the adult testis  <cit>  contained  <dig> conserved binding sites for mir- <dig>  therefore it affected the target coding genes expression of mir- <dig>  nevertheless, some circrnas had different conserved binding sites of different mirnas, yang  <cit>  has verified that circ-foxo <dig> could bind to eight mirnas, effectively changing the translation of the target mrna.
conclusively, circrnas function as a sponge to bind a certain mirna or multiple potential targeting mirnas, resulting in changed gene expression.
a global reduction of circrna abundance was reported in colorectal cancer  <cit> , esophageal squamous cell carcinoma   <cit> , gastric cancer  <cit> , rectal cancer and ovarian cancer  <cit>  compared to the peritumoral tissue, indicating that circrnas might exert their important action in cell proliferation and apoptosis.
cell proliferation was the most important physiological activity during rat lr  <cit> .
consequently, an intriguing possibility was raised that alternative circrna formation might constitute a novel regulatory layer in rat lr.
salmena et al.
hypothesized that these cernas , such as mrnas, pseudogenes and long non-coding rnas , could communicate with each other through their ability to compete for mirna binding.
the communication generated wide-ranging cis and trans regulatory crosstalk across the transcriptome as a whole  <cit> .
recently enhancing evidences have indicated that circrnas could function potent cerna molecules  <cit> .
much work has provided insights into the regulatory mechanism of non-coding rna in rat lr including mirna and lncrna, little is known about the circrna expression profile and the functions of circrnas in rat lr.
an in-depth study should be carried out to elucidate the potential role of circrna, one of the cernas, in rat lr.
in this paper, high-throughput rna sequencing technology was applied to detect the classification and expression level of circrnas during priming phase of rat lr.it was assumed that the circrna function would be related to the known function of the host gene, we performed go and kegg biological pathway analysis to predict potential functions of circrnas during priming phase of rat lr.
lr-related circrnas were further screened by analyzing the differentially expressed circrnas, and then were validated by qrt-pcr.
RESULTS
expression pattern of circrnas during priming phase of rat lr
high-throughput sequencing technology was used to detect the expression profile of circrnas using the pipeline described by memczak et al.
<cit> , and the result showed that  <dig>  circrnas were identified in the regenerating rat livers at  <dig>   <dig> and 6 h after ph.
the size of lr-related circrna candidates ranged from under 100 nt to over 2000 nt, most of which was not very long in length.
about 75% of circrnas had the predicted spliced length of less than 2000 nt, wherein circrnas with the length less than 500 nt were  <dig> % and the length from 500 nt to 1000 nt were  <dig> %.
.
among the  <dig>  circrnas,  <dig> circrnas were detected at 0 h after ph,  <dig> at 2 h,  <dig> at 6 h, and  <dig> at all three time points , respectively.
expression abundance of circrnas at three time-points after ph was measured based on rpm , and the result indicated that no abnormal expression was found in the three samples .fig.
<dig> expression pattern of circrnas was detected by high-throughput rna sequencing during priming phase of rat lr.
a the length distribution of circrnas.
b venn analysis of circrnas detected at each time points.
c box plots of rpm value of circrnas in three groups
annotation of host linear transcripts and identification of differentially expressed circrnas
biogenesis of circrnas via backsplicing was different from the canonical splicing of linear rnas, but they all generally formed by alternative splicing of pre-mrna.
to provide a comprehensive landscape of the origination of circrnas, linear transcripts of circrnas from the corresponding genes were annotated and the circrna distribution in the genome was also explored according to the location of the chromosome where the circrna sequence was overlapped .
it proved that there were several distribution patterns of circrnas in the gemome .
one hundred fifty-nine differentially expressed circrnas were selected during priming phase of rat lr.
among them, compared with control group ,  <dig> differentially expressed circrnas were selected by ma-plot with  <dig> up-regulated and  <dig> down-regulated at 2 h after ph , while  <dig> were selected with  <dig> up-regulated and  <dig> down-regulated at 6 h after ph  which elucidated that many circrnas were down-regulated at 6 h compared to 2 h after ph.
furthermore, at both  <dig> and 6 h compared with cg after ph,  <dig> circrnas were differentially expressed with  <dig> circrnas up-regulated and  <dig> circrnas down-regulated, while only two circrnas were regulated oppositely .fig.
<dig> annotation of linear transcripts and identification of differentially expressed circrnas.
a the distribution of circular rnas in the rat genome.
b ma-plot of differentially expressed circrnas at 2 h after ph compared with cg.
c differentially up-expressed circrnas during priming phase of rat lr.
d differentially down-expressed circrnas during priming phase of rat lr
the biomathematical predicted biological function of host linear transcripts
under the assumption that circrna function would be related to the known function of the host linear transcripts, differentially regulated linear transcripts were further analyzed by david bioinformatics resources  <dig>  .
go analysis were made on three different aspects namely biological process , cellular component  and molecular function  for host linear transcripts.
prediction terms with p-value less than  <dig>  were selected and ranked by p-value.
according to the results,  <dig> and  <dig> go terms were found up regulated  at  <dig> and 6 h after ph.
in contrast,  <dig> and  <dig> go terms were found down regulated .
top ten generally changed go terms in all comparison groups classified by bp, cc, mf and ranked by p-value were listed.
the consequence indicated that at 2 h after ph compared to gc, the most enriched and meaningful bp terms were superoxide metabolic process, cell aging, superoxide anion generation, and reactive oxygen species metabolic process.
as for cc, the most enriched terms were mostly about focal adhesion, stress fiber and endoplasmic reticulum membrane.
the most enriched mf terms were also closely related with the regulatory function of rna polymerase ii and enzyme activity involving in metabolic activity .
at 6 h after ph compared to gc, the most meaningful bp terms were cellular aldehyde metabolic process, long-chain fatty acid transport and lipid storage.
as for cc, the most enriched terms were mostly about lipid particle, apical plasma membrane and basolateral plasma membrane.
the most enriched mf terms were also closely related with enzyme activity involving in energy metabolism and substance metabolism, such as l-aspartate:2-oxoglutarate aminotransferase activity, hydroxymethylglutaryl-coa synthase activity, glyceryl-ether monooxygenase activity and so on .
the results showed that during the early phase of rat lr, highly expressed host linear transcripts were mainly functionally involved in the response to stress, energy metabolism and the regulation of cell proliferation; all of these responses were helpful for the repair of the injured liver.
moreover, kegg pathway analysis was made, and pathways  were also selected and ranked by p-value.
the top five were listed for differentially changed host linear transcripts at  <dig> and 6 h compared to gc.
kegg pathway analysis also showed that the top regulated ones were steroid hormone biosynthesis, inflammatory mediator regulation of trp channels, and biosynthesis of amino acids at 2 h after ph compared to gc .
the top regulated ones at 6 h after ph were bile secretion, tyrosine metabolism, and fatty acid degradation .table  <dig> go annotations of host linear transcripts at 2 h after ph compared to gc
construction of the circrna-mirna co-expression network
in an attempt to reveal the co-expression pattern of circrna-mirna, the circrna-mirna co-expression networks were constructed based on the high-throughput rna sequencing results.
a constructed network map contained top  <dig> relationships between circrnas and mirnas ranked by p-value of the hypergeometric distribution .
in the network of the circrna-mirna co-expression at 2 h compared with cg,  <dig> mirnas interacting with  <dig> circrnas among the top  <dig> relationships were prognosed to have closer connections during rat lr .
for example, mir-203a-3p was involved in liver regeneration, notably by inhibiting socs <dig>  one known regulator of hepatic cell proliferation.
likewise, in the network of the circrna-mirna co-expression at 6 h compared with cg, ten mirnas binding with  <dig> circrnas had closer connections among which mir-150-5p had been verified to play a vital part during rat lr   <cit> .fig.
<dig> the circrna-mirna co-expression network.
a network of the circrna-mirna co-expression at 2 h compared with cg during rat lr.
b network of the circrna-mirna co-expression at 6 h compared with cg during rat lr.
circle nodes represent circrnas and triangle nodes represent mirnas.
the size of circle and triangle represents p-value with larger size owing smaller p-value
screening of key circrnas during priming phase of rat lr
after all circrnas were screened during rat lr according to two methods, eight key circrnas  were selected according to method i , and four circrnas  according to method ii .
the key mirnas related with rat lr were suppled to screen key circrnas and were listed in table  <dig>  based on the outcome of rna high-throughput sequencing technology in rat liver, eight circrnas selected out by method i were all up-regulated, and three of four circrnas  identified by method ii were down-regulated during priming phase of rat lr.table  <dig> eight circrnas selected by method i
red and green colors denote the expression level higher and lower than the control, respectively
verification of the expression changes of key circrnas during rat lr by qrt-pcr
twelve selected circrnas were experimentally tested in 0 h ph group.
both outward-facing primers and opposite-directed primers were designed to detect the circular and linear form.
head-to-tail splicing existed only in circular form was assayed by quantitative polymerase chain reaction  after reverse transcription, with outward-facing primers and sanger sequencing.
the result showed that the circular form was only amplified using the outward-facing primers and cdna as templates, but it couldn’t be amplified using genomic dna.
pcr assays using both cdna and genomic dna could produce linear rna using opposite-directed set .fig.
<dig> outward-facing primers amplify circrnas in cdna but not genomic dna
to confirm further the circular characteristics and explore the expression changes of  <dig> key circrnas during priming phase of rat lr, six circrnas and four host linear transcripts were randomly selected and amplified using outward-facing primers by qrt-pcr with rnase r digestion at  <dig>   <dig> and 6 h after ph.
as expected, the circrnas were more resistant to rnase r treatment than linear control rnas .
interestingly, the relative quantitative expression of the circrnas and their host linear transcripts with or without rnase r digestion had the similar trends during priming phase of rat lr.
to further verify the expression changes of six key circrnas during rat lr, they were also amplified by qrt-pcr.
these data demonstrated that circ <dig>  circ <dig>  circ <dig> and circ <dig> selected by method i significantly changed during rat lr, as well as the consequence showed positive correlation between four circrnas and their corresponding origination genes, mapk <dig>  fn <dig> , tnfrsf <dig>  and got <dig> , respectively .
in addition, circ <dig> and circ <dig> selected by method ii were significantly down-regulated at ten time points during rat lr .
therein, four circrnas  selected by method i were up-regulated and two circrnas  selected by method ii were down-regulated during priming phase of rat lr, which had accordance with the high-throughput sequencing result even though the abundance relative expression values of the genes detected by above two methods were not all the same, demonstrating reliability of the rna high-throughput sequencing technology.fig.
<dig> qrt-pcr analysis of differential decay of linear and circrnas in the early phase of rat lr following the rnase r digestion.
expression levels were shown by ct values.
fig.
<dig> the expression changes of key circrnas during rat lr by qrt-pcr.
athe expression changes of four circrnas selected by method i and their host linear transcripts during rat lr.
b the expression changes of two circrnas selected by method ii during rat lr.
data represent the average value of at least three independent experiments
discussion
recently, the phenomenon of circrna has gone from being perceived as a rare curiosity to having a central regulatory role in rna metabolism  <cit> .
and the ever-growing discovery of circrna with functional capacity showed that circrna was a transcriptional product in various tissues and cell types of archaea  <cit> , human  <cit> , mouse  <cit> , drosophila  <cit>  and nematodes  <cit>  and in hundreds of cases constituted the dominant rna isoform.
some circrnas have been shown to affect microrna regulation.
hansen et al.
found that the circular transcripts of sry had  <dig> mir- <dig> binding sites and that cdr1as had  <dig> mir- <dig> binding sites  <cit> , so they proposed scientific hypothesis and demonstrated that circrna could function as mirna sponges to regulate the expression of target genes indirectly.
li et al.
pointed out that cir-itch interacted with  <dig> molecules of mir- <dig>  mir- <dig> and mir- <dig> to increase the level of itch and suppress the escc  <cit> .
wang et al.
proved that hrcr  could protect the heart from pathological hypertrophy and heart failure by targeting mir- <dig>  <cit> .
recently, yang et al.
demonstrated that circ-foxo <dig> could function as a sponge to bind multiple potential targeting mirnas, resulting in increased gene expression and inhibition of tumor growth and angiogenesis  <cit> .
thus, accumulating evidences suggested that circrnas could function as mirna sponges, and show distinct expression patterns coupled from their linear counterparts such as cir-itch and circ-foxo <dig>  or uncoupled from host gene expression such as cdr1as and hrcr.
to analyze the comprehensive circrna expression profile by high-throughput rna sequencing technology,  <dig>  circrnas were defined during priming phase of rat lr.
one hundred fifty-nine differentially expressed circrnas of them originated from  <dig> host linear transcripts.
on one hand,  <dig> of  <dig> circrnas were intergenic indicating that they did not have the corresponding linear transcripts.
on the other hand, a gene could be spliced into one or more circrnas  <cit> .
nineteen genes could produce more than one circrnas during priming phase of rat lr, such as four circrnas expressed from cpamd <dig>  the similar case had reported that anril  encoded multiple, non-abundant linear and circular species and the expression of circular anril isoforms  was correlated with ink4/arf transcription and atherosclerotic vascular disease  risk  <cit> .
furthermore, various circrna isoforms from the same host linear transcripts expressed at different time points during priming phase of rat lr, implying the important function of temporal regulation of lr-related circrnas.
to investigate the circrna regulatory role in rat lr further, go analysis was performed to annotate the biological functions of host linear transcripts during priming phase of rat lr, the result showed obviously that a significant amount of go terms were related with the response to stress, energy metabolism and regulation of cell proliferation, which had been acknowledged as the important activities in the early stage of rat lr  <cit> .
the most enriched cc terms were about different kinds of plasma membranes.
the plasma membranes were embedded with many proteins which could monitor and maintain the cell’s chemical climate and assist in the transfer of molecules across the membrane.
the most enriched mf terms were closely related with diverse enzymes participating in energy metabolism and protein binding which was similar with circrna function.
circ-foxo <dig> interacted with the anti-senescent protein id- <dig> and the transcription factor e2f <dig>  as well as the anti-stress proteins fak and hif1a and retained them in the cytoplasm.
as a result, these proteins could no longer exert their anti-senescent and anti-stress roles, resulting in increased cellular senescence  <cit> .
the cirna, ci-ankrd <dig>  largely accumulated to its sites of transcription, was associated with elongation pol ii machinery, and acted as a positive regulator of pol ii transcription  <cit> .
in this way, circrna might function as a scaffold for rna binding proteins exerting its biological functions including regulating tanscription and so on.
coinciding well with the results of go analysis, kegg pathway analysis showed that linear transcripts were related with the signaling pathways closely linked to energy metabolism and substance metabolism including amino acid metabolism, fatty acid metabolism and bile secretion.
all of them could provide a multitude of energy and materials to make preparation to trigger lr.
the amounting evidences have indicated that circrnas might regulate the function of mirnas acting as competing cernas  <cit> .
in order to explore the role of the huge amount of circrnas in rat lr, circrna-mirna co-expression networks were established to predict the relationships between circrnas and mirnas.
hitherto, there were no annotation of circrnas in the circrna-mirna co-expression network, but some of the co-expressed mirnas had already been proved functional crucially in rat lr.
exemplification showed that mir- <dig> regulated cell proliferation by targeting vegf  <cit> , besides mir- <dig> negatively regulated pten expression and increased akt phosphorylation during lr  <cit> .
from the co-expression network, the obvious conclusion was that mir- <dig> was related with nine circrnas, and mir- <dig> was related with  <dig> circrnas.
collectively, these results suggested that circrnas might regulate rat lr by binding up to mirnas that could regulate the associated gene expression.
examining the roles of individual circrnas will be the subject of future investigations.
we screened the key circrnas by two methods, and  <dig> key circrnas during priming phase of rat lr were confirmed.
then, six of them were randomly selected to examine by qrt-pcr and sequenced correctly.
moreover, the expression of four circrnas, circ <dig>  circ <dig>  circ <dig> and circ <dig> selected by method i was consistent with their linear transcripts mapk <dig>  fn <dig>  tnfrsf <dig> and got <dig>  respectively, affirming that our methods were creditable.
simultaneously, the host genes of these four circrnas have been interestingly substantiated to be vital for cell proliferation and energy metabolism.
for example, mapk <dig>  one member of the four p <dig> mapks, played an important place in the cascades of cellular responses evoked by extracellular stimuli such as proinflammatory cytokines or physical stress.
fn <dig> was a glycoprotein component of the extracellular matrix which functioned in embryogenesis, development, wound healing and metastasis.
blocking the adhesion function of fn <dig> by inhibiting its expression with mir-204-3p could promote the growth of hepatocellular carcinoma tumor endothelial cells  <cit> .
tumor necrosis factor  receptors were key factors in inflammation and immune regulation.
like other death receptors , tnfrsf <dig>  also known as dr <dig>  could interact with tradd, which had previously been shown to associate with tnfr <dig>  furthermore, ectopic expression of tnfrsf <dig> in mammalian cells induced apoptosis and activation of both nf-κb and jnk  <cit> .
got <dig> existed in cytoplasmic and mitochondrial forms and played a role in amino acid metabolism and the urea and tricarboxylic acid cycles.
hence, mapk <dig> was involved in a wide variety of cellular processes such as proliferation, differentiation, and development.
tnfrsf <dig> was strongly connected to tumorigenesis and apoptosis.
fn <dig> encoded structural protein involving in cell adhesion and migration processes.
got <dig> participated in energy and substance metabolism.
based on these investigation, a hypothesis was raised that circ <dig>  circ <dig>  circ <dig> and circ <dig> were novel and valuable parts in the process of hepatocyte proliferation, apoptosis, energy metabolism and substance metabolism during rat lr by altering the expression of their linear transcripts mapk <dig>  fn <dig>  tnfrsf <dig> and got <dig>  respectively.
in addition, circ <dig> and circ <dig> selected by method ii were both significantly down-regulated during rat lr.
the outcome of biomathematical prediction manifested that circ <dig> and circ <dig> could both couple with mir- <dig>  some experiments have demonstrated that mir- <dig> was capable of suppressing the hepatocyte proliferation, notably by inhibiting bcl <dig> and setd <dig>  <cit> .
thus, circ <dig> and circ <dig> might regulate the hepatocyte proliferation to control rat lr process by interacting with mir- <dig>  taken together, this paper provided the comprehensive landscape of circrna expression profile during priming phase of rat lr and determined six representative circrna candidates.
CONCLUSIONS
in this work,  <dig>  circrnas were detected and  <dig> of them were preliminarily determined changing remarkably during priming phase of rat lr indicating their potential roles in rat lr.
go and kegg biological pathway analysis indicated that host linear transcripts of circrnas during the early phase of rat lr critically contributed to the process of energy metabolism, substance metabolism and hepatocyte proliferation.
circ <dig>  circ <dig>  circ <dig> and circ <dig> might regulate the rat lr by controlling the expression level of mapk <dig>  fn <dig>  tnfrsf <dig> and got <dig>  respectively, involved in networks critical for hepatocyte proliferation, energy metabolism and substance metabolism.
it was also scientifically predicted that circ <dig> and circ <dig> could regulate the hepatocyte proliferation by interacting with mir- <dig>  so we suggest that expression of circrnas sponging up mirnas might play an important regulatory role as well.
the findings will undoubtedly facilitate the future analyses of the regulatory mechanism of circrna in rat lr, which may provide further insights into the comprehension of the regenerative process and have great promise to solve clinical problems.

 lr is of great clinical significance in various liver-associated diseases. as one of the vital organs in body, liver not only has the ability of detoxification, metabolism, biliation and immune defense, but has a high capacity to regenerate. following 2/ partial hepatectomy, hepatocytes can be activated, and then enter cell cycle and proliferate to restore the original mass and function, which is called lr. lr is a complex process regulated by growth factors, cytokines and non-coding rnas. circrnas, a novel type of non-coding rnas, compose a class of rna developing covalently closed loop structures without 5’–3’ polarities but with widespread, abundant and tissue-specific expression across animals. circrnas are generally formed by ‘backsplicing’ in which an upstream splice acceptor is joined to a downstream splice donor. although there isn’t consistent conclusion as to the function of circrnas, a number of studies have revealed that circrnas are classified into three types: circular exonic rnas, circular intronic rnas and exon-intron circrnas. in recent years, the roles of circrnas in the organism have been increasingly illuminated. circrnas can function as mirna sponges, as templates for viroid and viral replication, as intermediates in rna processing reactions, as regulators of transcription in cis, as snornas, and so on. different types of circrnas may have distinct properties. cirnas and eicirnas might tend to be enriched in the nucleus and associate with the polymerase ii elongation machinery to enhance expression of their corresponding parent genes. however, ecircrnas have been identified to act as mirna decoys to regulate the mirna effects on their coding rna targets by competing for mirna binding, which seems like playing a regulation for competing endogenous rna activities. hansen et al. attested that circrnas could act as mirna sponges to regulate the expression of corresponding genes at the post-transcription level. for example, cdr1as with circularization into circrna harboured conserved binding sites for targeting mir- so it interacted with mir- and strongly suppressed mir- activity with resistant to mirna-mediated target destabilization, resulting in increased levels of mir- targets. meanwhile, cdr1as was highly complementary to mature mir- and could be cleavaged by mir-. similarly, the transcript of sex-determining region y mainly in the form of circrna in the adult testis contained conserved binding sites for mir- therefore it affected the target coding genes expression of mir- nevertheless, some circrnas had different conserved binding sites of different mirnas, yang has verified that circ-foxo could bind to eight mirnas, effectively changing the translation of the target mrna. conclusively, circrnas function as a sponge to bind a certain mirna or multiple potential targeting mirnas, resulting in changed gene expression. a global reduction of circrna abundance was reported in colorectal cancer, esophageal squamous cell carcinoma, gastric cancer, rectal cancer and ovarian cancer compared to the peritumoral tissue, indicating that circrnas might exert their important action in cell proliferation and apoptosis. cell proliferation was the most important physiological activity during rat lr. consequently, an intriguing possibility was raised that alternative circrna formation might constitute a novel regulatory layer in rat lr. salmena et al. hypothesized that these cernas, such as mrnas, pseudogenes and long non-coding rnas, could communicate with each other through their ability to compete for mirna binding. the communication generated wide-ranging cis and trans regulatory crosstalk across the transcriptome as a whole. recently enhancing evidences have indicated that circrnas could function potent cerna molecules. much work has provided insights into the regulatory mechanism of non-coding rna in rat lr including mirna and lncrna, little is known about the circrna expression profile and the functions of circrnas in rat lr. an in-depth study should be carried out to elucidate the potential role of circrna, one of the cernas, in rat lr. in this paper, high-throughput rna sequencing technology was applied to detect the classification and expression level of circrnas during priming phase of rat lr.it was assumed that the circrna function would be related to the known function of the host gene, we performed go and kegg biological pathway analysis to predict potential functions of circrnas during priming phase of rat lr. lr-related circrnas were further screened by analyzing the differentially expressed circrnas, and then were validated by qrt-pcr.  expression pattern of circrnas during priming phase of rat lr high-throughput sequencing technology was used to detect the expression profile of circrnas using the pipeline described by memczak et al., and the result showed that circrnas were identified in the regenerating rat livers at and 6 h after ph. the size of lr-related circrna candidates ranged from under 100 nt to over 2000 nt, most of which was not very long in length. about 75% of circrnas had the predicted spliced length of less than 2000 nt, wherein circrnas with the length less than 500 nt were % and the length from 500 nt to 1000 nt were %.. among the circrnas, circrnas were detected at 0 h after ph, at 2 h, at 6 h, and at all three time points, respectively. expression abundance of circrnas at three time-points after ph was measured based on rpm, and the result indicated that no abnormal expression was found in the three samples.fig. expression pattern of circrnas was detected by high-throughput rna sequencing during priming phase of rat lr. a the length distribution of circrnas. b venn analysis of circrnas detected at each time points. c box plots of rpm value of circrnas in three groups annotation of host linear transcripts and identification of differentially expressed circrnas biogenesis of circrnas via backsplicing was different from the canonical splicing of linear rnas, but they all generally formed by alternative splicing of pre-mrna. to provide a comprehensive landscape of the origination of circrnas, linear transcripts of circrnas from the corresponding genes were annotated and the circrna distribution in the genome was also explored according to the location of the chromosome where the circrna sequence was overlapped. it proved that there were several distribution patterns of circrnas in the gemome. one hundred fifty-nine differentially expressed circrnas were selected during priming phase of rat lr. among them, compared with control group, differentially expressed circrnas were selected by ma-plot with up-regulated and down-regulated at 2 h after ph, while were selected with up-regulated and down-regulated at 6 h after ph which elucidated that many circrnas were down-regulated at 6 h compared to 2 h after ph. furthermore, at both and 6 h compared with cg after ph, circrnas were differentially expressed with circrnas up-regulated and circrnas down-regulated, while only two circrnas were regulated oppositely.fig. annotation of linear transcripts and identification of differentially expressed circrnas. a the distribution of circular rnas in the rat genome. b ma-plot of differentially expressed circrnas at 2 h after ph compared with cg. c differentially up-expressed circrnas during priming phase of rat lr. d differentially down-expressed circrnas during priming phase of rat lr the biomathematical predicted biological function of host linear transcripts under the assumption that circrna function would be related to the known function of the host linear transcripts, differentially regulated linear transcripts were further analyzed by david bioinformatics resources. go analysis were made on three different aspects namely biological process, cellular component and molecular function for host linear transcripts. prediction terms with p-value less than were selected and ranked by p-value. according to the results, and go terms were found up regulated at and 6 h after ph. in contrast, and go terms were found down regulated. top ten generally changed go terms in all comparison groups classified by bp, cc, mf and ranked by p-value were listed. the consequence indicated that at 2 h after ph compared to gc, the most enriched and meaningful bp terms were superoxide metabolic process, cell aging, superoxide anion generation, and reactive oxygen species metabolic process. as for cc, the most enriched terms were mostly about focal adhesion, stress fiber and endoplasmic reticulum membrane. the most enriched mf terms were also closely related with the regulatory function of rna polymerase ii and enzyme activity involving in metabolic activity. at 6 h after ph compared to gc, the most meaningful bp terms were cellular aldehyde metabolic process, long-chain fatty acid transport and lipid storage. as for cc, the most enriched terms were mostly about lipid particle, apical plasma membrane and basolateral plasma membrane. the most enriched mf terms were also closely related with enzyme activity involving in energy metabolism and substance metabolism, such as l-aspartate:2-oxoglutarate aminotransferase activity, hydroxymethylglutaryl-coa synthase activity, glyceryl-ether monooxygenase activity and so on. the results showed that during the early phase of rat lr, highly expressed host linear transcripts were mainly functionally involved in the response to stress, energy metabolism and the regulation of cell proliferation; all of these responses were helpful for the repair of the injured liver. moreover, kegg pathway analysis was made, and pathways were also selected and ranked by p-value. the top five were listed for differentially changed host linear transcripts at and 6 h compared to gc. kegg pathway analysis also showed that the top regulated ones were steroid hormone biosynthesis, inflammatory mediator regulation of trp channels, and biosynthesis of amino acids at 2 h after ph compared to gc. the top regulated ones at 6 h after ph were bile secretion, tyrosine metabolism, and fatty acid degradation.table go annotations of host linear transcripts at 2 h after ph compared to gc construction of the circrna-mirna co-expression network in an attempt to reveal the co-expression pattern of circrna-mirna, the circrna-mirna co-expression networks were constructed based on the high-throughput rna sequencing results. a constructed network map contained top relationships between circrnas and mirnas ranked by p-value of the hypergeometric distribution. in the network of the circrna-mirna co-expression at 2 h compared with cg, mirnas interacting with circrnas among the top relationships were prognosed to have closer connections during rat lr. for example, mir-203a-3p was involved in liver regeneration, notably by inhibiting socs one known regulator of hepatic cell proliferation. likewise, in the network of the circrna-mirna co-expression at 6 h compared with cg, ten mirnas binding with circrnas had closer connections among which mir-150-5p had been verified to play a vital part during rat lr.fig. the circrna-mirna co-expression network. a network of the circrna-mirna co-expression at 2 h compared with cg during rat lr. b network of the circrna-mirna co-expression at 6 h compared with cg during rat lr. circle nodes represent circrnas and triangle nodes represent mirnas. the size of circle and triangle represents p-value with larger size owing smaller p-value screening of key circrnas during priming phase of rat lr after all circrnas were screened during rat lr according to two methods, eight key circrnas were selected according to method i, and four circrnas according to method ii. the key mirnas related with rat lr were suppled to screen key circrnas and were listed in table  based on the outcome of rna high-throughput sequencing technology in rat liver, eight circrnas selected out by method i were all up-regulated, and three of four circrnas identified by method ii were down-regulated during priming phase of rat lr.table eight circrnas selected by method i red and green colors denote the expression level higher and lower than the control, respectively verification of the expression changes of key circrnas during rat lr by qrt-pcr twelve selected circrnas were experimentally tested in 0 h ph group. both outward-facing primers and opposite-directed primers were designed to detect the circular and linear form. head-to-tail splicing existed only in circular form was assayed by quantitative polymerase chain reaction after reverse transcription, with outward-facing primers and sanger sequencing. the result showed that the circular form was only amplified using the outward-facing primers and cdna as templates, but it couldn’t be amplified using genomic dna. pcr assays using both cdna and genomic dna could produce linear rna using opposite-directed set.fig. outward-facing primers amplify circrnas in cdna but not genomic dna to confirm further the circular characteristics and explore the expression changes of key circrnas during priming phase of rat lr, six circrnas and four host linear transcripts were randomly selected and amplified using outward-facing primers by qrt-pcr with rnase r digestion at and 6 h after ph. as expected, the circrnas were more resistant to rnase r treatment than linear control rnas. interestingly, the relative quantitative expression of the circrnas and their host linear transcripts with or without rnase r digestion had the similar trends during priming phase of rat lr. to further verify the expression changes of six key circrnas during rat lr, they were also amplified by qrt-pcr. these data demonstrated that circ circ circ and circ selected by method i significantly changed during rat lr, as well as the consequence showed positive correlation between four circrnas and their corresponding origination genes, mapk fn, tnfrsf and got, respectively. in addition, circ and circ selected by method ii were significantly down-regulated at ten time points during rat lr. therein, four circrnas selected by method i were up-regulated and two circrnas selected by method ii were down-regulated during priming phase of rat lr, which had accordance with the high-throughput sequencing result even though the abundance relative expression values of the genes detected by above two methods were not all the same, demonstrating reliability of the rna high-throughput sequencing technology.fig. qrt-pcr analysis of differential decay of linear and circrnas in the early phase of rat lr following the rnase r digestion. expression levels were shown by ct values. fig. the expression changes of key circrnas during rat lr by qrt-pcr. athe expression changes of four circrnas selected by method i and their host linear transcripts during rat lr. b the expression changes of two circrnas selected by method ii during rat lr. data represent the average value of at least three independent experiments discussion recently, the phenomenon of circrna has gone from being perceived as a rare curiosity to having a central regulatory role in rna metabolism. and the ever-growing discovery of circrna with functional capacity showed that circrna was a transcriptional product in various tissues and cell types of archaea, human, mouse, drosophila and nematodes and in hundreds of cases constituted the dominant rna isoform. some circrnas have been shown to affect microrna regulation. hansen et al. found that the circular transcripts of sry had mir- binding sites and that cdr1as had mir- binding sites, so they proposed scientific hypothesis and demonstrated that circrna could function as mirna sponges to regulate the expression of target genes indirectly. li et al. pointed out that cir-itch interacted with molecules of mir- mir- and mir- to increase the level of itch and suppress the escc. wang et al. proved that hrcr could protect the heart from pathological hypertrophy and heart failure by targeting mir-. recently, yang et al. demonstrated that circ-foxo could function as a sponge to bind multiple potential targeting mirnas, resulting in increased gene expression and inhibition of tumor growth and angiogenesis. thus, accumulating evidences suggested that circrnas could function as mirna sponges, and show distinct expression patterns coupled from their linear counterparts such as cir-itch and circ-foxo or uncoupled from host gene expression such as cdr1as and hrcr. to analyze the comprehensive circrna expression profile by high-throughput rna sequencing technology, circrnas were defined during priming phase of rat lr. one hundred fifty-nine differentially expressed circrnas of them originated from host linear transcripts. on one hand, of circrnas were intergenic indicating that they did not have the corresponding linear transcripts. on the other hand, a gene could be spliced into one or more circrnas. nineteen genes could produce more than one circrnas during priming phase of rat lr, such as four circrnas expressed from cpamd the similar case had reported that anril encoded multiple, non-abundant linear and circular species and the expression of circular anril isoforms was correlated with ink4/arf transcription and atherosclerotic vascular disease risk. furthermore, various circrna isoforms from the same host linear transcripts expressed at different time points during priming phase of rat lr, implying the important function of temporal regulation of lr-related circrnas. to investigate the circrna regulatory role in rat lr further, go analysis was performed to annotate the biological functions of host linear transcripts during priming phase of rat lr, the result showed obviously that a significant amount of go terms were related with the response to stress, energy metabolism and regulation of cell proliferation, which had been acknowledged as the important activities in the early stage of rat lr. the most enriched cc terms were about different kinds of plasma membranes. the plasma membranes were embedded with many proteins which could monitor and maintain the cell’s chemical climate and assist in the transfer of molecules across the membrane. the most enriched mf terms were closely related with diverse enzymes participating in energy metabolism and protein binding which was similar with circrna function. circ-foxo interacted with the anti-senescent protein id- and the transcription factor e2f as well as the anti-stress proteins fak and hif1a and retained them in the cytoplasm. as a result, these proteins could no longer exert their anti-senescent and anti-stress roles, resulting in increased cellular senescence. the cirna, ci-ankrd largely accumulated to its sites of transcription, was associated with elongation pol ii machinery, and acted as a positive regulator of pol ii transcription. in this way, circrna might function as a scaffold for rna binding proteins exerting its biological functions including regulating tanscription and so on. coinciding well with the results of go analysis, kegg pathway analysis showed that linear transcripts were related with the signaling pathways closely linked to energy metabolism and substance metabolism including amino acid metabolism, fatty acid metabolism and bile secretion. all of them could provide a multitude of energy and materials to make preparation to trigger lr. the amounting evidences have indicated that circrnas might regulate the function of mirnas acting as competing cernas. in order to explore the role of the huge amount of circrnas in rat lr, circrna-mirna co-expression networks were established to predict the relationships between circrnas and mirnas. hitherto, there were no annotation of circrnas in the circrna-mirna co-expression network, but some of the co-expressed mirnas had already been proved functional crucially in rat lr. exemplification showed that mir- regulated cell proliferation by targeting vegf, besides mir- negatively regulated pten expression and increased akt phosphorylation during lr. from the co-expression network, the obvious conclusion was that mir- was related with nine circrnas, and mir- was related with circrnas. collectively, these results suggested that circrnas might regulate rat lr by binding up to mirnas that could regulate the associated gene expression. examining the roles of individual circrnas will be the subject of future investigations. we screened the key circrnas by two methods, and key circrnas during priming phase of rat lr were confirmed. then, six of them were randomly selected to examine by qrt-pcr and sequenced correctly. moreover, the expression of four circrnas, circ circ circ and circ selected by method i was consistent with their linear transcripts mapk fn tnfrsf and got respectively, affirming that our methods were creditable. simultaneously, the host genes of these four circrnas have been interestingly substantiated to be vital for cell proliferation and energy metabolism. for example, mapk one member of the four p mapks, played an important place in the cascades of cellular responses evoked by extracellular stimuli such as proinflammatory cytokines or physical stress. fn was a glycoprotein component of the extracellular matrix which functioned in embryogenesis, development, wound healing and metastasis. blocking the adhesion function of fn by inhibiting its expression with mir-204-3p could promote the growth of hepatocellular carcinoma tumor endothelial cells. tumor necrosis factor receptors were key factors in inflammation and immune regulation. like other death receptors, tnfrsf also known as dr could interact with tradd, which had previously been shown to associate with tnfr furthermore, ectopic expression of tnfrsf in mammalian cells induced apoptosis and activation of both nf-κb and jnk. got existed in cytoplasmic and mitochondrial forms and played a role in amino acid metabolism and the urea and tricarboxylic acid cycles. hence, mapk was involved in a wide variety of cellular processes such as proliferation, differentiation, and development. tnfrsf was strongly connected to tumorigenesis and apoptosis. fn encoded structural protein involving in cell adhesion and migration processes. got participated in energy and substance metabolism. based on these investigation, a hypothesis was raised that circ circ circ and circ were novel and valuable parts in the process of hepatocyte proliferation, apoptosis, energy metabolism and substance metabolism during rat lr by altering the expression of their linear transcripts mapk fn tnfrsf and got respectively. in addition, circ and circ selected by method ii were both significantly down-regulated during rat lr. the outcome of biomathematical prediction manifested that circ and circ could both couple with mir- some experiments have demonstrated that mir- was capable of suppressing the hepatocyte proliferation, notably by inhibiting bcl and setd. thus, circ and circ might regulate the hepatocyte proliferation to control rat lr process by interacting with mir- taken together, this paper provided the comprehensive landscape of circrna expression profile during priming phase of rat lr and determined six representative circrna candidates.  in this work, circrnas were detected and of them were preliminarily determined changing remarkably during priming phase of rat lr indicating their potential roles in rat lr. go and kegg biological pathway analysis indicated that host linear transcripts of circrnas during the early phase of rat lr critically contributed to the process of energy metabolism, substance metabolism and hepatocyte proliferation. circ circ circ and circ might regulate the rat lr by controlling the expression level of mapk fn tnfrsf and got respectively, involved in networks critical for hepatocyte proliferation, energy metabolism and substance metabolism. it was also scientifically predicted that circ and circ could regulate the hepatocyte proliferation by interacting with mir- so we suggest that expression of circrnas sponging up mirnas might play an important regulatory role as well. the findings will undoubtedly facilitate the future analyses of the regulatory mechanism of circrna in rat lr, which may provide further insights into the comprehension of the regenerative process and have great promise to solve clinical problems. 
this study will lay the foundation for revealing the functional roles of circrnas during rat lr and help solve the remaining clinical problems. the molecular mechanism of hepatocyte proliferation, energy metabolism and substance metabolism during rat lr had been elucidated. high-throughput rna sequencing technologycircrnarat liver regenerationmirnahost linear transcriptshepatocyte proliferationenergy metabolismsubstance metabolismhttp://dx.doi.org/ /501100001809national natural science foundation of china31572270xu cunshuan project of fundamental research and advanced technology of henan162300410144guo jianlin issue-copyright-statement© the author 2017 to excavate the key circrnas in the early phase of rat lr, we comprehensively evaluated and integrated the relationship of expression level between the circrnas and the linear transcripts as well as the distribution of mirna binding sites in circrna sequences. to investigate the regulatory mechanism of circrna during priming phase of rat lr, high-throughput rna sequencing technology was performed to unbiasedly profile the expression of circrna during priming phase of rat lr. gene ontology and kyoto encyclopedia of genes and genomes biological pathway analysis was conducted to predict the functions of differentially expressed circrnas and their host linear transcripts. two thousand four hundred twelve circrnas were detected, and circrnas deriving from host linear transcripts differentially expressed. however, the regulatory capacity of circrna in rat lr remains a fascinating topic. this paper is the first to employ the comprehensive circrna expression profile and to investigate circrna-mirna interactions during priming phase of rat lr. six significantly changed circrnas during priming phase of rat lr were screened as key circle molecules, and then were validated by qrt-pcr.
 rat liver regeneration proceeds along a process of highly organized and ordered tissue growth in response to the loss or injury of liver tissue, during which many physiological processes may play important roles. the molecular mechanism of hepatocyte proliferation, energy metabolism and substance metabolism during rat lr had been elucidated. further, the correlation of circular rna abundance with proliferation has recently been clarified. however, the regulatory capacity of circrna in rat lr remains a fascinating topic.  to investigate the regulatory mechanism of circrna during priming phase of rat lr, high-throughput rna sequencing technology was performed to unbiasedly profile the expression of circrna during priming phase of rat lr. gene ontology and kyoto encyclopedia of genes and genomes biological pathway analysis was conducted to predict the functions of differentially expressed circrnas and their host linear transcripts. co-expression networks of circrna-mirna were constructed based on the correlation analysis between the differentially expressed lr-related circrnas and the condition of their mirna binding sites. to excavate the key circrnas in the early phase of rat lr, we comprehensively evaluated and integrated the relationship of expression level between the circrnas and the linear transcripts as well as the distribution of mirna binding sites in circrna sequences.  this paper is the first to employ the comprehensive circrna expression profile and to investigate circrna-mirna interactions during priming phase of rat lr. two thousand four hundred twelve circrnas were detected, and circrnas deriving from host linear transcripts differentially expressed. six significantly changed circrnas during priming phase of rat lr were screened as key circle molecules, and then were validated by qrt-pcr. this study will lay the foundation for revealing the functional roles of circrnas during rat lr and help solve the remaining clinical problems. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords high-throughput rna sequencing technologycircrnarat liver regenerationmirnahost linear transcriptshepatocyte proliferationenergy metabolismsubstance metabolismhttp://dx.doi.org/ /501100001809national natural science foundation of china31572270xu cunshuan project of fundamental research and advanced technology of henan162300410144guo jianlin issue-copyright-statement© the author 2017 