BACKGROUND
ionizing radiations  induce a large spectrum of damages that irradiated cells have to counteract by activating complex response pathways.
the gene networks mobilized in the cellular response to radiations are subtly regulated.
several molecular actors have been shown to be involved in the regulation of gene expression after irradiation including chromatin-remodeling factors  <cit> , transcription factors  <cit>  or factors controlling protein synthesis  <cit> , maturation  <cit>  and degradation  <cit> .
micrornas  are short non-coding rnas  which are predicted to control in mammals the activity of approximately 30% of all protein coding genes and have been shown to participate in the regulation of almost any cellular processes investigated so far  <cit> .
by base pairing to the 3′ untranslated regions of target mrnas, mirnas mediate translational repression during the initiation or elongation step, and proteolysis of the nascent peptide or mrna degradation after deadenylation  <cit> .
mirnas have been proposed to be important actors of the dna damage response  <cit> .
several mirnas expression studies have been conducted after irradiation in mice  <cit> , human tumor samples  <cit> , cancer cell lines  <cit>  or normal human cells  <cit> .
by crossing these studies, it was not possible to identify a short-list of universal mirnas responding to ir.
the radiation effect on mirna expression seems therefore to vary according to cell type, radiation dose and post-irradiation time point  <cit> .
skin is the first organ to be targeted by external radiations and among the most sensitive.
the epidermis is a stratified squamous epithelium that forms the protective covering of the skin.
it is composed of a basal layer of proliferating cells, a spinous postmitotic cell layer, a granular layer, and a stratum corneum of terminally differentiating keratinocytes.
high doses of radiation can induce various deleterious effects in skin, which can appear a short time after exposure, such as erythema and desquamation, and several years later, such as carcinoma and late complications  <cit> .
radiation-induced damages in epidermal proliferating cells have been suspected to be involved in late effects such as skin cancer whereas defects in differentiated cells could lead to early effects such as hyperkeratosis or desquamation.
it is therefore of great interest to investigate the role of mirnas in the radiation response in proliferating versus differentiated keratinocytes.
although the biological effects of low doses are controversial, we have previously shown that a very low dose of ir is able to induce a strong and specific mrna response in human keratinocytes  <cit> .
by providing a global view of the mirna response after strong and low doses of ir, our study should help answering in which condition a mirna response is induced after irradiation.
in this study, mirna expression profiling was investigated in human primary keratinocytes after acute exposure to a very low  or a strong  dose of gamma rays.
we showed that radiation induced changes in mirna levels varied according to the dose and the differentiation status of irradiated keratinocytes.
indeed, after a strong dose of 6 gy, a global repression of mirnas was observed in proliferating keratinocytes whereas a global induction was detected in differentiated keratinocytes.
the very low dose of 10 mgy was also able to modulate some mirnas in proliferating cells.
over-expression of the most stringently regulated mirnas in proliferating cells modified their survival after 6 gy irradiation.
in differentiated keratinocytes, repression of the  <dig> most activated mirnas had no detectable effect on survival.
our data suggest that the mirna response is globally modulated in irradiated keratinocytes and could be involved in cellular pathways that are highly dependent to the differentiation state of the ir-exposed cells.
RESULTS
characterization of the cellular model
the cellular model used in this study consisted of primary cultures of human keratinocytes  isolated from infant foreskin.
for modeling epidermal basal cells, second passage cultures were cultivated up to 50% of confluency where most of the cells are still proliferating.
to induce epidermal differentiation, cells were cultured at low calcium concentration up to confluence, and then incubated 3 days post-confluence in a culture medium containing  <dig>  mm of calcium.
differentiated cultures were subjected to expression analysis by real-time pcr of two well-known markers of epidermal suprabasal layers: keratin  <dig>  and involucrin  as well as markers of proliferating basal cells: keratin  <dig>  and pcna.
the expression was compared to that of proliferative cultures.
as expected, a strong expression of krt <dig> and ivl transcripts was observed in differentiated keratinocytes whereas pcna and krt <dig> were clearly repressed .
these results suggest that the majority of cells cultured 3 days post-confluence in a high-calcium medium exhibited a differentiation state close to that found in spinous and granular layers.
on the contrary, semi-confluent keratinocytes express markers typical to the basal layers of the human epidermis.
we next investigated the short-term radiosensitivity of proliferating and differentiated keratinocytes after the acute high dose of 6 gy.
the viability of irradiated cells was measured  <dig> to 72 hours after irradiation .
for proliferating cells , we observed a significant decrease in cell viability starting 24 h post irradiation  with a maximal effect intensity of 20% decrease 48 h and 72 h post-irradiation .
in differentiated cells , a weaker but significant effect was observed from 24 h to 72 h post-irradiation with a maximal decrease in cell viability of 10% 3 days post-irradiation .
these data confirmed, as previously reported by our group  <cit> , that differentiated keratinocytes are more radio-resistant than proliferative keratinocytes.
mirna expression profiling
we used taqman quantitative real-time pcr low density array  to profile mirnas expression in control and irradiated keratinocytes.
we investigated the mirna response to a high dose of 6 gy in proliferating and differentiated keratinocytes and to a very low dose of 10 mgy in proliferating cells, which are more radiosensitive.
mirnas expression was assessed 3 hours post-irradiation since this time-point corresponds to the immediate gene response in irradiated keratinocytes  <cit>  and mirnas might be involved in the regulation of this gene response.
three to four human keratinocytes cultures established from independent donors were analyzed at each dose.
due to the high repeatability of the tlda methodology  <cit> , we privileged in our experimental plan biological replicates from different donors than technical repeats.
it is important to note that the experimental plan is constructed on paired data: for each donor, a reference condition response and a response following ionizing irradiation were quantified.
out of  <dig> mirnas studied, only those with a cq  value below 32 cycles in all samples were considered as significantly expressed.
this threshold was calculated using mirna duplicate and snrna  tetraplicate repeatability, in agreement with literature  <cit> .
<dig> mirnas for  <dig> gy vs 6 gy  irradiated proliferating keratinocytes,  <dig> mirnas for 0/6 gy-irradiated differentiated keratinocytes, and  <dig> mirnas for 0 gy vs 10 mgy  irradiated proliferating keratinocytes were therefore considered for further analysis .
we first investigated if a bias exists in the average expression of mirnas between hpk cell samples sorted by differentiation state and irradiation dose .
we used all-paired tukey-kramer tests to compare cq means within hpk samples.
for 0/6 gy-irradiated proliferating keratinocytes, there was no expression bias between the cell samples .
for 0/6 gy-irradiated differentiated keratinocytes, significant differences were observed: samples from hpk <dig> revealed higher cq than the three others donors .
a difference was also observed for 0/10 mgy-irradiated proliferating keratinocytes: samples from donor hpk <dig> showed in average lower cq, than those from hpk <dig> and hpk <dig> .
however, these differences in the average expression of mirnas were similar for reference and irradiated conditions , and could be due to inter-individual variability in primary cells.
this observation underlined the importance to use paired data for statistical analyses.
it further confirmed that there was no sample processing bias due to irradiation state and that raw data normalization was not necessary as previously proposed  <cit> .
we then focused on mirna profiles after irradiation in proliferating and differentiated keratinocytes.
after the high dose of 6 gy, a global trend of repression was observed in proliferating keratinocytes whereas an opposite effect was observed in differentiated cells where most of the expressed mirnas were induced after irradiation .
after the very low dose, a slight increase of the mirna expression profile was observed in proliferating keratinocytes .
these data revealed that the mirna response follows a global trend that is highly dependent on the radiation dose and the differentiation status of irradiated cells.
individual mirnas significantly modulated after irradiation among the  <dig> or  <dig> independent cell samples were identified .
these mirnas covered a wide range of expression from highly  to weakly expressed .
after 6 gy,  <dig> and  <dig> mirnas were found to be significantly modulated in proliferating and differentiated cells respectively .
as expected with regards to the global response, mirnas modulated in proliferating cells were all repressed whereas  <dig> out  <dig> mirnas modulated in differentiated cells were induced.
the mirnas responding to the high dose of 6 gy in proliferating cells were all different from those induced in differentiated keratinocytes.
after 10 mgy, only  <dig> induced mirnas were identified so far in proliferating keratinocytes .
to strengthen the validity of the data obtained from the large-scale tlda analysis, we used individual qpcr to study the expression of  <dig> mirnas in proliferating or differentiated keratinocytes among a time course between  <dig> and 24 hours post 6 gy-irradiation.
in proliferating keratinocytes , we confirmed the down-regulation of the  <dig> tested mirnas  at 3 h whereas no modulation was detected at  <dig> or 24 h. in differentiated keratinocytes , we also observed a significant response at 3 h for two of the  <dig> tested mirnas: as observed after tlda analysis , let-7b-5p and mir-196b-5p were up-regulated 3 hours after irradiation.
globally, these data are in accordance with those obtained from the large-scale tlda analysis.
they also support the choice of the 3 hours time-point as a key moment in the mirna response of irradiated keratinocytes.
towards mechanisms of the mirna response
to go further into the mechanisms of this mirna response after ir, we analyzed the effect of radiations on two proteins known to involved in the processing of mirnas: dicer <dig> and ago <dig>  <cit> .
indeed, dicer <dig> is an rnase iii endonuclease that acts in the endonucleolytic processing of mirnas by cleaving their loop to release a duplex rna stem  <cit> .
this duplex rna is incorporated into the risc complex, a multi-protein complex that also contains the argonaute proteins .
therefore, we analyzed the mrna response of dicer <dig> and ago <dig> genes in proliferating and differentiated keratinocytes 3 hours after 6 gy irradiation.
in proliferating cells, we observed a faint but significant decrease of mrna expression level for both dicer <dig> and ago <dig>  , whereas protein amount was not significantly different .
in differentiated cells, no modulation was detected either at rna or protein level for both dicer <dig> and ago <dig> .
these data suggest that the late steps of the mirna-processing pathway, that require ago <dig> and dicer, are not directly impacted by ionizing radiations.
to have a look to the first step of mirna biogenesis, we analyzed the relative expression of the primary transcripts corresponding to  <dig> repressed mirnas in proliferating keratinocytes.
a significant down-regulation was observed for primir- <dig> and primir-200b in 6 gy-irradiated keratinocytes .
these results indicate that some ir-responding mirnas are directly regulated at the transcriptional level.
as mir-191-5p is down-regulated whereas the level of its precursor remains constant, those results suggest that the global modulation of the mirnas expression that we detected after irradiation  is the result of multiple and complementary levels of regulation within the mirna biogenesis pathway.
functional relevance of ir-responding mirnas
after identification of radiation-responsive mirnas, our aim was to test their individual or global impact on the survival of irradiated cells.
we decided to focus first on proliferating cells that are more radio-sensitive .
because of their higher level of repression in irradiated proliferating cells, mir-191-5p, mir-200b-3p and mir-331-3p were selected for further analysis.
these  <dig> mirnas were individually over-expressed before irradiation by transfection of the corresponding pre-mirna.
in parallel, a pool of the  <dig> mirnas was also over-expressed in proliferating keratinocytes prior to irradiation.
the experimental conditions were adapted to ensure a moderate over-expression  of the mirnas in transfected cells .
over-expression of individual mirnas had no effect on cell survival  whereas a combination of the  <dig> mirnas leads to a significant reduction of cell viability especially at 72 h post-irradiation .
we also observed that some combined over-expression of two of the  <dig> mirnas were also able to significantly reduced cell survival at 72 h .
these data suggest that the mirnas that are modulated in irradiated cells plays a role in the cellular radiation response through a combined mode of action.
this action is probably exerted through the regulation of downstream targets genes.
to go further into these mechanisms, we searched for putative gene targets of mir-191-5p, mir-200b-3p and mir-331-3p.
by crossing the results of five prediction programs , we identified hundreds of genes targets for each mirnas.
among these genes, the  <dig> mirnas share  <dig> targets.
<dig> genes are potentially regulated by at least  <dig> of the  <dig> mirnas .
a functional annotation of these  <dig> putative targets using gorilla  revealed an enrichment of genes involved in the modulation of g-protein coupled receptor-signaling pathway through adenylate cyclase .
the p-values of bilateral paired t-test used to assess the significance of the viability assays 24 h, 48 h or 72 h after irradiation are given.
we used the same strategy to explore the functional impact of the 6 gy-responding mirnas in differentiated cells.
a combined silencing of the  <dig> more induced sequences  by transfection of the corresponding antagomir had no effect on cell survival at 72 h post-irradiation .
discussion
in this work, we identified several mirnas responding to ir in human keratinocytes.
in proliferating cells exposed to 6 gy, we identified  <dig> mirnas that are repressed 3 hours after exposure.
among these  <dig> mirnas, only mir-17-5p  <cit>  and mir-31-5p  <cit>  have been previously found to be deregulated by ir in other primary or cancer cells.
moreover, mir-31-5p has been shown to repress the expression of several genes involved in dna repair in adenocarcinoma cell lines  <cit> .
its down-regulation in proliferating keratinocytes might contribute to increase the dna repair efficiency after irradiation.
mir-99b-5p was also described as playing a role in dna repair.
the mir- <dig> family, including mir-99b-5p, regulates the dna-damage response in breast and prostate cancer cells by targeting the chromatin remodeling factor snf2h  <cit> .
when the mir- <dig> cluster is over-expressed in irradiated cells, the rate and the overall efficiency of repair by both nhej and homologous recombination are reduced  <cit> .
in our study, the down-regulation of mir-99b-5p could contribute to an activation of double-strand breaks repair in irradiated keratinocytes.
irradiation of proliferating keratinocytes leads to a cell cycle arrest that is requested for dna repair  <cit> .
some of the mirnas repressed by ir in proliferating keratinocytes could be involved in this growth arrest: it is the case of mir-17-5p and mir-191-5p that are over-expressed in cancer cells where they contribute to activate cell proliferation  <cit> .
in differentiated keratinocytes exposed to 6 gy, we identified  <dig> responding mirnas, all except mir-125b-5p were induced in irradiated cells.
among these  <dig> mirnas only let-7b-5p has been previously described as being modulated by ionizing radiations in primary or cancer cells  <cit> .
this suggests that the mirna response elicited by ir in human keratinocytes is very specific to this cell type and also dependent to the differentiation status of irradiated keratinocytes.
in a recent study, zhou et al.
investigated the mirna profile of uvb-irradiated normal human keratinocytes.
among the  <dig> mirnas that they found modulated by uvb, only  <dig>  also responded to ir in our study  <cit> .
this could indicate that uvb and ir provoke specific effects in exposed keratinocytes that in turn elicit a stress-specific mirna response.
it has been previously observed that genotoxic stresses, such as uv or ir, induce a very specific response in differentiated keratinocytes including induction of terminal differentiation  <cit> : among the  <dig> mirnas that we found induced after irradiation in differentiated keratinocytes,  <dig> of them  have been previously observed as being also induced during the differentiation process of human keratinocytes  <cit> .
these  <dig> radiation-responding mirnas could be directly involved into the modification of the differentiation program that is elicited by gamma irradiation in differentiated keratinocytes.
the main question raised by our data is the biological significance of this mirna response.
we have re-expressed  <dig> down-regulated mirnas in irradiated proliferating keratinocytes.
we observed that a combined over-expression of these mirnas reduced cell viability 72 hours post-ir.
these results indicate that the down-regulation of these mirnas is important for cell survival after ir.
this could also evocate a coordinated function of these mirnas in the cellular radiation response probably through the regulation of common genes targets.
we identified more than  <dig> potential targets in common between at least two of these  <dig> responding mirnas.
functional annotation of this list revealed an over-representation of genes involved in the adenylate cyclase g-protein coupled receptor-signaling pathway.
the camp signaling pathway has been shown to modulate dna-damaging agents induced apoptosis and dna repair activity  <cit> .
this pathway could be one of the mechanisms downstream to ionizing-radiation-responding mirnas and necessary for the survival of irradiated cells.
in differentiated keratinocytes, the silencing of the  <dig> more responding mirnas had no effects on immediate cell viability: this suggests that the mirnas responding to ir in differentiated keratinocytes are not directly involved in immediate cell survival.
since these cells are already engaged in a differentiation process that will finally lead to cornification, a specialized form of programmed cell death, the activation of the differentiation process might be a way to eliminate damaged cells.
as discussed above, this hypothesis was supported by the fact that, in differentiated keratinocytes, several mirnas that we found induced by ir are also markers of epidermal differentiation.
several studies have been conducted to study the effects of low doses ranging from 50 mgy to  <dig> mgy on mirnas expression in various cellular models including fibroblasts  <cit> , blood cells  <cit>  or thyroid cells  <cit> .
however, to our knowledge, the present study is the first to address the mirna response of human cells after a very low dose of 10 mgy.
this dose is relevant in the context of medical imaging procedures where 20% of the patients can be potentially exposed to radiation between  <dig> and 20 mgy, especially after abdominal compute tomography  <cit> .
contrary to the high dose of 6 gy where all the responding mirnas are down-regulated in proliferating keratinocytes, the very low dose of 10 mgy leads to a particular response with only two up-regulated mirnas: mir-342-3p and mir-708-5p.
as previously observed for mrna  <cit> , a specific mirna response is then detected in human keratinocytes after the very low dose of 10 mgy.
this might reflect the emerging concept that the dose response is not linear and that very low doses of ir induce specific cellular mechanisms in irradiated cells  <cit> .
further experiments will be necessary to precise how mir-342-3p and mir-708-5p might be involved in this particular response.
CONCLUSIONS
our results suggest that in human keratinocytes the expression of mirnas in response to ionizing radiation is highly dependent to the differentiation status of irradiated cells.
it is also clear from our study that the mirnas response is dose dependent and that a very low dose of 10 mgy is able to modulate some mirnas.
since deregulation of specific mirnas has the consequence of changing cell survival after irradiation, this might represents a possible strategy to modulate cellular sensitivity to anti-cancer treatments.

 ionizing radiations induce a large spectrum of damages that irradiated cells have to counteract by activating complex response pathways. the gene networks mobilized in the cellular response to radiations are subtly regulated. several molecular actors have been shown to be involved in the regulation of gene expression after irradiation including chromatin-remodeling factors, transcription factors or factors controlling protein synthesis, maturation and degradation. micrornas are short non-coding rnas which are predicted to control in mammals the activity of approximately 30% of all protein coding genes and have been shown to participate in the regulation of almost any cellular processes investigated so far. by base pairing to the 3′ untranslated regions of target mrnas, mirnas mediate translational repression during the initiation or elongation step, and proteolysis of the nascent peptide or mrna degradation after deadenylation. mirnas have been proposed to be important actors of the dna damage response. several mirnas expression studies have been conducted after irradiation in mice, human tumor samples, cancer cell lines or normal human cells. by crossing these studies, it was not possible to identify a short-list of universal mirnas responding to ir. the radiation effect on mirna expression seems therefore to vary according to cell type, radiation dose and post-irradiation time point. skin is the first organ to be targeted by external radiations and among the most sensitive. the epidermis is a stratified squamous epithelium that forms the protective covering of the skin. it is composed of a basal layer of proliferating cells, a spinous postmitotic cell layer, a granular layer, and a stratum corneum of terminally differentiating keratinocytes. high doses of radiation can induce various deleterious effects in skin, which can appear a short time after exposure, such as erythema and desquamation, and several years later, such as carcinoma and late complications. radiation-induced damages in epidermal proliferating cells have been suspected to be involved in late effects such as skin cancer whereas defects in differentiated cells could lead to early effects such as hyperkeratosis or desquamation. it is therefore of great interest to investigate the role of mirnas in the radiation response in proliferating versus differentiated keratinocytes. although the biological effects of low doses are controversial, we have previously shown that a very low dose of ir is able to induce a strong and specific mrna response in human keratinocytes. by providing a global view of the mirna response after strong and low doses of ir, our study should help answering in which condition a mirna response is induced after irradiation. in this study, mirna expression profiling was investigated in human primary keratinocytes after acute exposure to a very low or a strong dose of gamma rays. we showed that radiation induced changes in mirna levels varied according to the dose and the differentiation status of irradiated keratinocytes. indeed, after a strong dose of 6 gy, a global repression of mirnas was observed in proliferating keratinocytes whereas a global induction was detected in differentiated keratinocytes. the very low dose of 10 mgy was also able to modulate some mirnas in proliferating cells. over-expression of the most stringently regulated mirnas in proliferating cells modified their survival after 6 gy irradiation. in differentiated keratinocytes, repression of the most activated mirnas had no detectable effect on survival. our data suggest that the mirna response is globally modulated in irradiated keratinocytes and could be involved in cellular pathways that are highly dependent to the differentiation state of the ir-exposed cells.  characterization of the cellular model the cellular model used in this study consisted of primary cultures of human keratinocytes isolated from infant foreskin. for modeling epidermal basal cells, second passage cultures were cultivated up to 50% of confluency where most of the cells are still proliferating. to induce epidermal differentiation, cells were cultured at low calcium concentration up to confluence, and then incubated 3 days post-confluence in a culture medium containing  mm of calcium. differentiated cultures were subjected to expression analysis by real-time pcr of two well-known markers of epidermal suprabasal layers: keratin and involucrin as well as markers of proliferating basal cells: keratin and pcna. the expression was compared to that of proliferative cultures. as expected, a strong expression of krt and ivl transcripts was observed in differentiated keratinocytes whereas pcna and krt were clearly repressed. these results suggest that the majority of cells cultured 3 days post-confluence in a high-calcium medium exhibited a differentiation state close to that found in spinous and granular layers. on the contrary, semi-confluent keratinocytes express markers typical to the basal layers of the human epidermis. we next investigated the short-term radiosensitivity of proliferating and differentiated keratinocytes after the acute high dose of 6 gy. the viability of irradiated cells was measured to 72 hours after irradiation. for proliferating cells, we observed a significant decrease in cell viability starting 24 h post irradiation with a maximal effect intensity of 20% decrease 48 h and 72 h post-irradiation. in differentiated cells, a weaker but significant effect was observed from 24 h to 72 h post-irradiation with a maximal decrease in cell viability of 10% 3 days post-irradiation. these data confirmed, as previously reported by our group, that differentiated keratinocytes are more radio-resistant than proliferative keratinocytes. mirna expression profiling we used taqman quantitative real-time pcr low density array to profile mirnas expression in control and irradiated keratinocytes. we investigated the mirna response to a high dose of 6 gy in proliferating and differentiated keratinocytes and to a very low dose of 10 mgy in proliferating cells, which are more radiosensitive. mirnas expression was assessed 3 hours post-irradiation since this time-point corresponds to the immediate gene response in irradiated keratinocytes and mirnas might be involved in the regulation of this gene response. three to four human keratinocytes cultures established from independent donors were analyzed at each dose. due to the high repeatability of the tlda methodology, we privileged in our experimental plan biological replicates from different donors than technical repeats. it is important to note that the experimental plan is constructed on paired data: for each donor, a reference condition response and a response following ionizing irradiation were quantified. out of mirnas studied, only those with a cq value below 32 cycles in all samples were considered as significantly expressed. this threshold was calculated using mirna duplicate and snrna tetraplicate repeatability, in agreement with literature. mirnas for gy vs 6 gy irradiated proliferating keratinocytes, mirnas for 0/6 gy-irradiated differentiated keratinocytes, and mirnas for 0 gy vs 10 mgy irradiated proliferating keratinocytes were therefore considered for further analysis. we first investigated if a bias exists in the average expression of mirnas between hpk cell samples sorted by differentiation state and irradiation dose. we used all-paired tukey-kramer tests to compare cq means within hpk samples. for 0/6 gy-irradiated proliferating keratinocytes, there was no expression bias between the cell samples. for 0/6 gy-irradiated differentiated keratinocytes, significant differences were observed: samples from hpk revealed higher cq than the three others donors. a difference was also observed for 0/10 mgy-irradiated proliferating keratinocytes: samples from donor hpk showed in average lower cq, than those from hpk and hpk. however, these differences in the average expression of mirnas were similar for reference and irradiated conditions, and could be due to inter-individual variability in primary cells. this observation underlined the importance to use paired data for statistical analyses. it further confirmed that there was no sample processing bias due to irradiation state and that raw data normalization was not necessary as previously proposed. we then focused on mirna profiles after irradiation in proliferating and differentiated keratinocytes. after the high dose of 6 gy, a global trend of repression was observed in proliferating keratinocytes whereas an opposite effect was observed in differentiated cells where most of the expressed mirnas were induced after irradiation. after the very low dose, a slight increase of the mirna expression profile was observed in proliferating keratinocytes. these data revealed that the mirna response follows a global trend that is highly dependent on the radiation dose and the differentiation status of irradiated cells. individual mirnas significantly modulated after irradiation among the or independent cell samples were identified. these mirnas covered a wide range of expression from highly to weakly expressed. after 6 gy, and mirnas were found to be significantly modulated in proliferating and differentiated cells respectively. as expected with regards to the global response, mirnas modulated in proliferating cells were all repressed whereas out mirnas modulated in differentiated cells were induced. the mirnas responding to the high dose of 6 gy in proliferating cells were all different from those induced in differentiated keratinocytes. after 10 mgy, only induced mirnas were identified so far in proliferating keratinocytes. to strengthen the validity of the data obtained from the large-scale tlda analysis, we used individual qpcr to study the expression of mirnas in proliferating or differentiated keratinocytes among a time course between and 24 hours post 6 gy-irradiation. in proliferating keratinocytes, we confirmed the down-regulation of the tested mirnas at 3 h whereas no modulation was detected at or 24 h. in differentiated keratinocytes, we also observed a significant response at 3 h for two of the tested mirnas: as observed after tlda analysis, let-7b-5p and mir-196b-5p were up-regulated 3 hours after irradiation. globally, these data are in accordance with those obtained from the large-scale tlda analysis. they also support the choice of the 3 hours time-point as a key moment in the mirna response of irradiated keratinocytes. towards mechanisms of the mirna response to go further into the mechanisms of this mirna response after ir, we analyzed the effect of radiations on two proteins known to involved in the processing of mirnas: dicer and ago. indeed, dicer is an rnase iii endonuclease that acts in the endonucleolytic processing of mirnas by cleaving their loop to release a duplex rna stem. this duplex rna is incorporated into the risc complex, a multi-protein complex that also contains the argonaute proteins. therefore, we analyzed the mrna response of dicer and ago genes in proliferating and differentiated keratinocytes 3 hours after 6 gy irradiation. in proliferating cells, we observed a faint but significant decrease of mrna expression level for both dicer and ago, whereas protein amount was not significantly different. in differentiated cells, no modulation was detected either at rna or protein level for both dicer and ago. these data suggest that the late steps of the mirna-processing pathway, that require ago and dicer, are not directly impacted by ionizing radiations. to have a look to the first step of mirna biogenesis, we analyzed the relative expression of the primary transcripts corresponding to repressed mirnas in proliferating keratinocytes. a significant down-regulation was observed for primir- and primir-200b in 6 gy-irradiated keratinocytes. these results indicate that some ir-responding mirnas are directly regulated at the transcriptional level. as mir-191-5p is down-regulated whereas the level of its precursor remains constant, those results suggest that the global modulation of the mirnas expression that we detected after irradiation is the result of multiple and complementary levels of regulation within the mirna biogenesis pathway. functional relevance of ir-responding mirnas after identification of radiation-responsive mirnas, our aim was to test their individual or global impact on the survival of irradiated cells. we decided to focus first on proliferating cells that are more radio-sensitive. because of their higher level of repression in irradiated proliferating cells, mir-191-5p, mir-200b-3p and mir-331-3p were selected for further analysis. these mirnas were individually over-expressed before irradiation by transfection of the corresponding pre-mirna. in parallel, a pool of the mirnas was also over-expressed in proliferating keratinocytes prior to irradiation. the experimental conditions were adapted to ensure a moderate over-expression of the mirnas in transfected cells. over-expression of individual mirnas had no effect on cell survival whereas a combination of the mirnas leads to a significant reduction of cell viability especially at 72 h post-irradiation. we also observed that some combined over-expression of two of the mirnas were also able to significantly reduced cell survival at 72 h. these data suggest that the mirnas that are modulated in irradiated cells plays a role in the cellular radiation response through a combined mode of action. this action is probably exerted through the regulation of downstream targets genes. to go further into these mechanisms, we searched for putative gene targets of mir-191-5p, mir-200b-3p and mir-331-3p. by crossing the results of five prediction programs, we identified hundreds of genes targets for each mirnas. among these genes, the mirnas share targets. genes are potentially regulated by at least of the mirnas. a functional annotation of these putative targets using gorilla revealed an enrichment of genes involved in the modulation of g-protein coupled receptor-signaling pathway through adenylate cyclase. the p-values of bilateral paired t-test used to assess the significance of the viability assays 24 h, 48 h or 72 h after irradiation are given. we used the same strategy to explore the functional impact of the 6 gy-responding mirnas in differentiated cells. a combined silencing of the more induced sequences by transfection of the corresponding antagomir had no effect on cell survival at 72 h post-irradiation. discussion in this work, we identified several mirnas responding to ir in human keratinocytes. in proliferating cells exposed to 6 gy, we identified mirnas that are repressed 3 hours after exposure. among these mirnas, only mir-17-5p and mir-31-5p have been previously found to be deregulated by ir in other primary or cancer cells. moreover, mir-31-5p has been shown to repress the expression of several genes involved in dna repair in adenocarcinoma cell lines. its down-regulation in proliferating keratinocytes might contribute to increase the dna repair efficiency after irradiation. mir-99b-5p was also described as playing a role in dna repair. the mir- family, including mir-99b-5p, regulates the dna-damage response in breast and prostate cancer cells by targeting the chromatin remodeling factor snf2h. when the mir- cluster is over-expressed in irradiated cells, the rate and the overall efficiency of repair by both nhej and homologous recombination are reduced. in our study, the down-regulation of mir-99b-5p could contribute to an activation of double-strand breaks repair in irradiated keratinocytes. irradiation of proliferating keratinocytes leads to a cell cycle arrest that is requested for dna repair. some of the mirnas repressed by ir in proliferating keratinocytes could be involved in this growth arrest: it is the case of mir-17-5p and mir-191-5p that are over-expressed in cancer cells where they contribute to activate cell proliferation. in differentiated keratinocytes exposed to 6 gy, we identified responding mirnas, all except mir-125b-5p were induced in irradiated cells. among these mirnas only let-7b-5p has been previously described as being modulated by ionizing radiations in primary or cancer cells. this suggests that the mirna response elicited by ir in human keratinocytes is very specific to this cell type and also dependent to the differentiation status of irradiated keratinocytes. in a recent study, zhou et al. investigated the mirna profile of uvb-irradiated normal human keratinocytes. among the mirnas that they found modulated by uvb, only also responded to ir in our study. this could indicate that uvb and ir provoke specific effects in exposed keratinocytes that in turn elicit a stress-specific mirna response. it has been previously observed that genotoxic stresses, such as uv or ir, induce a very specific response in differentiated keratinocytes including induction of terminal differentiation: among the mirnas that we found induced after irradiation in differentiated keratinocytes, of them have been previously observed as being also induced during the differentiation process of human keratinocytes. these radiation-responding mirnas could be directly involved into the modification of the differentiation program that is elicited by gamma irradiation in differentiated keratinocytes. the main question raised by our data is the biological significance of this mirna response. we have re-expressed down-regulated mirnas in irradiated proliferating keratinocytes. we observed that a combined over-expression of these mirnas reduced cell viability 72 hours post-ir. these results indicate that the down-regulation of these mirnas is important for cell survival after ir. this could also evocate a coordinated function of these mirnas in the cellular radiation response probably through the regulation of common genes targets. we identified more than potential targets in common between at least two of these responding mirnas. functional annotation of this list revealed an over-representation of genes involved in the adenylate cyclase g-protein coupled receptor-signaling pathway. the camp signaling pathway has been shown to modulate dna-damaging agents induced apoptosis and dna repair activity. this pathway could be one of the mechanisms downstream to ionizing-radiation-responding mirnas and necessary for the survival of irradiated cells. in differentiated keratinocytes, the silencing of the more responding mirnas had no effects on immediate cell viability: this suggests that the mirnas responding to ir in differentiated keratinocytes are not directly involved in immediate cell survival. since these cells are already engaged in a differentiation process that will finally lead to cornification, a specialized form of programmed cell death, the activation of the differentiation process might be a way to eliminate damaged cells. as discussed above, this hypothesis was supported by the fact that, in differentiated keratinocytes, several mirnas that we found induced by ir are also markers of epidermal differentiation. several studies have been conducted to study the effects of low doses ranging from 50 mgy to mgy on mirnas expression in various cellular models including fibroblasts, blood cells or thyroid cells. however, to our knowledge, the present study is the first to address the mirna response of human cells after a very low dose of 10 mgy. this dose is relevant in the context of medical imaging procedures where 20% of the patients can be potentially exposed to radiation between and 20 mgy, especially after abdominal compute tomography. contrary to the high dose of 6 gy where all the responding mirnas are down-regulated in proliferating keratinocytes, the very low dose of 10 mgy leads to a particular response with only two up-regulated mirnas: mir-342-3p and mir-708-5p. as previously observed for mrna, a specific mirna response is then detected in human keratinocytes after the very low dose of 10 mgy. this might reflect the emerging concept that the dose response is not linear and that very low doses of ir induce specific cellular mechanisms in irradiated cells. further experiments will be necessary to precise how mir-342-3p and mir-708-5p might be involved in this particular response.  our results suggest that in human keratinocytes the expression of mirnas in response to ionizing radiation is highly dependent to the differentiation status of irradiated cells. it is also clear from our study that the mirnas response is dose dependent and that a very low dose of 10 mgy is able to modulate some mirnas. since deregulation of specific mirnas has the consequence of changing cell survival after irradiation, this might represents a possible strategy to modulate cellular sensitivity to anti-cancer treatments. 
the keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. to go further into the biological meaning of this mirna response, we over-expressed some of the responding mirna in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. functional annotation of their predicted targets revealed that g-protein related pathways might be regulated by these responding mirnas. we found that proliferative cells irradiated at 6 gy display a global fall of mirna expression whereas differentiated cells exposed to the same dose display a global increase of mirnas expression. micrornas, a group of short non-coding rnas that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. however, in epidermal cells, the involvement of the mirna machinery in the cellular response to ionizing radiations remains to be clarified. our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a mirna pattern strongly related to the differentiation status of irradiated cells. we also demonstrate that some mirnas play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes. we identified twenty mirnas weakly but significantly modulated after 6 gy irradiation, whereas only mirnas were modulated after low-dose irradiation in proliferating cells.
 micrornas, a group of short non-coding rnas that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. however, in epidermal cells, the involvement of the mirna machinery in the cellular response to ionizing radiations remains to be clarified. indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive.  we settled up an expression study of mirnas in primary human skin keratinocytes using a microfluidic system of qpcr assay, which permits to assess the expression of almost annotated mirnas. the keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. these cells were irradiated at 10 mgy or 6 gy and rna was extracted 3 hours after irradiation. we found that proliferative cells irradiated at 6 gy display a global fall of mirna expression whereas differentiated cells exposed to the same dose display a global increase of mirnas expression. we identified twenty mirnas weakly but significantly modulated after 6 gy irradiation, whereas only mirnas were modulated after low-dose irradiation in proliferating cells. to go further into the biological meaning of this mirna response, we over-expressed some of the responding mirna in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. functional annotation of their predicted targets revealed that g-protein related pathways might be regulated by these responding mirnas.  our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a mirna pattern strongly related to the differentiation status of irradiated cells. we also demonstrate that some mirnas play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes. primary keratinocytesepidermal differentiationgamma irradiationmirnasexpression profiling 