extensive epidemiological investigations have indicated that the incidence of type 1 diabetes mellitus ( t1 dm ) in children and adolescents is increasing steadily year by year and is presumably to be doubled by 2020 [ 15 ] . however , the etiology and pathogenesis of t1 dm are complex and have not been fully elucidated . additionally , long - term insulin injection and acute and chronic clinical complications such as ketoacidosis , nephropathy , neuropathy , and retinopathy could cause significant physical and mental trauma on the children with this disease . therefore , exploring the pathogenesis of t1 dm and developing novel intervention strategies have become top priorities in the field of endocrine research . pancreatic beta cells secrete insulin to regulate blood glucose levels within a relatively narrow range . t1 dm is an organ - specific autoimmune disease characterized by chronic and progressive apoptotic destruction of pancreatic beta cells , resulting in severe insulin deficiency . autoimmunity against the beta cells is likely triggered by environmental risk factors that act in genetically susceptible individuals [ 79 ] . once stimulated , various immune cells , including t or b cells , macrophages , and dendritic cells , infiltrate the islets to induce beta cell death via mechanisms such as reactive oxygen species , perforin / granzymes , fas / fasl , nitrogen species , and proinflammatory cytokines , among which cytokines have been recognized to play key roles in the regulation of autoimmunity and beta cell loss . prolonged exposure to immune cell - secreted proinflammatory cytokines such as interleukin- ( il- ) 1 , tumor necrosis factor- ( tnf- )  , and interferon- ( ifn- )  is highly cytotoxic to pancreatic beta cells , which impairs insulin secretion and ultimately induces beta cell loss by apoptosis [ 1013 ] . during this inflammatory process , proinflammatory cytokines affect the expression of many gene networks and modulate pro- and antiapoptotic pathways [ 14 , 15 ] . to date , research has mainly focused on protein - coding genes , though recent studies have shown that protein - coding genes account for less than 2% of the actively transcribed genome , suggesting that the majority of transcripts are noncoding rnas ( ncrnas ) . ncrnas can be subdivided by length into small ncrnas ( shorter than 200 nucleotides ) and long ncrnas ( lncrnas ) , which are greater than 200 nucleotides in length , with little or no protein - coding capacity . in recent years , research in the field of rna biology has mainly focused on small ncrnas , largely ignoring lncrnas . for a long time , more and more evidence suggests that lncrnas play significant roles in genome regulation , such as in x chromosome inactivation , genomic imprinting , chromatin modification , transcription , splicing , translation , degradation , and transport processes that are involved in the regulation of the growth and development of individual cells in apoptosis , proliferation , differentiation , and other cellular activities [ 17 , 1922 ] . the dysregulation of these lncrnas has been associated with many human diseases , including different types of cancers , neurodegenerative diseases , and other disorders . with the development of lncrna microarrays , bioinformatics , and high - throughput sequencing , thousands of lncrnas currently , some studies have examined the roles of lncrnas in diabetes , and the emerging evidence indicates that lncrnas may be involved in maintaining pancreatic beta cell function and insulin signal transduction , which may affect diabetes development . in this study , we examined the expression profiles of lncrnas and mrnas in min6 cells after exposure to a combination of il-1 , tnf- , and ifn- using microarray technology . we found that the expression levels of many lncrnas and mrnas changed in response to proinflammatory cytokines . some of the interested lncrnas with remarkable regulation were further verified by qrt - pcr , and the gene ontology ( go ) and pathway analyses were used to analyze the biological roles of these differentially expressed lncrnas and mrnas . coding - noncoding gene coexpression networks were used to predict the potential interactions between these lncrnas and mrnas . pancreatic min6 -cells were grown in high - glucose dulbecco 's modified eagle medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 100  g / ml streptomycin , 100  g / ml penicillin , and 50  m -mercaptoethanol at 37c in an atmosphere of 5% co2 . min6 cells were seeded in 6-well plates to approximately 80% confluence and were then treated with or without 5  ng / ml il-1 , 25  ng / ml tnf- , and 25  ng / ml ifn- for 24  h. total rna was extracted from the min6 cells using trizol reagent ( invitrogen , usa ) and was then purified using an rneasy mini kit ( qiagen , hilden , germany ) according to the manufacturer 's protocol . an affymetrix genechip mouse transcriptome array 1.0 , which is designed for measuring a broad range of expression changes across the whole mouse transcriptome , was used to profile the ncrnas and protein - coding genes . the lncrnas were obtained from authoritative databases , including ensembl , refseq , ucsc known genes , vertebrate genome annotation ( vega ) database , mgc mammalian gene collection , mgi , noncode , and others . the sample preparation and labeling , microarray hybridization , and washing were performed using a genechip wt terminal labeling and controls kit , an ambion wt expression kit , and a genechip hybridization , wash , and stain kit , respectively , according to the manufacturers ' standard protocols . briefly , 1  g total rna , which was primed with primers containing a t7 promoter sequence , was used to synthesize single - stranded cdna . then , the single - stranded cdna was transformed into double - stranded cdna , which served as a template to synthesize and amplify the antisense rna ( complimentary rna ) from an in vitro transcription reaction . rnase h was used to hydrolyze the crna template , leaving single - stranded cdna . after hydrolysis , the 2nd - cycle single - stranded cdna , which was purified to remove salts , enzymes , and unincorporated dntps , was fragmented and labeled . the labeled cdnas were hybridized onto the affymetrix genechip mouse transcriptome array 1.0 using a genechip hybridization oven 645 . after washing the hybridized arrays , affymetrix expression console software ( version 1.3 ) was used to analyze the microarray data . the significant differentially expressed lncrnas and mrnas between the two groups were selected if the fold changes of the threshold values were 2.0 or 2.0 ( p0.05 ) . the three - step robust multichip analysis ( rma ) algorithm , which includes ( 1 ) background adjustment , ( 2 ) quantile normalization , and ( 3 ) summarization , was used to analyze the raw data . reaction cdna was synthesized using the primescript rt master mix ( perfect real time , takara , japan ) according to the manufacturer 's recommendations . reverse transcription of lncrnas was performed using the sybr green method in an applied biosystems 7500 system ( life technologies , us ) . the gene expression levels were quantified based on the cycle threshold ( ct ) values and were normalized to the internal control gene -actin . go , which describes gene and gene product attributes , contains three key domains named biological process , cellular component , and molecular function . the kyoto encyclopedia of genes and genomes ( kegg ) database , a bioinformatics resource for deciphering the genome , was used to identify the significant pathways of the targeted genes . an lncrna - mrna coexpression network , which can identify the interactions between the differentially expressed lncrnas and mrnas , was constructed according to the normalized signal intensities of specifically expressed genes and lncrnas . the pearson correlation was calculated for each lncrna - gene , gene - gene , or lncrna - lncrna pair , and significant correlation pairs were selected to construct the network . the random variance model t - test was employed to identify the differentially expressed genes and lncrnas between the control and cytokine - stimulated groups . an affymetrix genechip mouse transcriptome array 1.0 was designed to profile all mouse lncrnas and protein - coding transcripts . according to the microarray expression profiling data , 723 differently regulated lncrnas were identified in the cytokine - stimulated group compared with the control group with a set filter fold - change 2.0 , and 2180 differently regulated mrnas were identified with a set filter fold - change 1.5 . additionally , 444 upregulated and 279 downregulated lncrnas were detected and presented in table ( in supplementary material available online at http://dx.doi.org/10.1155/2016/9275106 ) . the result of the hierarchical clustering shows the lncrna and mrna expression patterns ( figures 1(a ) and 1(b ) ) . to further explore the potential functions of dysregulated lncrna in the cytokine - stimulated group the length distribution showed that the deregulated lncrnas were mostly less than 500  bp or longer than 5000  bp ( figure 2 ) . the chromosomal distribution showed that the upregulated lncrnas were mostly located on chromosome 1 and that the downregulated lncrnas were mostly located on chromosome 2 ( figure 3 ) . based on our go analysis , the following top 10 enriched go terms were associated with the upregulated lncrnas : ( 1 ) innate immune response , ( 2 ) inflammatory response , ( 3 ) apoptotic process , ( 4 ) positive regulation of i - kappa b kinase / nuclear factor- ( nf- ) kappa b cascade , ( 5 ) defense response to virus , ( 6 ) biological process , ( 7 ) response to virus , ( 8) immune response , ( 9 ) positive regulation of transcription from rna polymerase ii promoter , and ( 10 ) negative regulation of endopeptidase activity ( figure 4(a ) ) . the following top 10 enriched go terms were associated with the downregulated lncrnas : ( 1 ) transport , ( 2 ) cell cycle , ( 3 ) oxidation - reduction process , ( 4 ) nucleosome assembly , ( 5 ) cell division , ( 6 ) mitosis , ( 7 ) metabolic process , ( 8) biological process , ( 9 ) protein transport , and ( 10 ) ion transport ( figure 4(b ) ) . based on our pathway analysis , the following top 10 enriched pathways were associated with the upregulated lncrnas : ( 1 ) cytokine - cytokine receptor interaction , ( 2 ) nf - kappa b signaling pathway , ( 3 ) toll - like receptor signaling pathway , ( 4 ) nod - like receptor signaling pathway , ( 5 ) mapk signaling pathway , ( 6 ) rig - i - like receptor signaling pathway , ( 7 ) apoptosis , ( 8) jak - stat signaling pathway , ( 9 ) chemokine signaling pathway , and ( 10 ) hippo signaling pathway ( figure 5(a ) ) . additionally , the following top 10 enriched pathways were associated with the downregulated lncrnas : ( 1 ) metabolic pathways , ( 2 ) viral carcinogenesis , ( 3 ) protein processing in endoplasmic reticulum , ( 4 ) cell cycle , ( 5 ) n - glycan biosynthesis , ( 6 ) transcriptional misregulation in cancer , ( 7 ) purine metabolism , ( 8) insulin secretion , ( 9 ) biosynthesis of unsaturated fatty acids , and ( 10 ) maturity onset diabetes of the young ( figure 5(b ) ) . based on the correlations between the differentially expressed lncrnas and mrnas with significant correlation pearson coefficients , we developed a profile between the lncrnas and coding mrnas in the cytokine - stimulated and control groups ( figures 6(a ) and 6(b ) ) . the coexpression network showed that one lncrna was associated with one to dozens of mrnas and lncrnas . in a network analysis , degree centrality is used to measure a gene or lncrna centrality within a network , determining its relative importance . from the two networks , we identified the core regulatory lncrnas and mrnas , which had the largest degrees ( tables 2 and 3 ) . we randomly selected 15 interested lncrnas to validate the microarray analysis data using qrt - pcr . the results of the qrt - pcr were largely consistent with the data from the microarray ( figure 7 ) . during the initial phases of t1 dm , cytokines and other inflammatory mediators released by immune cells progressively infiltrate islets , contributing to the functional suppression and death of beta cells . in nonobese diabetic ( nod ) these observations led to the concept that proinflammatory mediators produced by infiltrating cells play key roles in inducing beta cell death . accordingly , prolonged exposure to proinflammatory cytokines is highly cytotoxic to pancreatic beta cell function [ 1013 ] and affects the expression of many gene networks [ 14 , 15 ] . little information exists on the lncrnas that regulate beta cell apoptosis and contribute to t1 dm development [ 2729 ] . compared with these studies , we use a new and different gene chip , expand the lncrna expression profile , and update the database . here , we report the lncrna expression profiles of min6 cells that have been exposed to the proinflammatory cytokines il-1 , tnf- , and ifn-. comprehensive analysis shows that lncrnas are generally expressed at lower levels compared with protein - coding genes and are most likely to display tissue - specific expression patterns [ 30 , 31 ] . the affymetrix genechip mouse transcriptome array 1.0 , which contains both lncrna and mrna probes , was employed to determine the expression profiles of lncrnas and mrnas in min6 cells . in this study , we identified 723 differently regulated lncrnas and 2180 differently regulated mrnas in the cytokine - stimulated group compared with the control cells , among which 444 upregulated and 279 downregulated lncrnas were detected with a set filter fold - change 2.0 . qrt - pcr was used to validate some of the microarray analysis data , and the results were consistent with the microarray data . the mostly upregulated lncrna , nonmmut036704 , was found to exhibit sense overlap with the lipocalin 2 ( lnc2 ) , which is also known as neutrophil gelatinase - associated lipocalin ( ngal ) . found that il-10 could mediate ngal expression in breast cancer cells , which might contribute to tumor progression . in addition , ngal could create the local and systemic proinflammatory environment for atherosclerosis by induction of proinflammatory mediators . and , ngal was overexpression in subcutaneous adipose tissue in overweight women with gestational diabetes mellitus ( gdm ) , which suggests that it may play a role in the development of insulin resistance in gdm . currently , there are no studies about ngal in the apoptosis of  cells . but in view of its role in the parenchyma cells , we surmise that lncrna nonmmut036704 may play an important role in the development of t1 dm through the regulation of ngal . this transcript was found to sense overlap cd274 antigen ( cd274 ) , which is also known as programmed death-1 ligand-1 ( pd - l1 ) . a recent study shows that forced expression of pd-1 in transgenic mice significantly decreased the incidence of autoimmune diabetes . another study also shows that pd-1 or pd - l1 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic ( nod ) mice regardless of age . pd - l1 deficiency could increase susceptibility to diabetes via a direct effect on pathogenic t cells , while regulatory t cells and b cells prevented autoimmune diabetes via a mechanism independent of pd-1/pd - l1 pathway . therefore , we surmise that lncrnanonmmut034373 may contribute to the development of t1 dm via regulating pd - l1 . studies have reported that there are susceptible genes to t1 dm in some chromosomes , such as ptpn22 on chromosome 1 , ctla4 on chromosome 2 , ifih1 on chromosome 2 , and il2ra on chromosome 10 [ 3841 ] . in this study , compared with the other chromosomes , chromosome 1 had a higher percentage of upregulated lncrnas , and chromosome 2 had a higher percentage of downregulated lncrnas . this finding suggests that we may have a higher chance to find the susceptible lncrnas that contribute to beta cell apoptosis on chromosomes 1 and 2 . to evaluate the potential biological functions of the differentially expressed lncrnas , go enrichment and pathway analyses were used to analyze the differentially expressed protein - coding genes associated with these lncrnas . in our existing data , we found that the main biological processes that were enriched for the dysregulated lncrnas were  innate immune response ,   inflammatory response , apoptotic process ,  and  positive regulation of i - kappa b kinase / nf - kappa b cascade . these findings are in agreement with one current concept that immune - mediated inflammation may serve as a pathogenic mechanism of type-1 diabetes . the released inflammatory cytokines bind to their receptors on beta cells and could activate the transcription factors p38 , c - jun n - terminal kinase ( jnk ) , signal transducer and activator of transcription-1 ( stat-1 ) , and nf - kappa b and the mitogen - activated protein- ( map- ) kinase signaling pathways , provoking endoplasmic reticulum stress and leading to functional impairment and ultimately apoptosis . here the pathway analysis showed that the identified lncrnas are mainly involved in the nf - kappa b signaling pathway , toll - like receptor signaling pathway , mapk signaling pathway , and jak - stat signaling pathway , indirectly indicating that these differentially expressed lncrnas play important roles in cytokine - induced islet cell injury . pathway analysis also showed that the identified lncrnas are involved in the hippo signaling pathway , rig - i - like receptor signaling pathway . the hippo signaling pathway plays critical roles in organ size control and tissue regeneration by inhibiting cell proliferation and promoting apoptosis . after partial hepatectomy ( ph ) in rats , the hippo signaling pathway is inhibited early during liver regeneration . in addition , inactivation of the hippo pathway or activation of its downstream effector , the yap transcription coactivator , improves cardiac regeneration . therefore , we assumed that activation of the hippo signaling pathway may induce  cells apoptosis and inactivation of the hippo signaling pathway may induce  cells proliferation . rig - i - like receptors are key cytoplasmic pathogen recognition receptors that are involved in the recognition of viruses , including functioning as major sensors of rna viruses , and promoting recognition of some dna viruses . although there are no evidence showing that rig - i - like receptors are associated with type 1 diabetes , environmental factors such as virus infection have been thought to trigger t1 dm . therefore , we speculate that activation of rig - i - like receptors may cause innate immune - related inflammatory response to promote  cells apoptosis , resulting in t1 dm . our current study reveals many differentially expressed lncrnas and their related mrnas in cytokine - induced min6 cell apoptosis using a microarray assay . these differentially expressed lncrnas may play key or partial roles in cytokine - mediated beta cell dysfunction . understanding the functions of these lncrnas could help us to identify new diagnostic and therapy targets for t1 dm . thus , further studies to identify the contributions of some interested targets in the pathogenesis of t1 dm based on current study would be important and valuable .