after informed consent was given and under the institutional review board approval , human hcc and paired normal liver tissues were obtained immediately after surgery at the johns hopkins hospital ( baltimore , us ) , the 3rd affiliated hospital of sun yat - sen university ( guangzhou , china ) , fundeni clinical institute ( bucharest , romania ) , and ion chiricuta comprehensive cancer center ( clui nanoca , romania ) .
tissues were snap frozen upon acquisition and stored in a   80  c freezer until use .
the arraystar human lncrna array v2.0 was used to profile both lncrnas and messenger rnas ( mrnas ) in human genome of 3 pairs of human hcc and the matched normal tissues .
33,045 lncrnas were collected from the authoritative data sources including refseq , ucsc knowngenes , ensembl and many related literatures .
sample labeling and array hybridization were performed according to the agilent one - color microarray - based gene expression analysis protocol ( agilent technology ) with minor modifications .
briefly , mrna was purified from 1  g total rna after the removal of rrna ( mrna - only eukaryotic mrna isolation kit , epicentre ) .
then , each sample was amplified and transcribed into fluorescent crna along the entire length of the transcripts without 3 bias utilizing a random priming method .
the concentration and specific activity of the labeled crnas ( pmol cy3/g crna ) were measured by using nanodrop nd-1000 .
1  g of each labeled crna is fragmented by adding 11  l 10    blocking agent and 2.2  l of 25    fragmentation buffer , and then heated the mixture at 60  c for 30  min ; finally 55  l 2    ge hybridization buffer is added to dilute the labeled crna
. 100  l of hybridization solution was dispensed into the gasket slide and assembled to the lncrna expression microarray slide .
the slides were incubated for 17  h at 65  c in an agilent hybridization oven .
the hybridized arrays were washed , fixed and scanned by using the agilent dna microarray scanner ( part number g2505b ) .
mircury hy3/hy5 power labeling kit ( exiqon ) was used according to the manufacturer 's guideline for mir labeling of the same 3 pairs of hcc and matched normal liver tissues .
1.0  mg of each sample was 3-end - labeled with hy3 fluorescent label using t4 rna ligase by the following procedure : rna in 2.0  l of water was combined with 1.0  l of cip buffer and cip ( exiqon ) .
the mixture was incubated for 30  min at 37  c , and was terminated by incubation for 5  min at 95  c .
then 3.0  l of labeling buffer , 1.5  l of fluorescent label ( hy3 ) , 2.0  l of dmso , and 2.0  l of labeling enzyme were added into the mixture .
the labeling reaction was incubated for 1  h at 16  c , and terminated by incubation for 15  min at 65  c . after stopping the labeling procedure ,
the hy3-labeled samples were hybridized on the mircury lna array ( v.18.0 ) ( exiqon ) according to the array manual .
the total 25  l mixture from hy3-labeled samples with 25  l hybridization buffer was first denatured for 2  min at 95  c , incubated on ice for 2  min and then hybridized to the microarray for 1620  h at 56  c in a 12-bay hybridization systems ( hybridization system  nimblegen systems ) , which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal .
following hybridization , the slides were achieved , washed several times using wash buffer kit ( exiqon ) , and finally dried by centrifugation for 5  min at 400  rpm .
then the slides were scanned using the axon genepix 4000b microarray scanner ( axon instruments ) .
agilent feature extraction software ( version 10.7.3.1 ) was used to analyze lncrna and mrna array images .
quantile normalization and subsequent data processing were performed by using the genespring gx v11.5.1 software package ( agilent technologies ) .
after quantile normalization of the raw data , lncrnas and mrnas that at least 1 out of 6 samples have flags in present or marginal were chosen for further data analysis .
for microrna array analysis , the scanned images were then imported into genepix pro 6.0 software ( axon ) for grid alignment and data extraction .
replicated mirnas were averaged and mirnas that intensities    50 in all samples were chosen for calculating normalization factor .
after informed consent was given and under the institutional review board approval , human hcc and paired normal liver tissues were obtained immediately after surgery at the johns hopkins hospital ( baltimore , us ) , the 3rd affiliated hospital of sun yat - sen university ( guangzhou , china ) , fundeni clinical institute ( bucharest , romania ) , and ion chiricuta comprehensive cancer center ( clui nanoca , romania ) .
tissues were snap frozen upon acquisition and stored in a   80  c freezer until use .
the arraystar human lncrna array v2.0 was used to profile both lncrnas and messenger rnas ( mrnas ) in human genome of 3 pairs of human hcc and the matched normal tissues .
33,045 lncrnas were collected from the authoritative data sources including refseq , ucsc knowngenes , ensembl and many related literatures .
sample labeling and array hybridization were performed according to the agilent one - color microarray - based gene expression analysis protocol ( agilent technology ) with minor modifications .
briefly , mrna was purified from 1  g total rna after the removal of rrna ( mrna - only eukaryotic mrna isolation kit , epicentre ) .
then , each sample was amplified and transcribed into fluorescent crna along the entire length of the transcripts without 3 bias utilizing a random priming method .
the concentration and specific activity of the labeled crnas ( pmol cy3/g crna ) were measured by using nanodrop nd-1000 .
1  g of each labeled crna is fragmented by adding 11  l 10    blocking agent and 2.2  l of 25    fragmentation buffer , and then heated the mixture at 60  c for 30  min ; finally 55  l 2    ge hybridization buffer is added to dilute the labeled crna
. 100  l of hybridization solution was dispensed into the gasket slide and assembled to the lncrna expression microarray slide .
the slides were incubated for 17  h at 65  c in an agilent hybridization oven .
the hybridized arrays were washed , fixed and scanned by using the agilent dna microarray scanner ( part number g2505b ) .
mircury hy3/hy5 power labeling kit ( exiqon ) was used according to the manufacturer 's guideline for mir labeling of the same 3 pairs of hcc and matched normal liver tissues .
1.0  mg of each sample was 3-end - labeled with hy3 fluorescent label using t4 rna ligase by the following procedure : rna in 2.0  l of water was combined with 1.0  l of cip buffer and cip ( exiqon ) .
the mixture was incubated for 30  min at 37  c , and was terminated by incubation for 5  min at 95  c .
then 3.0  l of labeling buffer , 1.5  l of fluorescent label ( hy3 ) , 2.0  l of dmso , and 2.0  l of labeling enzyme were added into the mixture .
the labeling reaction was incubated for 1  h at 16  c , and terminated by incubation for 15  min at 65  c . after stopping the labeling procedure ,
the hy3-labeled samples were hybridized on the mircury lna array ( v.18.0 ) ( exiqon ) according to the array manual .
the total 25  l mixture from hy3-labeled samples with 25  l hybridization buffer was first denatured for 2  min at 95  c , incubated on ice for 2  min and then hybridized to the microarray for 1620  h at 56  c in a 12-bay hybridization systems ( hybridization system  nimblegen systems ) , which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal .
following hybridization , the slides were achieved , washed several times using wash buffer kit ( exiqon ) , and finally dried by centrifugation for 5  min at 400  rpm .
then the slides were scanned using the axon genepix 4000b microarray scanner ( axon instruments ) .
agilent feature extraction software ( version 10.7.3.1 ) was used to analyze lncrna and mrna array images .
quantile normalization and subsequent data processing were performed by using the genespring gx v11.5.1 software package ( agilent technologies ) .
after quantile normalization of the raw data , lncrnas and mrnas that at least 1 out of 6 samples have flags in present or marginal were chosen for further data analysis . differentially expressed lncrnas and mrnas with statistical significance were identified through volcano plot filtering .
go analysis and pathway analysis were performed by using the standard enrichment computation method . for microrna array analysis
, the scanned images were then imported into genepix pro 6.0 software ( axon ) for grid alignment and data extraction .
replicated mirnas were averaged and mirnas that intensities    50 in all samples were chosen for calculating normalization factor . expressed data were normalized using the median normalization .
according to the cerna paradigm , long non - coding transcripts share mir responsive elements ( mres ) with protein - coding genes , and compete with these protein - coding genes for the pool of shared mirs .
thus , cerna would play a crucial role in the regulation of their cognate genes , , . here
, we described a dataset composed of lncrna , mrna and microrna microarray gene expression profiling for hcc and normal liver tissue . with this dataset
we believe that this dataset would be particularly valuable for investigating the interplay among lncrna , mrna , and microrna in hcc .
for example , we have found that ints6p1 and ints6 are part of the same competitive tumor suppressor network that includes oncomir-17 - 5p ( fig .  1 ) .
this finding provides a new insight in understanding the underlying mechanisms of hepatocarcinogenesis and uncovers the potential therapeutic targets .
 to assess the potential competitive endogenous rna ( cerna ) network in hepatocellular cancer ( hcc ) , the lncrna , mrna , and microrna microarrays were conducted on 3 pairs of hcc and paired normal liver tissue . after that , the arrays were normalized and analyzed with gene oncology ( go ) and pathway analysis . 
 next , we screened out the pseudogenes and their cognate protein coding genes which are both down - regulated in hcc . 
 finally , the up - regulated microrna binding sites were predicted on the most down - regulated pseudogene and its cognate protein - coding gene . 
 all the array data were uploaded to gene expression omnibus ( accession number gse64633 ) . 
Direct link to deposited data
Experimental design, materials and methods
Human tissues
LncRNA, mRNA labeling and array hybridization
MicroRNA labeling and array hybridization
Data analysis
Discussion