merkel cell carcinoma ( mcc ) is a primary neuroendocrine carcinoma of the skin of uncertain origin .
although not as prevalent as other skin cancers , mcc is aggressive and has a high mortality rate , with an overall five - year survival of sixty percent .
median survival for patients with and without regional lymph node involvement at presentation is thirteen and forty months , respectively , and up to fifty percent of patients eventually develop systemic disease with metastases to liver , bone , and brain .
unfortunately , while the incidence of mcc increases , our knowledge of these tumors remains limited .
several factors are implicated in its pathogenesis , including uv radiation exposure , an associated polyomavirus ( mcpyv ) , and immunosuppression [ 4 , 5 ] , but the lack of effective treatment options available for mcc reflects our limited knowledge . in an attempt to expand our understanding , we focus our attention on micrornas , small single - stranded rna molecules that participate in the negative regulation of gene expression . with average lengths of roughly twenty - two nucleotides ,
these molecules serve as guides of the rna - induced silencing complex ( risc ) .
micrornas regulate the expression of genes by binding to partially complementary target sites in mrna transcripts and inhibiting their translation .
a wealth of data has accumulated in recent years implicating micrornas as significant modulators of gene expression , and through their unique role in posttranscriptional regulation , they can function as important regulators of tumor growth and metastasis .
the study of micrornas holds much promise for improving the diagnosis and treatment of cancer .
recent progress in our understanding of the role of micrornas in disease has excited oncology .
an effective antiviral therapy was recently developed based on microrna biology , and micrornas are promising new tools and targets in cancer research .
indeed , micrornas have been demonstrated to play significant roles in the pathogenesis of other skin cancers , such as squamous cell carcinoma ( scc ) and melanoma .
they have also been implicated in basal cell carcinoma ( bcc ) and may even be employed in the accurate identification of cancer subtype , as demonstrated for melanoma and cutaneous t - cell lymphoma ( ctcl ) . however , despite the volume of literature on micrornas in skin cancer , little is yet known about the role that these molecules play in mcc .
thus , we set out to close this knowledge gap and define the micrornas involved in mcc biology .
frozen tissue samples of various skin cancers ( mcc , melanoma , scc , and bcc ) , normal skin , and normal lymph node were obtained from the cooperative human tissue network ( chtn ) and stored at 80c .
formalin - fixed paraffin - embedded ( ffpe ) tissue samples of various skin cancers ( mcc , melanoma , scc , and bcc ) and normal skin were obtained from vanderbilt pathology and dermatopathology ( nashville , tn ) . clinical information corresponding to the ffpe mcc samples is provided ( table 1 ) .
total rna , including micrornas , was isolated from frozen tissue and cell culture with the mirneasy mini kit and from ffpe tissue with the mirneasy ffpe kit ( qiagen ; hilden , germany ) , according to the manufacturer 's protocols .
the concentration and integrity of the extracted total rna were estimated by qubit 2.0 fluorometer ( invitrogen , carlsbad , ca , usa ) and agilent 2100 bioanalyzer ( applied biosystems , carlsbad , ca , usa ) , respectively .
rna samples with a rna integrity number ( rin ) value of at least 7.0 or higher was used for further processing .
approximately 1  g of total rna from each sample was taken into small rna library preparation protocol using nebnext small rna library prep set for illumina ( new england biolabs inc . ,
ipswich , ma , usa ) according to manufacturer 's protocol . briefly , 3 adapters were ligated to total input rna followed by hybridization of multiplex sr rt primers and ligation of multiplex 5 sr adapters .
reverse transcription ( rt ) was done using superscript iii rt ( life technologies , grand island , ny , usa ) for 1 hour at 50c . immediately after rt reaction , pcr amplification was performed for 15 cycles using longamp taq 2x master mix .
post - pcr material was purified using qiaquick pcr purification kit ( qiagen inc . ,
post - pcr yield and concentration of the prepared libraries were assessed using qubit 2.0 fluorometer and dna 1000 chip on agilent 2100 bioanalyzer .
size selection of small rna with a target size range of 140  bp was done by running samples on a 6% page gel for 1 hour at 120  v. accurate quantification for sequencing applications was performed using the qpcr - based kapa biosystems library quantification kit .
each library was diluted to a final concentration of 12.5  nm and pooled in equimolar ratios prior to clustering .
cluster generation was carried out on a cbot v1.4.36.0 using illumina 's truseq single read ( sr ) cluster kit v3.0 .
single end ( se ) sequencing was performed to generate at least 15 million reads per sample on an illumina hiseq2000 , running hiseq control software ( hcs ) v1.5.15.1 , using a 50-cycle truseq sbs hs v3 reagent kit .
the clustered flowcells were sequenced for 56 cycles , consisting of a 50-cycle read , followed by a 6-cycle index read .
image analysis and base calling was performed using the standard illumina pipeline consisting of real time analysis ( rta ) version v1.13 and demultiplexed using bcl2fastq converter with default settings .
at least 15 million , 50  bp , se reads were generated from each sample .
further downstream analysis of the sequenced reads from each sample was performed as per our unique in - house pipeline .
briefly , quality control checks on raw sequence data from each sample will be performed using fastqc ( babraham bioinformatics , london , uk ) .
raw reads were then imported on a commercial data analysis platform clcbio ( clcbio , ma , usa ) .
adapter trimming was done to remove ligated adapter from 3 end of the sequenced reads with only one mismatch allowed ; poorly aligned 3 ends were also trimmed .
were then used to extract and count the small rna which were then annotated with micrornas in mirbase release 18 database . samples were grouped as per their types identifiers and quantification of mirna abundance was done .
differential expression of mirna was calculated on the basis of their fold change ( default cut - off 2.0 ) between mapped counts observed between individual groups . to validate the ngs data , the expression of micrornas was analyzed via quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) with the rotor - gene sybr green pcr kit , miscript primer assays , and miscript universal primer , following reverse transcription of total rna with the miscript ii rt kit ( qiagen ; hilden , germany ) .
the qrt - pcr analysis was performed in technical replicates according to the manufacturer 's instructions using the rotor - gene sybr green pcr master mix ( qiagen ; hilden , germany ) .
the packaged operating software was utilized for instrument control , data acquisition , and raw data analysis .
amplification curves were analyzed using the packaged operating software , and assays were inspected for distinct melting curves .
in addition , only assays detected with ct < 35 were included in the data analysis . to calculate the relative expression levels of target micrornas ,
mir-423 - 3p and mir-423 - 5p were stably expressed across all samples , thus the average of their cts in each sample was used as the normalization factor .
assays were calibrated to the same normal skin sample . 
 in situ detection of mir-182
was carried out using the accurish service of orior lab , llc ( rockville , md ) .
an rnase - free tissue sectioning environment was created by treating the microtome , blades , water bath , ice bucket , forceps , and slide tray with rnasezap , followed by rnase - free water .
each 5-m tissue section was dewaxed , rehydrated , and demasked , and protease treatment was optimized for each tissue block with gapdh probe and differing concentrations of protease k. after treatment , tumor and normal tissue sections were stained side - by - side using an anti - mir-182 oligonucleotide probe of same concentration with same hybridization and stringent washing temperature of 5560c . the final color development for all sections of each batch
sequencing of small rna libraries was performed for the following frozen tissue samples : three mccs , one melanoma , one scc , one bcc , and one normal skin .
the mcc and melanoma were lymph node metastases , while the scc and bcc were primary cutaneous lesions .
comparison of the sequencing profiles identified several micrornas upregulated and downregulated in mcc versus other tissues ( table 2 ) . to validate the next generation sequencing ( ngs ) data ,
several micrornas were evaluated via qrt - pcr in larger cohorts of ffpe tissue samples .
the mcc cohort consisted of a mixture of primary cutaneous lesions and metastases ( table 1 ) .
the tumor group consisted of primary cutaneous lesions of melanoma , scc , and bcc ( figure 1 ) .
the qrt - pcr results confirmed the upregulation ( 2-fold ) of eight micrornas in mcc : mir-502 - 3p , mir-9 , mir-7 , mir-340 , mir-182 , mir-190b , mir-873 , and mir-183 .
in addition , three micrornas were found to be downregulated ( 2-fold ) in mcc : mir-3170 , mir-125b , and mir-374c .
the data also identified the slight upregulation ( 1.5-fold ) of mir-96a , another member of the mir-183/96/182 cluster . to assess whether these micrornas are specific tumor markers for mcc , the expression of each of the eight mcc - mirs was evaluated via qrt - pcr in several frozen mcc lymph node metastases and compared to a human tissue panel consisting of twelve different organs ( figure 2 ) .
three of the mcc - mirs demonstrated higher expression ( 2-fold ) in the mcc cohort versus all other organs : mir-183 , mir-182 , and mir-190b . to assess the potential of these findings for future functional studies , ngs of small rna libraries was performed for the patient derived mcpyv - positive cell line , ms-1 ( table 2 ) .
several of the same mcc - mirs found to be upregulated in the tissue samples also demonstrated high absolute expression values in ms-1 .
thus , the expression of each of the mcc - mirs was evaluated via qrt - pcr in ms-1 and compared to that of various non - mcc cell lines ( figure 3 ) .
the following mcc - mirs were confirmed to be elevated ( 2-fold ) in ms-1 versus a set of sixteen non - mcc cell lines : mir-183 , mir-182 , mir-340 , and mir-190b  interestingly , the same micrornas were found to be upregulated in mcc versus the tissue panel .
together , these data indicate that we have identified a set of high quality mcc - mirs .
the expression of each of the four micrornas was also evaluated via qrt - pcr in the mcpyv - negative cell line , mcc13 , but with different results ( figure 3 ) .
in contrast to ms-1 , the four micrornas , mir-183 , mir-182 , mir-340 , and mir190b demonstrated low expression levels in mcc13 .
the levels of these micrornas were instead comparable to that of the other cell lines .
explanations for this discrepancy are provided in the discussion . to support the notion that these micrornas play a role in the actual tumor cells in lieu of the surrounding tissue , ish
was performed for one of the more highly expressed mcc - mirs , mir-182 , on a sample of mcc of the cheek and on a sample of normal skin ( figure 4 ) .
as expected , mir-182 was localized to mcc cells , and , as expected from the qrt - pcr data , expression in surrounding tissue and normal skin was low compared to that in mcc cells .
micrornas are a relatively young field of biomedical research , born in 2000 with the detection of let-7 in humans , with potential for applications in other pathologies [ 6 , 7 ] .
we believe this will hold true in mcc as well , for which we have identified eight upregulated and three downregulated micrornas .
while these mcc - mirs are highly expressed in mcc , some of them have been demonstrated to play significant roles in the pathogenesis of other cancers as well . in particular , the mir-183/96/182 cluster , at chromosomal locus 7q32 ,
is expressed in a diversity of cancers and may contribute to their pathogenesis by targeting multiple components of the cell cycle , dna damage response , and homologous recombination pathways , and by enriching pathways associated with metastasis , migration , and epithelial - mesenchymal transition . here
we review significant findings in the literature concerning this cluster . regarding skin cancer , the mir-183 cluster is frequently overexpressed in melanoma .
our work confirms this observation , inasmuch as mir-182 and mir-183 were upregulated in melanoma versus scc , bcc , and normal skin ( although still not as highly expressed as in mcc ) . in melanoma ,
overexpression of mir-182 promotes survival , migration , and metastasis by directly repressing the tumor suppressors foxo3 and microphthalmia - associated transcription factor - m ; and expression of mir-182 increases with progression from primary to metastatic melanoma .
this correlation between mir-182 level and aggressiveness is interesting , when noting that mcc , in general , is widely considered to be a tumor with a similar level of aggressiveness as melanoma .
it is tempting to entertain the notion that a common pathway may perhaps exist within the two and that a greater dysregulation of said pathway in mcc could account for its high rate of metastasis , morbidity , and mortality .
downregulation of fox transcription factors is a common theme of this cluster , as mir-183/96/182 have been demonstrated to inhibit foxo1 in classical hodgkin lymphoma   and in endometrial cancer as well , resulting in decreased g1 cell cycle arrest and cell death .
aberrations of fox transcription factors have yet to be evaluated in mcc and may be an avenue worth exploring , considering this new information .
the mir-183/96/182 cluster serves essential functions in various noncutaneous carcinomas as well , with much research focused on its role in breast cancer , particularly with invasion and metastasis . for example , in mammary ductal carcinoma in situ , both mir-182 and mir-183 have been demonstrated to target cbx7 , a regulator of e - cadherin expression .
mir-182 , activated by -catenin , also targets the matrix metalloproteinase inhibitor reck , resulting in increased mmp-9 activity , as well as mim , which normally suppresses metastasis by inhibition of rhoa .
dysregulation of these pathways shares the common result of increasing tumor motility and colony formation , and indeed , overexpression of mir-182 in breast cancer cell xenografts results in increased pulmonary colonization by cancer cells .
the molecular pathways altered in mcc pathogenesis have yet to be fully characterized , but a literature review reveals that some connections to well - known tumorigenic pathways have been made .
for example , multiple research groups have discovered that the pi3k / akt / mtor pathway is activated , independent of mcpyv - status , in the majority of human mccs , identifying it as a potential new therapeutic target [ 15 , 16 ] .
interestingly , the mir-183/96/182 cluster has been demonstrated to enhance pi3k / akt / mtor signaling and promote cell migration in medulloblastoma , with the majority of this effect attributed to mir-182 .
knockdown of the full cluster in medulloblastoma cells results in dysregulation of the pi3k / akt / mtor signaling axis and enhancement of genes related to apoptosis ; thus , the mir-183/96/182 cluster again appears as an attractive target for potential therapeutic applications in mcc . as an additional note
, data also suggest that inactivation of pten may play a role in mcc pathogenesis ; however , mutation and homozygous deletion screening of the pten gene in tumor samples reveals nonsense mutations and homozygous deletions in only a small subset of patients .
the mir-183/96/182 cluster may provide an explanation to this puzzle , inasmuch as mir-183 has been demonstrated to target the tumor suppressor gene , egr1 , and participate in a mir-183-egr1-pten network that promotes tumorigenesis and cell migration in synovial sarcoma , rhabdomyosarcoma , and colon cancer .
furthermore , some of these mcc - mirs , when employed in combination , could be potentially useful in the diagnosis of mcc .
misdiagnosis is high on the list of issues associated with this cancer ; however , unlike bcc , for which it may often be confused [ 2022 ] , a delay in diagnosis could prove to be fatal .
cytokeratin ( ck ) 20 has demonstrated its usefulness in the immunohistochemical ( ihc ) diagnosis of this cancer ; however , there have been reports of ck20/ck7 + variants of mcc [ 23 , 24 ] ; thus , another tumor marker may aid pathologists .
we have demonstrated , via ish , that mir-182 is indeed localized to the tumor cells ( figure 4 ) .
recently , renwick et al . demonstrated that multicolor microrna fish can be utilized to effectively differentiate between mcc and bcc in ffpe tissues .
the researchers employed mir-205 and mir-375 , which were shown to be tumor - specific for bcc and mcc , respectively .
similar methods could be applied with other micrornas highly expressed in mcc , for example mir-182 and the other identified mcc - mirs in this work , in order to increase the specificity of such a diagnostic test .
although mir-375 was not identified in our study , our mcc - mirs were identified using a larger cohort of mcc samples and have greater versatility as diagnostic markers , inasmuch as they were measured in a wider range of tumors , cell lines , and normal tissues .
in addition , our results from the tissue panel demonstrate that the mcc - mirs could also potentially serve as markers for tumor metastasis ( figure 2 ) .
since baseline levels of these micrornas are relatively low in target organs such as liver , bone , and brain , among other sites of metastatic involvement , they could potentially serve as markers for mcc cells disseminated in these tissues . employing microrna signatures as diagnostic tools
has been successfully carried out for other skin cancers . in 2011 , ralfkiaer et al .
developed a qrt - pcr - based classifier consisting of three micrornas capable of differentiating ctcl from other cutaneous pathologies with high accuracy .
finally , we demonstrated that several of these mcc - mirs are highly expressed in the patient - derived mcc cell line , ms-1 .
the same findings were not demonstrated with the mcpyv - negative cell line , mcc13 ; however , we are not the first to experience such findings . in their previously mentioned study , renwick et al .
, upon clustering samples via comparison of microrna profiles , found that mcpyv - positive cell lines ( ms-1 , mkl-1 , mkl-2 ) clustered in the mcc group , while mcpyv - negative cell lines ( mcc13 , mcc26 , uiso ) clustered in the non - mcc group .
their findings , in addition to ours , support the notion that intrinsic mirnome differences exist between the two cell lines .
this raises the question of which cell line , ms-1 or mcc13 , holds more validity as a surrogate of mcc in vivo . to address this problem
, we refer to the recent work of guastafierro et al . , which characterizes the intrinsic cellular , immunohistochemical , and virological differences between ms-1 and mcc13 in detail .
morphologically , ms-1 and other mcpyv - positive cell lines ( mkl-1 , mkl-2 ) grow as floating aggregates in suspension , while mcc13 and other mcpyv - negative cell lines ( uiso , mcc26 ) grow as adherent monolayers in culture .
immunohistochemically , ms-1 is positive for the traditional mcc - markers , ck20 and synaptophysin , and negative for ck7 , but in contrast , mcc13 is the exact opposite : negative for ck20 and synaptophysin and positive for ck7 . and
virologically , ms-1 harbors the integrated viral sequence within its genome and consequently expresses antigens associated with mcpyv - infection and tumorigenesis ( e.g. , large t antigen ) , while mcc13 does neither . taking these differences into account , guastafierro
raise the legitimate question of whether or not the mcpyv - negative cell lines ( mcc13 , uiso , mcc26 , and mati ) even stem from accurately diagnosed mcc tumors . based upon these findings , we believe that our results in the ms-1 and mcc13 cell lines corroborate the existing literature that suggests that only the mcpyv - positive cell lines truly mimic mcc .
its cellular , immunohistochemical , and virological features , along with its high expression of mcc - mirs , showcase ms-1 as an attractive candidate for future studies .
further evaluation of the mirnomes of other mcpyv - positive cell lines ( mkl-1 , mkl-2 ) would be valuable in supporting this notion .
 micrornas have been implicated in various skin cancers , including melanoma , squamous cell carcinoma , and basal cell carcinoma ; however , the expression of micrornas and their role in merkel cell carcinoma ( mcc ) have yet to be explored in depth . to identify micrornas specific to mcc ( mcc - mirs ) , next - generation sequencing ( ngs ) of small rna libraries 
 was performed on different tissue samples including mccs , other cutaneous tumors , and normal skin . 
 comparison of the profiles identified several micrornas upregulated and downregulated in mcc . for validation , 
 their expression was measured via qrt - pcr in a larger group of mcc and in a comparison group of non - mcc cutaneous tumors and normal skin . 
 eight micrornas were upregulated in mcc : mir-502 - 3p , mir-9 , mir-7 , mir-340 , mir-182 , mir-190b , mir-873 , and mir-183 . 
 three micrornas were downregulated : mir-3170 , mir-125b , and mir-374c . 
 many of these mcc - mirs , the mir-183/182/96a cistron in particular , have connections to tumorigenic pathways implicated in mcc pathogenesis . in situ hybridization confirmed that the highly expressed mcc - mir , mir-182 , is localized within tumor cells . 
 furthermore , ngs and qrt - pcr reveal that several of these mcc - mirs are highly expressed in the patient - derived mcc cell line , ms-1 . 
 these data indicate that we have identified a set of mcc - mirs with important implications for mcc research . 
1. Introduction
2. Methods
3. Results
4. Discussion