In the present study, we determined the expression pattern of a collection of intronic lncRNAs in clear cell RCC patients and identified candidates that might play a role in renal cancer biology. There are only two published studies of lncRNAs in RCC so far: our previous study  that identified for the first time seven intronic lncRNAs differentially expressed in RCC among a protein-coding gene signature; and the work of Yu et al. that identified 626 lncRNAs differentially expressed between tumor and nontumor tissue in 6 clear cell RCC patients. These authors used a microarray that essentially probed intergenic lncRNAs  and they validated by qPCR four transcripts, being three intergenic lncRNAs (ENST00000456816, X91348 and NR_024418); one was not a lncRNA, but rather the non-coding 3′-end portion of the TMEM72 protein-coding gene (BC029135).
We identified 29 lncRNA transcripts originated from intronic regions and additionally 11 from intergenic regions, resulting in a ccRCC-associated gene expression profile comprised exclusively of lncRNAs. From this set, there are three intronic lncRNAs from the ACTN4, HDAC5 and SLC2A1 loci identified as down-regulated both here and in our previous study  using the same microarray platform. This partial overlap (3 out of the 6 intronic lncRNAs described in Ref. ) is possibly related to the more stringent statistical criteria presently used, namely the leave-one-out approach that minimizes the contribution of each individual patient to the set of significantly altered genes when a small patient cohort is analyzed [51, 72].
The comparison of our 217 protein-coding gene profile with nine published studies of differentially expressed protein-coding genes in ccRCC [5, 49, 52–58] verified that the vast majority (83%) of the genes in common (142/170) presented a concordant pattern of expression (Table 2), thus validating the present analysis as representative of the ccRCC biology.
Besides a set of intronic lncRNAs potentially involved in carcinogenesis, the present study identified a set of 26 intronic lncRNAs that were correlated to the survival of ccRCC patients. From this set, eight lncRNAs were identified as altered in both the malignancy and the survival outcome expression profiles, and they are transcribed from the loci: ACTN4, CSNK1D, DNAJC3, GIGYF2, HDAC5, PTPN3, RAB25 and VPS13B. To the best of our knowledge, this is the first study suggesting lncRNAs as correlated to the patient survival outcome in RCC. Regarding other types of ncRNAs, there are at least two miRNA expression studies that had identified candidates correlated with patient survival outcome in RCC [21, 73]. The lncRNAs identified in the present work may contribute to future studies focusing on lncRNAs as molecular markers in RCC oncology.
There are few examples of well-characterized lncRNAs associated with RCC. The lincRNA GAS5 is a well described tumor suppressor in breast cancer , and very recently it was described in prostate cancer cell lines  and in RCC . A decreased expression of the lincRNA GAS5 is associated to RCC genesis and progression, and its overexpression is associated to cell proliferation inhibition and apoptosis induction . Another example includes two antisense lncRNAs at the 5′ (5′aHIF-1α) and 3′ (3′aHIF-1α) ends of the human HIF-1α gene that are expressed in human kidney cancer tissues .
In cancer, there are a few examples of the mechanisms of action of intronic lncRNAs. Our group described the intronic antisense and unspliced lncRNA ANRASSF1 that causes the epigenetic in cis downregulation of the tumor suppressor RASSF1A gene and increases cell proliferation , and its expression is higher in prostate and breast cancer cell lines compared with nontumor cells . Guil et al. identified that overexpression of the sense intronic lncRNA from the SMYD3 locus caused the epigenetic in cis regulation of SMYD3 and a decrease in colorectal cancer cell line proliferation . The androgen-regulated intronic antisense lncRNA CTBP1-AS appears to be a key antisense ncRNA that acts as both cis- and trans- regulator of gene expression. The CTBP1-AS lncRNA promotes prostate cancer growth through sense-antisense repression of the transcriptional co-regulator CTBP1 transcribed from the same locus (cis- regulation), and through a global epigenetic regulation of tumor suppressor genes (trans- regulation) . In fact, the intronic and also the intergenic lncRNAs play important epigenetic roles in cancer .
We decided to study the intronic lncRNA ncHDAC5 in more detail because it showed a decreased expression in ccRCC tumor compared with nontumor tissue that was confirmed by qPCR, and because its increased expression seems to be associated to the cancer-related death after surgery in RCC, as suggested by our patient survival outcome analysis. We determined that ncHDAC5 is an unspliced long transcript (1.7 kb long), detected in the antisense and sense directions relative to the protein-coding gene histone deacetylase 5 (HDAC5). It has a short half-life of 42 min compared with other well studied lncRNAs, such as Air, Kcnq1ot1 and Xist, which have half-lives of 2.1, 3.4 and 4.6 h, respectively , with an evolutionarily conserved secondary structure. The absence of association between the expression of ncHDAC5 and the protein-coding mRNA HDAC5, determined by qPCR and by a meta-analysis of five kidney cancer gene expression studies (Table 1), suggests a locus independent function, with the ncHDAC5 possibly acting in trans to regulate protein-coding genes (see the discussion on trans regulation below). Unfortunately, a probe for this ncHDAC5 was not present in the 44 k oligoarray that was used for assessing the trans correlation of expressed lncRNAs/mRNAs, and it was not possible to determine the ncHDAC5 candidate target mRNAs by our co-expression analysis.
An in silico analysis indicated the presence of RNA Pol II binding and of the histone marks H3K27ac and H3K4me3 at ~1.5 kb upstream of the putative TSS of an antisense ncHDAC5 transcript in the HDAC5 locus. Considering the lack of methylation marks in the vicinity of the lncRNA, this observation opens an interesting possibility of transcriptional regulation of the antisense lncRNA ncHDAC5 by histone acetylation. It is in line with the result recently described for the lncRNA-LET, a lncRNA generally downregulated in carcinomas, that was shown to be repressed by histone deacetylase 3 under hypoxic conditions . Interestingly, the transcriptional-activation-associated H3K4me1 and H3K27ac histone modification marks at human enhancers have been described as related to a cell-type specific protein-coding gene expression . The TSSs at the lncRNA ncHDAC5 locus as well as at the loci of the other intronic antisense lncRNAs expressed in RCC were enriched with both histone marks, in agreement with the fact that the intronic lncRNAs tend to have a tissue-specific pattern of expression , thus supporting a possible cell-type specific modulation of intronic antisense lncRNAs by histone methylation and acetylation.
Because the intronic lncRNAs revealed a promising well-defined pattern of altered expression in RCC, and there is scarce data about this ncRNA class in RCC, we extended our study to the antisense intronic lncRNAs using a custom-designed strand-specific 44 k-element microarray that contained 15-fold more probes for lncRNAs than the 4 k-array that we had previously used. With this new platform, we identified 4303 antisense intronic lncRNAs expressed in RCC; we found that 4061 out of the 4303 antisense lncRNAs have not been previously reported in the Yu et al. study  as being expressed in RCC, which is in agreement with the fact that Yu et al.  used a microarray that probed mostly intergenic lncRNAs. In addition, only six lncRNAs are already annotated as RefSeq noncoding RNAs (Additional file 8: Table S6). In fact, the most recent catalog of human intronic lncRNAs comes from the GENCODE project , which documented the intronic lncRNAs expressed in 12 human normal tissues. Thus, the present study is a contribution towards the generation of a catalog of intronic antisense lncRNAs expressed in renal cancer.
The set of 4303 intronic antisense lncRNAs expressed in renal cancer identified in the present study probably has diverse functions, other than being precursors of small RNAs, because only one lncRNA mapped to a known small RNA sequence (U99, Additional file 8: Table S6). We found that 22% of the intronic antisense lncRNAs have expression levels in RCC, normal kidney, normal liver and tumor prostate that are correlated in cis to the expression levels of the mRNA from the same locus. These lncRNAs correlated in cis are transcribed from loci enriched with genes related to regulation, including the term “Regulation of Transcription from RNA polymerase II”, as seen when analyzing together the positively and negatively cis-correlated antisense lncRNA/mRNA as well as when analyzing only the positively cis-correlated transcripts (Additional file 9: Figure S3). Our group has described a similarly enriched GO term when analyzing the host gene loci of the 30% most abundant intronic antisense lncRNAs, without considering any expression correlation between the ncRNAs and the mRNAs . Now we point to this GO term enrichment for those loci expressing the antisense lncRNAs and the mRNAs in a correlated manner, reinforcing the suggestion that the lncRNAs might cis-regulate the expression of the genes involved in “Regulation of Transcription” and/or that the antisense lncRNAs and the mRNAs might be controlled by a similar regulatory event in these loci.
We found that the expression of the majority of the intronic antisense lncRNAs was not correlated to the expression of the mRNA from the same locus, and those are most likely regulated in an independent way of the mRNAs. Among these, we identified a set of antisense lncRNAs whose expression in RCC, normal liver, prostate tumor and kidney nontumor tissues was positively or negatively correlated in trans to the expression levels of sets of mRNAs belonging to enriched GO terms such as “Inflammatory response” and “Response to stress”; these protein-coding genes may be related to the cellular renal cancer context, and the correlated lncRNAs are candidates to be acting in trans to regulate their expression. The present GO analyses support the proposal that ncRNAs might be part of a fine-tuning regulatory network in the cells [82–84].
Our computational analysis has generated a list of 4303 intronic antisense lncRNAs expressed in RCC (Additional file 8: Table S6) that includes subsets associated to CpG islands, CAGE tag marks, RNA pol II binding site, promoter-associated chromatin marks, tissue-specificity and evolutionary conservation. The set of 53 intronic antisense lncRNAs expressed in common at syntenic loci in human and mouse represent good candidates for subsequent in-depth biological follow up work; the low overlap may be related to the known tissue-specific expression of lncRNAs [8, 41] and to the known tissue-pattern of expression conservation among different species , considering that StLaurent et al.  used mouse lung tissues and we have used human kidney tumor tissues. Although lncRNAs are much less conserved than other functional ncRNAs such as miRNAs and snoRNAs , there is good evidence in the literature regarding the presence among the intronic lncRNAs of evolutionarily conserved regions spanning 400 nt or more [39, 85, 87]. Our recent work with pancreatic cancer has identified an enrichment of conserved regions within intronic and intergenic lncRNAs , and here we extend the identification of conserved regions to the intronic antisense lncRNAs expressed in RCC. Although some of the introns could contain regulatory sequences, or yet undiscovered coding exons overlapped by the intronic RNAs, thus accounting for part of the enrichment signal, the observed primary and secondary structure conservation suggests that the intronic lncRNAs are under the influence of evolutionary constraints.
In silico approaches have been successfully used to characterize sets of lncRNAs expressed in other tissues or cell lineages [9, 28, 29, 46, 69]. Here, we used them to obtain new data indicating that intronic lncRNAs should not be regarded simply as by-products of random transcription , but rather as a diverse and heterogeneous class of cellular transcripts that may comprise yet uncharacterized regulatory RNAs. The intronic lncRNAs identified here as expressed in RCC may have several mechanism of action, both positively and negatively regulating gene expression, and as a consequence, may constitute a promising starting point for further functional investigations.