BACKGROUND
kaposi’s sarcoma-associated herpesvirus , also known as human herpesvirus  <dig> , is associated with kaposi’s sarcoma   <cit> .
ks is the most common cancer in individuals with aids and one of the most common cancers in patients who have immunosuppression  <cit> .
ks typically appears as colored lesions or blotches on the skin, although it can spread to internal organs  <cit> .
this disease has been recognized as a peculiar neoplasm that has a diverse cellular makeup and an extensive neovasculature.
in host endothelial cells, kshv infection alters the expression of mrnas and micrornas required for many aspects of tumorigenesis, including angiogenesis  <cit> .
therefore, ks and kshv are recognized as an excellent model to study angiogenesis and to discover new angiogenic mechanisms, including coding and non-coding rnas.
recently, the effect of kshv infection on cellular microrna  expression has been reported  <cit> .
it was found that pre-mirna signatures delineate the stages of kshv-associated huvec transformation  <cit> .
mirnas are small rnas of 18– <dig> nucleotides in length that have emerged as master regulators of cancer and key modulators of angiogenesis .
an increasing number of studies have shown that some mirnas, called angiomirs , play a crucial role in regulating angiogenesis  <cit> .
for example, mir- <dig> and mir- <dig>  two closely related mirnas encoded in cluster from a genomic region on chromosome x, can modulate the angiogenic properties of human umbilical vein endothelial cells  by targeting c-kit  <cit>  and endothelial nitric oxide synthase   <cit> .
mir- <dig> and mir- <dig> also regulate lymphatic endothelial cell  motility by targeting ets <dig> and ets <dig> transcription factors, respectively  <cit> .
loss of mir- <dig> expression demarks the transition from merely immortalized to fully tumorigenic huvecs  <cit> .
kshv also induces endothelial cell motility by inducing mir- <dig> expression while suppressing that of mir- <dig> and mir- <dig>  <cit> .
kaposin b, one of the three kaposin proteins encoded from the kshv genome k <dig> locus, is one of the viral genes expressed during latency and is therefore expressed in all kshv tumor cells  <cit> .
kaposin b stabilizes various cytokine  mrna containing au-rich elements  via the p38/mk <dig> pathway.
in response to lps, kaposin b and mk <dig> were shown to be exported to the cytoplasm, where mrna stability is regulated  <cit> .
kaposin b also enhances the prox <dig> mrna stability during lymphatic reprogramming of vascular endothelial cells  <cit> .
kaposin b can influence cellular gene expression by regulating promoter activities of host genes: both kaposin b and kshv viral latency-associated nuclear antigen  proteins can down-regulate mir- <dig> and mir- <dig> levels by repressing the activity of mir-221/- <dig> cluster promoter  <cit> .
since there is no predictable dna-binding domain on kaposin b, how this nuclear protein can regulate mrna and mirna expression remains unclear.
c-myc achieves its oncogenic effects by regulating transcription of protein-coding genes as well as microrna genes such as mir-29b-1/mir-29a  <cit> .
c-myc is also essential for vasculogenesis and angiogenesis during development and tumor progression  <cit>  via inducing the expression of mir-17 ~  <dig> angiogenic mirna cluster  <cit> .
revealing the angiomirs regulated by c-myc and the underlying regulatory mechanisms will help to further understand c-myc and endothelial cell biology.
here, we showed that kaposin b and c-myc are in the same transcription complex that directly regulates the mir-221/- <dig> cluster promoter activity.
a c-myc-oriented circuit is therefore formed in the presence of kaposin b in kshv-infected endothelial cells.
furthermore, we also provide a global microrna signature which is regulated by c-myc and kaposin b. we hope our roadmap will help the search and development of new therapeutic targets for virus- or cancer-induced angiogenesis, cancer formation and metastasis.
methods
cell culture and kshv infection
human primary umbilical vein endothelial cells  were purchased from clonetics inc.  and were cultured as described  <cit> .
hmec <dig>  an immortalized human microvascular endothelial cell line, was cultured in endothelial cell growth medium mv .
a recombinant virus, rkshv.
<dig>  that expresses the red fluorescent protein  from the kshv lytic pan promoter, the green fluorescent protein  from the ef-1alpha promoter, and with a gene for puromycin resistance as a selectable marker, was constructed using jsc- <dig> cells as described previously  <cit> .
plasmid construction
plasmids expressing kshv kaposin b and mir- <dig>  mir- <dig>  or mir-221/- <dig> were constructed as described previously  <cit> .
c-myc expression constructs  and knock down construct  was kindly provided by prof. kenneth cw wu  <cit> .
the full-length mir-221/- <dig> promoter reporter plasmid was constructed as described previously  <cit> , and primers for cloning mir-221/- <dig> promoter mutants are listed in additional file  <dig>
immunofluorescence assay
cells were fixed with 4 % paraformaldehyde , permeabilized with  <dig>  % triton x- <dig> , and then blocked with pbs containing 1 % bovine serum albumin .
for kaposin b staining, cells were incubated with the monoclonal antibody anti-flag m <dig> at a 1: <dig> dilution for  <dig> to 120 min at 25 °c followed by incubation with fitc-conjugated goat anti-mouse igg  for  <dig> to 120 min at 25 °c.
rhodamine-phalloidin  was used to label actin cytoskeleton.
cell nuclei were counterstained with hoechst  <dig> , and examined by fluorescence confocal microscopy .
transwell cell migration and endothelial cell tube formation assays
cell migration ability was evaluated using costar transwell® polycarbonate permeable supports  as described previously  <cit> .
in brief, 5 ×  <dig> cells in 500 μl of culture medium were applied to the upper chamber of the device, and 750 μl of medium containing 10 ng/ml human vegf  was added to the lower chamber.
a polycarbonate membrane with a pore size of 8 μm was placed in between the two chambers.
after 6 h of incubation at 37 °c for hmec <dig> and huvec cells, the membrane was fixed in 4 % paraformaldehyde  for 20 min at room temperature and then stained with hoechst  <dig> solution  for 30 min.
on the upper side of membrane were identified un-migrated cells and removed.
the cells under the membrane were counted under a microscope.
in vitro angiogenic activity of vascular endothelial cells was examined by tube formation assays: primary huvecs were placed onto matrigel® basement membrane matrix gel  for 6 h at 37 °c.
the formation of tubule structure was observed every hour under a microscope.
small rna sequencing  and data analysis
total rna was collected and small rna fractions were sequenced using illumina hiseq <dig> sequencer  according to the manufacturer’s instructions.
for data analysis, quality fastq sequences, which were without poly-a, ambiguous nucleotides or a 5′ adapter, yet flanking 6–18 nt of 3′ adapter sequence, had the adapter sequences trimmed and the identical sequences were then collapsed to unique sequences.
the resulting unique sequences that did not align to the mrna database  but were aligned to known microrna sequences  were subjected to further quantification analysis.
sequencing reads were used to obtain a rpm   <cit>  value calculated as c/ ×  <dig>  where c is “read numbers aligned to a given mirna chromosomal region”, m is “multiple mapping numbers across all mirna regions” and n is “total read numbers that map to human genome sequence”.
rna extraction, real-time quantitative polymerase chain reaction  and microrna expression
rna extraction and reverse transcription were performed as previously described  <cit> .
the expression of mature human mir- <dig> and mir- <dig> was determined by a stem-loop real-time pcr system using the primer pairs previously published  <cit> .
microrna expression data were normalized to that of u <dig> snrna.
details of qpcr primers are in additional file  <dig>
coimmunoprecipitation , chromatin immunoprecipitation  and immunoblotting
co-ip and chip assays were performed as previously described  <cit> .
briefly, kaposin b or c-myc expression plasmid was co-transfected into 293t cells using turbofect™ .
after 48 h, cells were lyzed in net lysis buffer containing protease and phosphatase inhibitors and cell lysate  was immunoprecipitated with anti-flag m <dig> , anti-ha or isotype igg antibody.
the immunoprecipitates were probed with anti-flag m <dig>  and anti-c-myc tag  monoclonal antibodies, and visualized with horseradish peroxidase-conjugated secondary antibodies using the ecl reagent .
for chromatin immunoprecipitation assay, chromosomal dna fragments were prepared as described  <cit> .
briefly, lysates were incubated with isotype igg or anti-flag antibody.
the approximately 100-base pair products from mir-221/- <dig> proximal promoter ; e2/ <dig> region: − <dig> ; nc region: − <dig> ) were detected by real-time pcr.
luciferase reporter assays
for reporter assays, 293t cells  in 24-well plates were transfected with either kaposin b or c-myc expression plasmid, along with the firefly luciferase reporter gene construct  and 50 ng prl-tk renilla luciferase construct  using turbofect™.
cells were harvested 48 h after transfection, and the luciferase activity was measured using the dual-luciferase reporter assay system .
RESULTS
kaposin b binds to c-myc for regulating endothelial cell angiogenic activities
kaposin b is a nuclear protein which can regulate mir- <dig> and mir- <dig> expressions without any predictable dna-binding domain  <cit> .
therefore, we hypothesized that kaposin b may be a novel transcription cofactor which regulates promoter activities by binding to other transcription factor.
to investigate, we overexpressed kaposin b in hmec <dig> and primary huvec cells and tested their migration and tube formation abilities.
we verified expression of kaposin b protein in lentivirus-transduced huvec using immunoblotting , and immunofluorescence staining showed that localization of kaposin b was in nucleus .
expression of kaposin b induced an increase in cell migration rate in hmec <dig> and primary huvec cells .
kaposin b also induced more microvascular structures formation by huvec in the in vitro tube formation assay .fig.
<dig> kaposin b binds to c-myc for regulating endothelial cell angiogenic activities.
a nuclear distribution pattern of kshv kaposin b protein in cells.
immunofluorescence staining for kaposin b proteins.
hmec <dig> cells with stable kaposin b  expression were fixed, and kaposin b proteins were detected using anti-flag mab, followed by anti–mouse igg secondary antibody conjugated with fitc .
cell nuclei were counterstained with hoechst  <dig>  while actin filaments with texas red phalloidin .
b hmec <dig> cells stably expressing kaposin b were subjected to the transwell cell-migration assay .
c kaposin b increases cell motility in huvec.
primary huvec stably transduced with kaposin b or the vector control by lentivirus were used for transwell cell-migration assays .
d kaposin b enhances microvascular formation of huvec in an in vitro matrigel angiogenesis assay.
pictures were taken after 6 h of incubation , and tube length was then measured and compared .
e schematic representation of mir-221/- <dig> proximal promoter.
three e-boxes  were found.
f co-immunoprecipitation assays show kaposin b and c-myc form a protein complex.
cell lysates were prepared from hmec <dig> cells stably expressing kaposin b. five micrograms of anti-flag , anti-ha  or isotype igg control were incubated with 500 μl of cell extracts and then analyzed by western blotting with indicated mabs.
g the interaction between c-myc and kaposin b was independent of promoter dna.
co-immunoprecipitation assays were performed with or without dnase pre-treatment on cell lysates prepared from hmec <dig> cells stably expressing kaposin b. anti-flag  or isotype igg control were incubated with cell extracts, and pull-down products were analyzed by western blotting with anti-ha mab for c-myc  or anti-flag m <dig> mab for kaposin b .
h-i knockdown of endogenous c-myc levels in kaposin b huvecs inhibits kaposin b-induced cell migration  and microvasculature formation
to maintain kshv latency and oncogenesis, kshv-latency proteins, lana and virf <dig>  stabilized and activated c-myc proteins  and c-myc was shown to reduce mir- <dig> expression in mouse mammary tumors  <cit> .
therefore, we examined the proximal promoter region of mir-221/- <dig> and identified three putative c-myc binding sites  .
we hypothesized that kaposin b may associate with c-myc in regulating host gene expression.
we therefore tested whether kaposin b and c-myc are in the same protein complex by co-transfecting flag-tagged kaposin b and ha-tagged c-myc into 293t cells.
c-myc was detected in immunoprecipitation  assays using anti-flag monoclonal antibody  against kaposin b. on the other hand, kaposin b was detected in ip assay using anti-ha mab against c-myc .
the interaction between c-myc and kaposin b was independent of promoter dna since c-myc was still detected in the anti-flag mab pull-down products even when cell lysates were pre-treated with dnase digestion for removal of genomic dna fragments .
we further checked whether c-myc is required for kaposin b functions.
knockdown of endogenous c-myc in kaposin b-expressing huvec cells abolished kaposin b-induced cell migration and microvasculature formation.
.
we also knocked down c-myc in kaposin b-expressing hmec <dig> cells and found that cell migration ability was down-regulated.
.
small rna sequencing reveals known mirnas co-regulated by c-myc and kaposin b in primary endothelial cells
previous study showed that kaposin b can down-regulate mir- <dig> and mir- <dig> levels by repressing the activity of mir-221/- <dig> cluster promoter  <cit> .
however, there are few studies exploring kaposin b-induced global mirnas alteration.
therefore, we overexpressed kaposin b in huvec to examine its regulated mirna expression.
we have demonstrated that c-myc binds to kaposin b  and is required for its functions , hence we overexpressed c-myc in kaposin b huvec to examine its ability to augment kaposin b-regulated mirna expression.
we analyzed small rna sequencing  data according to an in-house bioinformatics pipeline  <cit> .
the illumina next generation sequencing platform generated more than  <dig> million high-quality sequencing reads for huvec transduced with empty lentiviral vector alone, kaposin b alone and kaposin b with c-myc.
the initial operations included identifying sequence matches to the coding rna database for eliminating reads of degraded mrnas.
non-coding rna reads that match previously annotated mirnas deposited in the mirbase database  were subjected to normalization and quantitative profiling.
firstly, we explored known mirnas expression.
when we overexpressed kaposin b alone , we found  <dig> up-regulated and  <dig> down-regulated known mirnas.
in contrast,  <dig> up-regulated and  <dig> down-regulated mirnas  were identified upon simultaneous overexpression of kaposin b and c-myc .
we further interrogated the contribution of c-myc in kaposin b-regulated mirnome changes.
comparison of the c-myckaposin b mirna signature with that triggered by kaposin b alone in huvec indicated that c-myc helped to further induced the expression of  <dig>  %  of kapsoin b up-regulated mirnas .
similarly, the mirnas down-regulated by kaposin b and c-myc were  <dig>  % ; fig.
2b, lower panel, fold change ≥ <dig> ).
c-myc and kaposin b-regulated mirnas are enumerated in tables  <dig> and  <dig> kaposin b mirna are listed).
gene set enrichment analysis  revealed that the expression of up- or down-regulated mirnas by c-myc correlated with those induced or repressed by kaposin b in huvec .
we also performed qpcr to detect mirnas expression in kaposin b and kaposin bc-myc cells.
we identified that mir-193b-5p, mir-197-5p, mir- <dig>  and mir- <dig> were up-regulated in kaposin b and kaposin bc-myc cells.
in contrast, mir- <dig>  mir-146a-3p and mir-1271-5p were down-regulated .
rt-qpcr also demonstrated that mir- <dig> and mir- <dig> were down-regulated in kaposinb and kaposin bc-myc cells .
the mirnas detected in rt-qpcr, except mir- <dig> and mir-1271-5p, were similar to the ones detected in kshv-infected huvecs .
moreover, we had used up to  <dig> different kind of tools , mirdb  and targetminer ) to analyze the targeted genes of c-myc and kaposin b induced/reduced top  <dig> mirnas .
based on our results,  <dig> genes were targeted by the up-regulated mirnas  and  <dig> genes were targeted by the down-regulated mirnas , respectively.
the biological pathways were further analyzed, genes targeted by the up-regulated mirnas contribute to axonal guidance, cardiac β-adrenergic signaling and α-adrenergic signaling; while the genes targeted by the down-regulated mirnas contribute to wnt/β signaling, reelin signaling and molecular mechanisms of cancer .
this indicates kaposin b + c-myc regulated mirnas may trigger cells to more tumorigenic stage.fig.
<dig> c-myc cooperates with kaposin b to regulate cellular known mirnas.
a smrna-seq revealed differentially expressed mirnas between kaposin b and huvecs , and between kaposin bc-myc and huvecs .
b venn diagrams summarizing significant overlap between c-myc and kaposin b known mirnas signature.
up-regulated mirnas;  down-regulated mirnas.
c gene set enrichment analysis  verified the significant overlap between c-myc and kaposin b mirnas signature.
d rt-qpcr detected various known mirnas expression.
mean expression levels of the target mirnas are compared with the u <dig> control .
*: p < .
<dig>  e rt-qpcr detected mir-221/mir- <dig> expression in kaposin b and kaposin bc-myc cells.
f the biological pathway of genes which were targeted by the kaposin bc-myc induced/reduced mirnas.
the p value were marked on the figure
small rna sequencing identifies novel mirnas co-regulated by c-myc and kaposin b in primary endothelial cells
to identify novel mirnas, we first removed mrna contamination and known mirna loci.
using three independent bioinformatics algorithms , 20– <dig> unique genomic loci yielded at the same time in kaposin b-, kaposin bc-myc-expressing cells and huvec control cells , and we listed all novel mirna candidates in .
we found  <dig> and  <dig> novel mirna candidates in kaposin b-expressing cells and kaposin bc-myc cells  that were up-regulated, respectively.
in contrast, we identified  <dig> and  <dig> novel mirna candidates in kaposin b-expressing cells and down-regulated novel mirna candidates in kaposin bc-myc cells  that were down-regulated, respectively.
to characterize c-myc regulated novel mirna candidates in kaposin b cells, we compared the mirna profiles between kaposin bc-myc cells and kaposin b cells.
the mirna profiles revealed that out of  <dig> mirnas,  <dig>  were up-regulated and out of  <dig>   <dig>  were down-regulated novel mature mirna candidates correlated with c-myc expression in kaposin b cells .
to investigate the risc-binding properties of the novel mirna candidates, we acquired nine public ago1/2-mediated rna-immunoprecipitation sequencing  datasets from the geo database and re-analyzed using our in-house pipelines  <cit> .
the ago1/2-rna-ip-seq datasets verified  <dig> out of  <dig> up-regulated and  <dig> out of  <dig> down-regulated novel mirna candidates .fig.
<dig> c-myc cooperates with kaposin b to regulate cellular novel mirnas.
a the analysis pipeline for identification of known and novel mirnas from smrna-seq data.
reads or sequences pass each filtration process are indicated.
b venn diagrams summarizing significant overlap between c-myc and kaposin b signature novel mirnas.
up-regulated mirnas;  down-regulated mirnas.
c venn diagrams summarizing significant overlap between up-regulated or down-regulated novel mirna candidates and ago1/ <dig> rna-ip-seq data.
d detailed information of  <dig> c-myc-promoting and -reducing novel mirnas that expressed higher reads in different groups.
e deduced rna secondary structures of a set of newly discovered mirnas
to investigate c-myc-promoting or -reducing novel mirnas expression in kaposin b cells, we found that  <dig> out of  <dig> were up-regulated and  <dig> out of  <dig> were down-regulated novel mature mirna candidates that were c-myc-promoting or -reducing novel mirnas expression in kaposin b cells .
we showed  <dig> up-regulated or down-regulated novel c-myc-regulating mirnas candidates  that expressed higher read numbers in kaposin b and kaposin bc-myc cells  and their detailed information, including precursor locus, strand and mature sequence.
based on rnafold analysis , we demonstrated that these novel mirnas could fold into hairpin secondary structure, which is another mirna property .
c-myc enhances kaposin b association with and regulation of mirna promoters
previous studies showed that mir-221/- <dig> promoted angiogenesis and metastasis in various cancers, including gliomas, colon cancers and breast cancers  and that c-myc reduces mir- <dig> expression in mouse mammary tumors  <cit> .
furthermore, mir-221/- <dig> were crucial in kshv-mediated endothelial cell motility  <cit> .
therefore, to investigate whether c-myc can enhance kaposin b association with and regulation of mir-221/- <dig> promoters, we knocked down c-myc expression and we found that mir-221/- <dig> level were rescued .fig.
<dig> c-myc enhances kaposin b to regulate microrna promoter activity.
a knockdown of endogenous c-myc levels in kaposin b huvecs rescues mir- <dig>  and mir- <dig>  expression.
b chip analysis with immunoglobulin g control or anti-flag antibody .
schematic representation of mir-221/- <dig> promoter region and pcr fragments.
hmec <dig> cells stably expressing empty vector, kaposin b, or kaposin b + c-myc were subjected into chip assays.
the − <dig> promoter region pcr product was used as a negative control.
c wild type  promoter construct or double mutant construct  was transfected into a hmec <dig> cell line stably expressing kaposin b , and chip-qpcr assays were performed on transfectants using anti-flag mab.
d endogenous c-myc was stably knocked down using shrna in a hmec1-kaposin b stable cell line, and cells with or without c-myc knockdown were subjected to chip assays and qpcr for indicated regions .
e reporter activity of tested reporter plasmids after co-transfection with kaposin b, c-myc, or c-myc + kaposin b. schematic representation of reporter constructs  and reporter assay results  .
*: p < .
<dig>  f reporter assays on e <dig> or e2/ <dig> single mutants, and on the e <dig> and e2/e <dig> e-boxes double mutant.
only the double mutant showed significant promoter activity restoration.
*: p < .05
next, we performed chromatin immunoprecipitation  assays using an anti-flag mab to pull down kaposin b and its associated genomic dna fragments.
chip data identified the presence of c-myc-interacting mir-221/- <dig> promoter fragments containing the e <dig>  or e2/e <dig>  e-boxes in hmec <dig> cells stably expressing kaposin b .
overexpressing c-myc further enhanced the association between kaposin b and the mir-221/- <dig> promoter .
a negative control pcr containing a region with no putative e-box motif  showed no off-target enrichment from the same chip set .
direct involvement of e <dig> and e2/ <dig> motifs in kaposin b-c-myc complex binding was verified by chip assays using the site-directed mutagenesis of all the e <dig> and e2/e <dig> e-boxes plasmids.
a wild type − <dig> promoter construct, double mutant construct and kaposin b  were co-transfected into 293t cells, and chip assays were performed using anti-flag mab.
qpcr on ip-purified dna fragments showed that these e-box motifs were essential for kaposin b to associate with mir-221/- <dig> promoter .
we next examined the critical role of endogenous c-myc in kaposin b function.
knockdown of endogenous c-myc in kaposin b hmec <dig> cells significantly reduced kaposin b and mir-221/- <dig> promoter association .
repression of mir-221/- <dig> promoter activity was observed after transient co-transfection of kaposin b- or c-myc-expressing plasmids with the full-length reporter construct into 293t cells .
ectopic expression of both kaposin b and c-myc repressed mir-221/- <dig> promoter activity significantly more than expression of kaposin b or c-myc alone , suggesting c-myc enhances the capability of kaposin b to regulate the mir-221/- <dig> promoter.
deleting the region containing e2/ <dig> e-boxes did not dramatically influence kaposin b or c-myc function , but additional deletion of the e <dig> e-box significantly abolished the repression of the mir-221/- <dig> promoter by kaposin b, c-myc or both kaposin b and c-myc together .
site-directed mutagenesis of all the e <dig> and e2/e <dig> e-boxes, but not e <dig> or e2/ <dig> alone, hampered kaposin b- and/or c-myc-mediated repression, indicating c-myc could bind to all the  <dig> e-boxes to direct kaposin b to the mir-221/- <dig> promoter .
figure  <dig> is an illustration of the mechanism whereby kaposin b and c-myc in the same transcription complex can directly regulate the mir-221/- <dig> cluster promoter activity.fig.
<dig> a proposed model of regulation of endothelial cell activities by the kaposin b-c-myc circuit
discussion
kaposin b is known to stabilize various au-rich elements -containing cytokine mrnas, such as il <dig>  via the p38/mk <dig> pathway  <cit> .
ares are usually  <dig> to  <dig> nucleotides in length and located in the 3′ untranslated region  of short half-life mrnas.
ares serve as an mrna-destabilizing determinant by promoting degradation of mrnas.
ares are now grouped into three classes based on the number and distribution of the core auuua pentamers: cluster i are genes, such as cell cycle regulators c-myc and cyclin d <dig>  contain several dispersed copies of the auuua motif within the au-rich region  <cit> .
cluster ii are genes ) have at least  <dig> overlapping uuauuua motifs, and cluster iii are genes  are less characterized and do not contain the canonical auuua motif  <cit> .
database search revealed that there is no are on mir-221- <dig> pri-mirna.
whether kaposin b collaborates with c-myc to regulate promoter activities of these are genes is an interesting direction to follow and can be answered by global genomics approaches, such as chip-seq.
in this study, we use mir-221/- <dig> as a model to elucidate how kaposin b regulates promoter activity.
we demonstrate that c-myc together with kaposin b regulate the activity of mir-221/- <dig> cluster promoter.
c-myc is known to maintain kshv latency  <cit> .
kshv latency proteins, such as lana and virf <dig>  have been demonstrated to regulate c-myc.
lana stabilizes and activates c-myc through inhibition of gsk- <dig> phosphorylation and c-myc ubiquitination  <cit> .
lana protein that lacks a known dna-binding domain, also represses the expression of mir-221/- <dig> cluster  <cit> .
nevertheless, pilot studies showed that the interaction of kaposin b and c-myc is independent of lana .
virf <dig> associated with c-myc suppressor, mm-1α, to stimulate c-myc function  <cit> .
a c-myc-oriented angiogenesis program is therefore activated and plays a crucial role in kshv-infected cells and even in ks.
since ks is a neoplasm with an extensive neovasculature, the induction of endothelial cell migration and microvascular formation by c-myc and kaposin b may eventually benefit ks formation and progression.
myc protein, including c-myc, n-myc and l-myc, have been shown to be involved in pathogenesis of most cancers, such as burkitt’s lymphoma, breast cancer, prostate cancer, gastrointestinal cancer etc.
<cit> .
c-myc affects most important oncogenic programs through regulating several genes and mirnas expression  <cit> .
in c-myc-induced mouse mammary tumors,  <dig> and  <dig> mirnas show increased and decreased expression, respectively.
the expression of mir-20a, −20b, and mir- <dig> were increased, while that of mir- <dig> was decreased  <cit> .
in angiogenesis, previous report showed that c-myc activated mir- <dig> cluster expression to attenuate clusterin expression, thereby promoting angiogenesis  <cit> .
in this report, we provide a comprehensive c-myc-regulated mirome trait in primary human endothelial cells.
most of the c-myc and/or kaposin b-affected mirnas have not been linked to angiogenesis, cell motility or tumorigenesis.
among mirnas induced by both c-myc and kaposin b , mir- <dig>  the most prominent hypoxia-induced microrna, modulates cellular response to hypoxia through mnt, a max-interacting transcriptional repressor that functions as a c-myc antagonist  <cit> .
mir-483-5p, a microrna embedded in the intron of insulin-like growth factor  <dig> , inhibits angiogenesis via targeting serum response factor  <cit> .
mir- <dig> is involved in cell migration, proliferation, apoptosis, and globally affect the regulation circuits centered on mapk/erk signaling in colon cancer cells  <cit> .
knockdown of mir- <dig> in huvec significantly suppressed cell viability  <cit> .
mir- <dig> promotes cell growth and regulates g/s transition by targeting p21cip1/waf <dig> in hepatocellular carcinoma  <cit> .
CONCLUSIONS
in this study, we provide a global picture of kshv- and/or kaposin b-induced micrornas, especially those co-regulated by c-myc.
we have demonstrated that c-myc is essential for kaposin b to stimulate endothelial cell activity and to activate mirna promoters.
in addition to providing a global picture, this study also benefits the development of new therapeutic approaches to treat kshv-induced tumor progression in future.
additional files
additional file 1:
list of primers used in the study.
additional file 2:
primary huvec stably transduced with kaposin b or the vector control by lentivirus.
immunoblotting of kaposin b proteins with anti-flag mab .
β-actin was used as an internal control.
dnase treatment effects were verified by agarose gel electrophoresis.
knockdown of endogenous c-myc levels with shrna in kaposin b huvec  and kaposin b hmec <dig> cells  were validated by immunoblotting and qrt-pcr.
knockdown of endogenous c-myc levels with shrna in kaposin b hmec <dig> cells repressed kaposin b-induced cellular migration .
additional file 3:
validation of
smrna-seq
data by
rt-qpcr
in
kshv-infected
cells.
mean expression levels of the target mirnas are compared with the u <dig> control .
*: p < .
<dig>
additional file 4:
genes were targeted by the
up-regulated
mirnas  and
down-regulated
mirnas .
the genes interactions were analyzed by string .
additional file 5:
detail information of all novel mirnas.
additional file 6:
list of kaposin b and
c-myc
down-regulated
novel mirnas which exist in ago1/2-ip-seq data.
additional file 7:
list of kaposin b and
c-myc
up-regulated
novel mirnas which exist in ago1/2-ip-seq data.
competing interests
the authors declare that they no competing interests.
authors’ contributions
hww and ccc designed the analysis approach; hcc, thh, cft and ynt performed the research and analyzed the data; hww provided biologic guidance during the analysis process; hww and hcc wrote the manuscript; and all authors read and approved the final manuscript.

 kaposi’s sarcoma-associated herpesvirus, also known as human herpesvirus, is associated with kaposi’s sarcoma. ks is the most common cancer in individuals with aids and one of the most common cancers in patients who have immunosuppression. ks typically appears as colored lesions or blotches on the skin, although it can spread to internal organs. this disease has been recognized as a peculiar neoplasm that has a diverse cellular makeup and an extensive neovasculature. in host endothelial cells, kshv infection alters the expression of mrnas and micrornas required for many aspects of tumorigenesis, including angiogenesis. therefore, ks and kshv are recognized as an excellent model to study angiogenesis and to discover new angiogenic mechanisms, including coding and non-coding rnas. recently, the effect of kshv infection on cellular microrna expression has been reported. it was found that pre-mirna signatures delineate the stages of kshv-associated huvec transformation. mirnas are small rnas of 18– nucleotides in length that have emerged as master regulators of cancer and key modulators of angiogenesis. an increasing number of studies have shown that some mirnas, called angiomirs, play a crucial role in regulating angiogenesis. for example, mir- and mir- two closely related mirnas encoded in cluster from a genomic region on chromosome x, can modulate the angiogenic properties of human umbilical vein endothelial cells by targeting c-kit and endothelial nitric oxide synthase. mir- and mir- also regulate lymphatic endothelial cell motility by targeting ets and ets transcription factors, respectively. loss of mir- expression demarks the transition from merely immortalized to fully tumorigenic huvecs. kshv also induces endothelial cell motility by inducing mir- expression while suppressing that of mir- and mir-. kaposin b, one of the three kaposin proteins encoded from the kshv genome k locus, is one of the viral genes expressed during latency and is therefore expressed in all kshv tumor cells. kaposin b stabilizes various cytokine mrna containing au-rich elements via the p38/mk pathway. in response to lps, kaposin b and mk were shown to be exported to the cytoplasm, where mrna stability is regulated. kaposin b also enhances the prox mrna stability during lymphatic reprogramming of vascular endothelial cells. kaposin b can influence cellular gene expression by regulating promoter activities of host genes: both kaposin b and kshv viral latency-associated nuclear antigen proteins can down-regulate mir- and mir- levels by repressing the activity of mir-221/- cluster promoter. since there is no predictable dna-binding domain on kaposin b, how this nuclear protein can regulate mrna and mirna expression remains unclear. c-myc achieves its oncogenic effects by regulating transcription of protein-coding genes as well as microrna genes such as mir-29b-1/mir-29a. c-myc is also essential for vasculogenesis and angiogenesis during development and tumor progression via inducing the expression of mir-17 ~  angiogenic mirna cluster. revealing the angiomirs regulated by c-myc and the underlying regulatory mechanisms will help to further understand c-myc and endothelial cell biology. here, we showed that kaposin b and c-myc are in the same transcription complex that directly regulates the mir-221/- cluster promoter activity. a c-myc-oriented circuit is therefore formed in the presence of kaposin b in kshv-infected endothelial cells. furthermore, we also provide a global microrna signature which is regulated by c-myc and kaposin b. we hope our roadmap will help the search and development of new therapeutic targets for virus- or cancer-induced angiogenesis, cancer formation and metastasis. methods cell culture and kshv infection human primary umbilical vein endothelial cells were purchased from clonetics inc. and were cultured as described. hmec an immortalized human microvascular endothelial cell line, was cultured in endothelial cell growth medium mv. a recombinant virus, rkshv. that expresses the red fluorescent protein from the kshv lytic pan promoter, the green fluorescent protein from the ef-1alpha promoter, and with a gene for puromycin resistance as a selectable marker, was constructed using jsc- cells as described previously. plasmid construction plasmids expressing kshv kaposin b and mir- mir- or mir-221/- were constructed as described previously. c-myc expression constructs and knock down construct was kindly provided by prof. kenneth cw wu. the full-length mir-221/- promoter reporter plasmid was constructed as described previously, and primers for cloning mir-221/- promoter mutants are listed in additional file immunofluorescence assay cells were fixed with 4 % paraformaldehyde, permeabilized with  % triton x-, and then blocked with pbs containing 1 % bovine serum albumin. for kaposin b staining, cells were incubated with the monoclonal antibody anti-flag m at a 1: dilution for to 120 min at 25 °c followed by incubation with fitc-conjugated goat anti-mouse igg for to 120 min at 25 °c. rhodamine-phalloidin was used to label actin cytoskeleton. cell nuclei were counterstained with hoechst, and examined by fluorescence confocal microscopy. transwell cell migration and endothelial cell tube formation assays cell migration ability was evaluated using costar transwell® polycarbonate permeable supports as described previously. in brief, 5 ×  cells in 500 μl of culture medium were applied to the upper chamber of the device, and 750 μl of medium containing 10 ng/ml human vegf was added to the lower chamber. a polycarbonate membrane with a pore size of 8 μm was placed in between the two chambers. after 6 h of incubation at 37 °c for hmec and huvec cells, the membrane was fixed in 4 % paraformaldehyde for 20 min at room temperature and then stained with hoechst solution for 30 min. on the upper side of membrane were identified un-migrated cells and removed. the cells under the membrane were counted under a microscope. in vitro angiogenic activity of vascular endothelial cells was examined by tube formation assays: primary huvecs were placed onto matrigel® basement membrane matrix gel for 6 h at 37 °c. the formation of tubule structure was observed every hour under a microscope. small rna sequencing and data analysis total rna was collected and small rna fractions were sequenced using illumina hiseq sequencer according to the manufacturer’s instructions. for data analysis, quality fastq sequences, which were without poly-a, ambiguous nucleotides or a 5′ adapter, yet flanking 6–18 nt of 3′ adapter sequence, had the adapter sequences trimmed and the identical sequences were then collapsed to unique sequences. the resulting unique sequences that did not align to the mrna database but were aligned to known microrna sequences were subjected to further quantification analysis. sequencing reads were used to obtain a rpm value calculated as c/ ×  where c is “read numbers aligned to a given mirna chromosomal region”, m is “multiple mapping numbers across all mirna regions” and n is “total read numbers that map to human genome sequence”. rna extraction, real-time quantitative polymerase chain reaction and microrna expression rna extraction and reverse transcription were performed as previously described. the expression of mature human mir- and mir- was determined by a stem-loop real-time pcr system using the primer pairs previously published. microrna expression data were normalized to that of u snrna. details of qpcr primers are in additional file coimmunoprecipitation, chromatin immunoprecipitation and immunoblotting co-ip and chip assays were performed as previously described. briefly, kaposin b or c-myc expression plasmid was co-transfected into 293t cells using turbofect™. after 48 h, cells were lyzed in net lysis buffer containing protease and phosphatase inhibitors and cell lysate was immunoprecipitated with anti-flag m, anti-ha or isotype igg antibody. the immunoprecipitates were probed with anti-flag m and anti-c-myc tag monoclonal antibodies, and visualized with horseradish peroxidase-conjugated secondary antibodies using the ecl reagent. for chromatin immunoprecipitation assay, chromosomal dna fragments were prepared as described. briefly, lysates were incubated with isotype igg or anti-flag antibody. the approximately 100-base pair products from mir-221/- proximal promoter; e2/ region: −; nc region: − ) were detected by real-time pcr. luciferase reporter assays for reporter assays, 293t cells in 24-well plates were transfected with either kaposin b or c-myc expression plasmid, along with the firefly luciferase reporter gene construct and 50 ng prl-tk renilla luciferase construct using turbofect™. cells were harvested 48 h after transfection, and the luciferase activity was measured using the dual-luciferase reporter assay system.  kaposin b binds to c-myc for regulating endothelial cell angiogenic activities kaposin b is a nuclear protein which can regulate mir- and mir- expressions without any predictable dna-binding domain. therefore, we hypothesized that kaposin b may be a novel transcription cofactor which regulates promoter activities by binding to other transcription factor. to investigate, we overexpressed kaposin b in hmec and primary huvec cells and tested their migration and tube formation abilities. we verified expression of kaposin b protein in lentivirus-transduced huvec using immunoblotting, and immunofluorescence staining showed that localization of kaposin b was in nucleus. expression of kaposin b induced an increase in cell migration rate in hmec and primary huvec cells. kaposin b also induced more microvascular structures formation by huvec in the in vitro tube formation assay.fig. kaposin b binds to c-myc for regulating endothelial cell angiogenic activities. a nuclear distribution pattern of kshv kaposin b protein in cells. immunofluorescence staining for kaposin b proteins. hmec cells with stable kaposin b expression were fixed, and kaposin b proteins were detected using anti-flag mab, followed by anti–mouse igg secondary antibody conjugated with fitc. cell nuclei were counterstained with hoechst while actin filaments with texas red phalloidin. b hmec cells stably expressing kaposin b were subjected to the transwell cell-migration assay. c kaposin b increases cell motility in huvec. primary huvec stably transduced with kaposin b or the vector control by lentivirus were used for transwell cell-migration assays. d kaposin b enhances microvascular formation of huvec in an in vitro matrigel angiogenesis assay. pictures were taken after 6 h of incubation, and tube length was then measured and compared. e schematic representation of mir-221/- proximal promoter. three e-boxes were found. f co-immunoprecipitation assays show kaposin b and c-myc form a protein complex. cell lysates were prepared from hmec cells stably expressing kaposin b. five micrograms of anti-flag, anti-ha or isotype igg control were incubated with 500 μl of cell extracts and then analyzed by western blotting with indicated mabs. g the interaction between c-myc and kaposin b was independent of promoter dna. co-immunoprecipitation assays were performed with or without dnase pre-treatment on cell lysates prepared from hmec cells stably expressing kaposin b. anti-flag or isotype igg control were incubated with cell extracts, and pull-down products were analyzed by western blotting with anti-ha mab for c-myc or anti-flag m mab for kaposin b. h-i knockdown of endogenous c-myc levels in kaposin b huvecs inhibits kaposin b-induced cell migration and microvasculature formation to maintain kshv latency and oncogenesis, kshv-latency proteins, lana and virf stabilized and activated c-myc proteins and c-myc was shown to reduce mir- expression in mouse mammary tumors. therefore, we examined the proximal promoter region of mir-221/- and identified three putative c-myc binding sites. we hypothesized that kaposin b may associate with c-myc in regulating host gene expression. we therefore tested whether kaposin b and c-myc are in the same protein complex by co-transfecting flag-tagged kaposin b and ha-tagged c-myc into 293t cells. c-myc was detected in immunoprecipitation assays using anti-flag monoclonal antibody against kaposin b. on the other hand, kaposin b was detected in ip assay using anti-ha mab against c-myc. the interaction between c-myc and kaposin b was independent of promoter dna since c-myc was still detected in the anti-flag mab pull-down products even when cell lysates were pre-treated with dnase digestion for removal of genomic dna fragments. we further checked whether c-myc is required for kaposin b functions. knockdown of endogenous c-myc in kaposin b-expressing huvec cells abolished kaposin b-induced cell migration and microvasculature formation.. we also knocked down c-myc in kaposin b-expressing hmec cells and found that cell migration ability was down-regulated.. small rna sequencing reveals known mirnas co-regulated by c-myc and kaposin b in primary endothelial cells previous study showed that kaposin b can down-regulate mir- and mir- levels by repressing the activity of mir-221/- cluster promoter. however, there are few studies exploring kaposin b-induced global mirnas alteration. therefore, we overexpressed kaposin b in huvec to examine its regulated mirna expression. we have demonstrated that c-myc binds to kaposin b and is required for its functions, hence we overexpressed c-myc in kaposin b huvec to examine its ability to augment kaposin b-regulated mirna expression. we analyzed small rna sequencing data according to an in-house bioinformatics pipeline. the illumina next generation sequencing platform generated more than million high-quality sequencing reads for huvec transduced with empty lentiviral vector alone, kaposin b alone and kaposin b with c-myc. the initial operations included identifying sequence matches to the coding rna database for eliminating reads of degraded mrnas. non-coding rna reads that match previously annotated mirnas deposited in the mirbase database were subjected to normalization and quantitative profiling. firstly, we explored known mirnas expression. when we overexpressed kaposin b alone, we found up-regulated and down-regulated known mirnas. in contrast, up-regulated and down-regulated mirnas were identified upon simultaneous overexpression of kaposin b and c-myc. we further interrogated the contribution of c-myc in kaposin b-regulated mirnome changes. comparison of the c-myckaposin b mirna signature with that triggered by kaposin b alone in huvec indicated that c-myc helped to further induced the expression of  % of kapsoin b up-regulated mirnas. similarly, the mirnas down-regulated by kaposin b and c-myc were  %; fig. 2b, lower panel, fold change ≥ ). c-myc and kaposin b-regulated mirnas are enumerated in tables  and kaposin b mirna are listed). gene set enrichment analysis revealed that the expression of up- or down-regulated mirnas by c-myc correlated with those induced or repressed by kaposin b in huvec. we also performed qpcr to detect mirnas expression in kaposin b and kaposin bc-myc cells. we identified that mir-193b-5p, mir-197-5p, mir- and mir- were up-regulated in kaposin b and kaposin bc-myc cells. in contrast, mir- mir-146a-3p and mir-1271-5p were down-regulated. rt-qpcr also demonstrated that mir- and mir- were down-regulated in kaposinb and kaposin bc-myc cells. the mirnas detected in rt-qpcr, except mir- and mir-1271-5p, were similar to the ones detected in kshv-infected huvecs. moreover, we had used up to different kind of tools, mirdb and targetminer ) to analyze the targeted genes of c-myc and kaposin b induced/reduced top mirnas. based on our results, genes were targeted by the up-regulated mirnas and genes were targeted by the down-regulated mirnas, respectively. the biological pathways were further analyzed, genes targeted by the up-regulated mirnas contribute to axonal guidance, cardiac β-adrenergic signaling and α-adrenergic signaling; while the genes targeted by the down-regulated mirnas contribute to wnt/β signaling, reelin signaling and molecular mechanisms of cancer. this indicates kaposin b + c-myc regulated mirnas may trigger cells to more tumorigenic stage.fig. c-myc cooperates with kaposin b to regulate cellular known mirnas. a smrna-seq revealed differentially expressed mirnas between kaposin b and huvecs, and between kaposin bc-myc and huvecs. b venn diagrams summarizing significant overlap between c-myc and kaposin b known mirnas signature. up-regulated mirnas; down-regulated mirnas. c gene set enrichment analysis verified the significant overlap between c-myc and kaposin b mirnas signature. d rt-qpcr detected various known mirnas expression. mean expression levels of the target mirnas are compared with the u control. *: p <. e rt-qpcr detected mir-221/mir- expression in kaposin b and kaposin bc-myc cells. f the biological pathway of genes which were targeted by the kaposin bc-myc induced/reduced mirnas. the p value were marked on the figure small rna sequencing identifies novel mirnas co-regulated by c-myc and kaposin b in primary endothelial cells to identify novel mirnas, we first removed mrna contamination and known mirna loci. using three independent bioinformatics algorithms, 20– unique genomic loci yielded at the same time in kaposin b-, kaposin bc-myc-expressing cells and huvec control cells, and we listed all novel mirna candidates in. we found and novel mirna candidates in kaposin b-expressing cells and kaposin bc-myc cells that were up-regulated, respectively. in contrast, we identified and novel mirna candidates in kaposin b-expressing cells and down-regulated novel mirna candidates in kaposin bc-myc cells that were down-regulated, respectively. to characterize c-myc regulated novel mirna candidates in kaposin b cells, we compared the mirna profiles between kaposin bc-myc cells and kaposin b cells. the mirna profiles revealed that out of mirnas, were up-regulated and out of were down-regulated novel mature mirna candidates correlated with c-myc expression in kaposin b cells. to investigate the risc-binding properties of the novel mirna candidates, we acquired nine public ago1/2-mediated rna-immunoprecipitation sequencing datasets from the geo database and re-analyzed using our in-house pipelines. the ago1/2-rna-ip-seq datasets verified out of up-regulated and out of down-regulated novel mirna candidates.fig. c-myc cooperates with kaposin b to regulate cellular novel mirnas. a the analysis pipeline for identification of known and novel mirnas from smrna-seq data. reads or sequences pass each filtration process are indicated. b venn diagrams summarizing significant overlap between c-myc and kaposin b signature novel mirnas. up-regulated mirnas; down-regulated mirnas. c venn diagrams summarizing significant overlap between up-regulated or down-regulated novel mirna candidates and ago1/ rna-ip-seq data. d detailed information of c-myc-promoting and -reducing novel mirnas that expressed higher reads in different groups. e deduced rna secondary structures of a set of newly discovered mirnas to investigate c-myc-promoting or -reducing novel mirnas expression in kaposin b cells, we found that out of were up-regulated and out of were down-regulated novel mature mirna candidates that were c-myc-promoting or -reducing novel mirnas expression in kaposin b cells. we showed up-regulated or down-regulated novel c-myc-regulating mirnas candidates that expressed higher read numbers in kaposin b and kaposin bc-myc cells and their detailed information, including precursor locus, strand and mature sequence. based on rnafold analysis, we demonstrated that these novel mirnas could fold into hairpin secondary structure, which is another mirna property. c-myc enhances kaposin b association with and regulation of mirna promoters previous studies showed that mir-221/- promoted angiogenesis and metastasis in various cancers, including gliomas, colon cancers and breast cancers and that c-myc reduces mir- expression in mouse mammary tumors. furthermore, mir-221/- were crucial in kshv-mediated endothelial cell motility. therefore, to investigate whether c-myc can enhance kaposin b association with and regulation of mir-221/- promoters, we knocked down c-myc expression and we found that mir-221/- level were rescued.fig. c-myc enhances kaposin b to regulate microrna promoter activity. a knockdown of endogenous c-myc levels in kaposin b huvecs rescues mir- and mir- expression. b chip analysis with immunoglobulin g control or anti-flag antibody. schematic representation of mir-221/- promoter region and pcr fragments. hmec cells stably expressing empty vector, kaposin b, or kaposin b + c-myc were subjected into chip assays. the − promoter region pcr product was used as a negative control. c wild type promoter construct or double mutant construct was transfected into a hmec cell line stably expressing kaposin b, and chip-qpcr assays were performed on transfectants using anti-flag mab. d endogenous c-myc was stably knocked down using shrna in a hmec1-kaposin b stable cell line, and cells with or without c-myc knockdown were subjected to chip assays and qpcr for indicated regions. e reporter activity of tested reporter plasmids after co-transfection with kaposin b, c-myc, or c-myc + kaposin b. schematic representation of reporter constructs and reporter assay results. *: p <. f reporter assays on e or e2/ single mutants, and on the e and e2/e e-boxes double mutant. only the double mutant showed significant promoter activity restoration. *: p <.05 next, we performed chromatin immunoprecipitation assays using an anti-flag mab to pull down kaposin b and its associated genomic dna fragments. chip data identified the presence of c-myc-interacting mir-221/- promoter fragments containing the e or e2/e e-boxes in hmec cells stably expressing kaposin b. overexpressing c-myc further enhanced the association between kaposin b and the mir-221/- promoter. a negative control pcr containing a region with no putative e-box motif showed no off-target enrichment from the same chip set. direct involvement of e and e2/ motifs in kaposin b-c-myc complex binding was verified by chip assays using the site-directed mutagenesis of all the e and e2/e e-boxes plasmids. a wild type − promoter construct, double mutant construct and kaposin b were co-transfected into 293t cells, and chip assays were performed using anti-flag mab. qpcr on ip-purified dna fragments showed that these e-box motifs were essential for kaposin b to associate with mir-221/- promoter. we next examined the critical role of endogenous c-myc in kaposin b function. knockdown of endogenous c-myc in kaposin b hmec cells significantly reduced kaposin b and mir-221/- promoter association. repression of mir-221/- promoter activity was observed after transient co-transfection of kaposin b- or c-myc-expressing plasmids with the full-length reporter construct into 293t cells. ectopic expression of both kaposin b and c-myc repressed mir-221/- promoter activity significantly more than expression of kaposin b or c-myc alone, suggesting c-myc enhances the capability of kaposin b to regulate the mir-221/- promoter. deleting the region containing e2/ e-boxes did not dramatically influence kaposin b or c-myc function, but additional deletion of the e e-box significantly abolished the repression of the mir-221/- promoter by kaposin b, c-myc or both kaposin b and c-myc together. site-directed mutagenesis of all the e and e2/e e-boxes, but not e or e2/ alone, hampered kaposin b- and/or c-myc-mediated repression, indicating c-myc could bind to all the e-boxes to direct kaposin b to the mir-221/- promoter. figure  is an illustration of the mechanism whereby kaposin b and c-myc in the same transcription complex can directly regulate the mir-221/- cluster promoter activity.fig. a proposed model of regulation of endothelial cell activities by the kaposin b-c-myc circuit discussion kaposin b is known to stabilize various au-rich elements -containing cytokine mrnas, such as il via the p38/mk pathway. ares are usually to nucleotides in length and located in the 3′ untranslated region of short half-life mrnas. ares serve as an mrna-destabilizing determinant by promoting degradation of mrnas. ares are now grouped into three classes based on the number and distribution of the core auuua pentamers: cluster i are genes, such as cell cycle regulators c-myc and cyclin d contain several dispersed copies of the auuua motif within the au-rich region. cluster ii are genes ) have at least overlapping uuauuua motifs, and cluster iii are genes are less characterized and do not contain the canonical auuua motif. database search revealed that there is no are on mir-221- pri-mirna. whether kaposin b collaborates with c-myc to regulate promoter activities of these are genes is an interesting direction to follow and can be answered by global genomics approaches, such as chip-seq. in this study, we use mir-221/- as a model to elucidate how kaposin b regulates promoter activity. we demonstrate that c-myc together with kaposin b regulate the activity of mir-221/- cluster promoter. c-myc is known to maintain kshv latency. kshv latency proteins, such as lana and virf have been demonstrated to regulate c-myc. lana stabilizes and activates c-myc through inhibition of gsk- phosphorylation and c-myc ubiquitination. lana protein that lacks a known dna-binding domain, also represses the expression of mir-221/- cluster. nevertheless, pilot studies showed that the interaction of kaposin b and c-myc is independent of lana. virf associated with c-myc suppressor, mm-1α, to stimulate c-myc function. a c-myc-oriented angiogenesis program is therefore activated and plays a crucial role in kshv-infected cells and even in ks. since ks is a neoplasm with an extensive neovasculature, the induction of endothelial cell migration and microvascular formation by c-myc and kaposin b may eventually benefit ks formation and progression. myc protein, including c-myc, n-myc and l-myc, have been shown to be involved in pathogenesis of most cancers, such as burkitt’s lymphoma, breast cancer, prostate cancer, gastrointestinal cancer etc.. c-myc affects most important oncogenic programs through regulating several genes and mirnas expression. in c-myc-induced mouse mammary tumors, and mirnas show increased and decreased expression, respectively. the expression of mir-20a, −20b, and mir- were increased, while that of mir- was decreased. in angiogenesis, previous report showed that c-myc activated mir- cluster expression to attenuate clusterin expression, thereby promoting angiogenesis. in this report, we provide a comprehensive c-myc-regulated mirome trait in primary human endothelial cells. most of the c-myc and/or kaposin b-affected mirnas have not been linked to angiogenesis, cell motility or tumorigenesis. among mirnas induced by both c-myc and kaposin b, mir- the most prominent hypoxia-induced microrna, modulates cellular response to hypoxia through mnt, a max-interacting transcriptional repressor that functions as a c-myc antagonist. mir-483-5p, a microrna embedded in the intron of insulin-like growth factor, inhibits angiogenesis via targeting serum response factor. mir- is involved in cell migration, proliferation, apoptosis, and globally affect the regulation circuits centered on mapk/erk signaling in colon cancer cells. knockdown of mir- in huvec significantly suppressed cell viability. mir- promotes cell growth and regulates g/s transition by targeting p21cip1/waf in hepatocellular carcinoma.  in this study, we provide a global picture of kshv- and/or kaposin b-induced micrornas, especially those co-regulated by c-myc. we have demonstrated that c-myc is essential for kaposin b to stimulate endothelial cell activity and to activate mirna promoters. in addition to providing a global picture, this study also benefits the development of new therapeutic approaches to treat kshv-induced tumor progression in future. additional files additional file 1: list of primers used in the study. additional file 2: primary huvec stably transduced with kaposin b or the vector control by lentivirus. immunoblotting of kaposin b proteins with anti-flag mab. β-actin was used as an internal control. dnase treatment effects were verified by agarose gel electrophoresis. knockdown of endogenous c-myc levels with shrna in kaposin b huvec and kaposin b hmec cells were validated by immunoblotting and qrt-pcr. knockdown of endogenous c-myc levels with shrna in kaposin b hmec cells repressed kaposin b-induced cellular migration. additional file 3: validation of smrna-seq data by rt-qpcr in kshv-infected cells. mean expression levels of the target mirnas are compared with the u control. *: p <. additional file 4: genes were targeted by the up-regulated mirnas and down-regulated mirnas. the genes interactions were analyzed by string. additional file 5: detail information of all novel mirnas. additional file 6: list of kaposin b and c-myc down-regulated novel mirnas which exist in ago1/2-ip-seq data. additional file 7: list of kaposin b and c-myc up-regulated novel mirnas which exist in ago1/2-ip-seq data. competing interests the authors declare that they no competing interests. authors’ contributions hww and ccc designed the analysis approach; hcc, thh, cft and ynt performed the research and analyzed the data; hww provided biologic guidance during the analysis process; hww and hcc wrote the manuscript; and all authors read and approved the final manuscript. 
kaposi’s sarcoma, which is induced by the etiological infectious agent ks-associated herpesvirus, is a peculiar neoplasm that expresses both blood and lymphatic endothelial markers and possesses extensive neovasculature. by small rna sequencing, we revealed that  % of kaposin b up-regulated and  % of kaposin b down-regulated known mirnas were regulated by c-myc. micrornasangiogenesisks-associated herpesvirusc-myckaposin bsmall rna sequencingthe fourteenth asia pacific bioinformatics conference apbc 2016san francisco, ca, usa - january http://www.sfasa.org/apbc2016/apbc htmlissue-copyright-statement© the author 2016 since kaposin b is a nuclear protein with no dna-binding domain, it may regulate gene expression by incorporating itself into a transcription complex. micrornas have emerged as master regulators of angiogenesis and other cancer-related events. kaposin b is one of the latent viral genes and is expressed in all kshv tumor cells. we demonstrated that c-myc and kaposin b form a transcription complex and bind to the mir-221/- promoter, thereby affecting their expression and anti-angiogenic ability. we also found that novel mirna were up-regulated and novel mirnas were down-regulated in cells expressing both c-myc and kaposin b compared with cells expressing kaposin b only. our study identifies known and novel c-myc-regulated micrornas and reveals that a c-myc-oriented program is coordinated by kaposin b in kshv-infected cells. using kshv and its proteins as baits will be an efficient way to discover new angiomirs in endothelial cells.
 micrornas have emerged as master regulators of angiogenesis and other cancer-related events. discovering new angiogenesis-regulating micrornas will eventually help in developing new therapeutic strategies for tumor angiogenesis and cardiovascular diseases. kaposi’s sarcoma, which is induced by the etiological infectious agent ks-associated herpesvirus, is a peculiar neoplasm that expresses both blood and lymphatic endothelial markers and possesses extensive neovasculature. using kshv and its proteins as baits will be an efficient way to discover new angiomirs in endothelial cells. kaposin b is one of the latent viral genes and is expressed in all kshv tumor cells. since kaposin b is a nuclear protein with no dna-binding domain, it may regulate gene expression by incorporating itself into a transcription complex.  we demonstrated that c-myc and kaposin b form a transcription complex and bind to the mir-221/- promoter, thereby affecting their expression and anti-angiogenic ability. by small rna sequencing, we revealed that  % of kaposin b up-regulated and  % of kaposin b down-regulated known mirnas were regulated by c-myc. we also found that novel mirna were up-regulated and novel mirnas were down-regulated in cells expressing both c-myc and kaposin b compared with cells expressing kaposin b only. the result was confirmed by rna-ip-seq data.  our study identifies known and novel c-myc-regulated micrornas and reveals that a c-myc-oriented program is coordinated by kaposin b in kshv-infected cells. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords micrornasangiogenesisks-associated herpesvirusc-myckaposin bsmall rna sequencingthe fourteenth asia pacific bioinformatics conference apbc 2016san francisco, ca, usa - january http://www.sfasa.org/apbc2016/apbc htmlissue-copyright-statement© the author 2016 