Cultured rat MCs were purchased from the China Center for Type Culture Collection (Wuhan, China). MCs from passages 7–9 were used in the experiments. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humid atmosphere containing 5% CO₂ at 37°C. MCs were grown to 75% confluence in 100 cm² flasks (BD Biosciences, Franklin Lakes, NJ, USA) were incubated in serum-free medium for 24 h and then treated with ALD (R&D Systems, Inc., Minneapolis, MN, USA) at a concentration of 10⁻⁶ M in a 96-well plate for 24 h at 37°C.

Performed as previously described by Zhang et al (29), with 10⁻⁶ M ALD-treated MCs being used as the experimental cells. An equal amount of solvent (DMEM+10% FCS) was added to the solvent control group. Cells were cultured in 6-well plates for 24 h. MCs were washed with PBS three times, then 200 µl EDTA (Gibco; Thermo Fisher Scientific, Inc.) was added to each well followed by 750 µl TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were stored at −80°C and sent to the LncRNA Expression Microarray (Arraystar, Rockville, MD, USA).

The lncRNAs were constructed using public transcriptome databases (Refseq, www.ncbi.nlm.nih.gov/refseq; University of California Santa Cruz knowngenes, genome.ucsc.edu; and Gencode, www.gencodegenes.org), in addition to a publication (30). The lncRNA expression microarray used in the present study classifies its probes as the following subtypes: i) Enhancer lncRNAs: Contains profiling data of all lncRNAs with enhancer-like function; ii) Rinn lncRNAs: Contains profiling data of all lncRNAs based on studies by Khalil et al (31) and Guttman et al (32); iii) homeobox protein (HOX) cluster: Contains profiling data of all probes in the four HOX loci, targeting 407 discrete transcribed regions, lncRNAs and coding transcripts; iv) lncRNAs located near coding genes: These contain differentially expressed lncRNAs and nearby coding gene pairs (distance, 300 kb); and v) enhancer lncRNAs located near coding genes: These contain differentially expressed enhancer-like lncRNAs and their nearby coding genes (distance, 300 kb). Following hybridization and washing, processed slides were scanned with an Agilent DNA Microarray Scanner (part no. G2505B; Agilent Technologies, Inc., Santa Clara, CA, USA). Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies, Inc.) was used to analyze all acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX version 11.5.1 software package (Agilent Technologies, Inc.) (33). The profile of microarray data of the 8,459 lncRNAs was detected by third-generation lncRNA microarray. The general characteristics of the differentially-expressed lncRNAs were summarized, including chromosomal, source, relationship and fold-change distribution using the most widely-used public transcriptome databases (Ensembl, www.ensembl.org/index.html; UCSC, genome.ucsc.edu/index.html; NONCODE, www.noncode.org; NCBI, www.ncbi.nlm.nih.gov). The source of the lncRNA was collected from RefSeq_NR (RefSeq validated non-coding RNA), RefSeq_XR (RefSeq un-validated non-coding RNA), mouse_ortholog (rat lncRNAs which are obtained by sequence comparison with mouse lncRNAs), ‘ultra-conserved regions’ among human, mouse and rat (users.soe.ucsc.edu/~jill/ultra.html), and misc_lncRNA (other sources).

RT-qPCR was used to verify the differential expression of seven lncRNAs and six associated genes that were detected by the lncRNA and mRNA expression microarray. The cDNA was synthesized using reverse transcriptase (Takara Bio, Inc., Otsu, Japan) and oligo (dTs) primers with 1 µl RNA from the same samples as those used in the microarray. The reaction consisted of 2 µl 5X PrimeScript buffer, 0.5 µl PrimeScript RT Enzyme Mix I, 0.5 µl Oligo dT Primer, 0.5 µl Random 6 mers, 500 ng total RNA and RNase free dH₂O up to 10 µl, and was performed for 15 min at 37°C. Primers for each lncRNA and mRNA are reported in Table I. qPCR was performed on an Applied Biosystems ViiA™ 7 Dx (Thermo Fisher Scientific, Inc.) using the SYBR-Green method, according to the manufacturer's protocol. Each RT-qPCR reaction (in 10 µl) contained 5 µl SYBR⁻Green real-time PCR Master mix (Thermo Fisher Scientific, Inc.), 1.0 mM primer and 1 µl template cDNA. The cycling conditions consisted of an initial single cycle of 2 min at 50°C; 2 min at 95°C; followed by 40 cycles of 15 sec at 95°C, 15 sec at 56°C and 60 sec at 72°C. PCR amplifications were performed in triplicate for each sample. Gene expression levels were quantified relative to the expression of GAPDH (primers: Sense, 5′-CAAGTTCAACGGCACAGTCAA-3′; antisense, 5′-TGGTGAAGACGCCAGTAGACTC-3′) using an optimized comparative 2^−ΔΔCq method (34). RT-qPCR was performed in triplicate on the diluted cDNA and the experiments were performed twice in the control and ALD-treated MC groups.

Previous studies have demonstrated that lncRNAs are preferentially located next to genes with developmental functions (26). For each lncRNA locus the nearest protein-coding neighbor within 100 kb was identified. For antisense overlapping and intronic overlapping lncRNAs, the overlapping gene was identified. GO (http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses (http://www.genome.jp/kegg) were applied to determine the roles of these closest coding genes in GO terms or biological pathways.

GO analysis was used to assess the main function of the closest coding genes according to the GO database, which provides the key functional classifications for the National Center for Biotechnology Information (35). The ontology covers two domains: Biological processes and molecular function. Fisher's exact test is used to identify if there is more overlap between the differentially expressed list and the GO annotation list than would be expected by chance. The P-value denotes the significance of GO term enrichment in the differentially expressed genes. Pathway analysis is a functional analysis mapping genes to the KEGG pathways. The P-value (expression analysis systematic explorer-score, Fisher-P-value or Hypergeometric-P-value) denotes the significance of the pathway correlated with the conditions. P<0.05 was considered to indicate a statistically significant difference.

Each qPCR experiment was performed at least three times. Numerical data were presented as the mean ± standard error of the mean. Relative expression levels of lncRNAs between the two groups were analyzed using the Student's t-test. All statistical analyses were performed using SPSS software (version 18; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

A gene chip study was performed in the normal and ALD-treated MC group to investigate the possible lncRNA alteration in expression using the Arraystar probe dataset, which included 8,459 lncRNAs. The lncRNAs were constructed using public transcriptome databases (Refseq, University of California Santa Cruz knowngenes and Gencode), in addition to a publication (30). The scatterplot is useful for assessing the variation in the expression of lncRNAs and coding transcripts between the two MCs (Fig. 1A); the dot above the green line represents a difference of >2 times. The number of points above the top and below the bottom green lines indicated lncRNAs that exhibit >2.0 fold-change when comparing the control and ALD-treated MC groups. By setting a filter of fold-change >3.0, raw >100 of the expression level between the ALD-treated group and control group, 5 upregulated and 2 downregulated lncRNAs were identified (Fig. 1B).

An Affymetrics gene array containing 13,214 gene transcripts was used to perform a comprehensive analysis of mRNA expression in control compared with ALD-treated rat MCs group. Comparing the rat MCs treated with ALD with the control group, 180 genes were differentially expressed (fold-change >3.0; raw >100), of which 36 genes were upregulated and 144 genes were downregulated (Fig. 1C). A scatter-plot illustrating the expression patterns of these differentially expressed mRNAs between control and ALD-treated rat MCs is exhibited in Fig. 1D.

To quantify the microarray hybridization results, RT-qPCR was performed on five upregulated lncRNAs (BC168211, BC088254, AF336872, AY325162 and BC168687), two downregulated lncRNAs (AF230638 and BC167085) (Fig. 2A), three upregulated mRNAs (NM_001108598, NM_001109190 and NM_001101018) (Fig. 2B) and three downregulated mRNAs (NM_019347, NM_177962 and NM_001108823) (Fig. 2C), selected on the basis of their levels of expression on the microarray and their biological significance. The RT-qPCR data was demonstrated to be consistent with the microarray results, with BC168211, BC088254, AF336872, AY325162 and BC168687 being upregulated and AF230638, BC167085 being downregulated compared with the control. The RT-qPCR data were again consistent with the microarray results for the mRNAs with NM_001108598, NM_001109190 and NM_001101018 being upregulated and NM_019347, NM_177962 and NM_001108823 being downregulated (P<0.05 vs. the control group).

The general characteristics of the differentially expressed lncRNAs were summarized, including chromosomal, source, relationship and fold-change distribution. Chromosomal distribution of the number of up- or downregulated lncRNAs located on different chromosomes was demonstrated (Fig. 3A and B). Fold-change distribution demonstrated the differential expression of up- and downregulated lncRNAs (Fig. 3C and D), respectively. Source distribution respectively demonstrated the percentages of up- and downregulated lncRNAs collected from different sources (Fig. 4A and B), including misc_lncRNA, ultra-conserved region, Refseq-XR and mouse_ortholog. Relationship distribution demonstrated the association of up- and downregulated lncRNAs (Fig. 4C and D), respectively.

To elucidate the biological processes and functional classification of differentially expressed lncRNAs, GO and pathway analyses were performed. The functions of coding genes adjacent to dysregulated lncRNAs included (in size order, most prevalent first, the top ten): i) Response to external stimuli; ii) response to stress; iii) positive regulation of biological processes; iv) cyclic nucleotide metabolic process; v) regulation of multicellular organism processes; vi) positive regulation of the apoptotic process; vii) cell division; viii) M phase; ix) mitosis; and x) nuclear division (Fig. 5A). The cellular component containing dysregulated lncRNAs included the following (in size order, most prevalent first, the top ten): i) Cell periphery; ii) dendrite; iii) chloride channel complex; iv) neuron projection; v) extracellular region part; vi) hemoglobin complex; vii) extracellular space; viii) plasma membrane; ix) mitotic spindle; and x) spindle (Fig. 5B). The molecular function of dysregulated lncRNAs mainly consisted of the following (in size order, most prevalent first, the top ten): i) Ion channel activity; ii) potassium ion transmembrane transporter activity; iii) potassium channel activity; iv) transporter activity; v) passive transmembrane transporter activity; vi) ion transmembrane transporter activity; vii) protein binding; viii) oxygen transporter activity; ix) receptor binding; and x) substrate-specific transmembrane transporter activity (Fig. 5C). Pathway analysis is a functional analysis process that maps genes to KEGG pathways. In the present study, the top 10 pathways that were associated with coding genes of dysregulated lncRNAs involved: i) Estrogen signaling pathway; ii) peroxisome proliferator-activated receptor (PPAR) signaling pathway; iii) cell cycle; iv) retrograde endocannabinoid signaling; v) circadian entrainment; vi) hypertrophic cardiomyopathy; vii) gastric acid secretion; viii) microRNAs in cancer; ix) cardiac muscle contraction; and x) dilated cardiomyopathy-all Rattus norvegicus (Fig. 5D).

As the transcription of non-coding genes can affect the expression of their flanking coding genes, relatively lowly and highly expressed lncRNAs were selected with a < and >3-fold-change, respectively, as well as an raw >100 in the MCs treated with ALD and control group, and an associated coding gene with a function in a developmental processes, including cell cycle and PPAR signaling. It was demonstrated that dual specificity phosphatase 15 (Dusp15) and acyl-coenzyme A thioesterase Them4 (Them4) were associated with the lncRNAs BC168211 and BC168687, respectively (data not shown).