Abstract
Long non-coding RNAs represent a fraction of the transcriptome that is being increasingly recognized. For most of them no function has been allocated so far. Here, we describe the nature and function of a novel non-protein-coding transcript, named WISP1-AS1, discovered in human renal proximal tubule cells exposed to the carcinogenic nephrotoxin ochratoxin A. WISP1-AS1 overlaps parts of the fourth intron and fifth exon of the Wnt1-inducible signaling pathway protein 1 (WISP1) gene. The transcript is 2922 nucleotides long, transcribed in antisense direction and predominantly localized in the nucleus. WISP1-AS1 is expressed in all 20 samples of a human tissue RNA panel with the highest expression levels detected in uterus, kidney and adrenal gland. Its expression was confirmed in primary tissues of human kidneys. In addition, WISP1-AS1 is expressed at higher levels in renal cell carcinoma (RCC) cell lines compared to primary proximal tubule cells as well as in RCC lesions than in the adjacent healthy control tissue from the same patient. Using specific gapmer antisense oligonucleotides to prevent its upregulation, we show that WISP1-AS1 (1) does not influence the mRNA expression of WISP1, (2) affects transcriptional regulation by Egr-1 and E2F as revealed by RNA-sequencing, enrichment analysis and reporter assays, and (3) modulates the apoptosis-necrosis balance. In summary, WISP1-AS1 is a novel lncRNA with modulatory transcriptional function and the potential to alter the cellular phenotype in situations of stress or oncogenic transformation. However, its precise mode of action and impact on cellular functions require further investigations.
Similar content being viewed by others
References
Mercer TR, Dinger ME, Mattick JS (2009) Long non-coding RNAs: insights into functions. Nat Rev Genet 10:155–159
Kung JT, Colognori D, Lee JT (2013) Long noncoding RNAs: past, present, and future. Genetics 193:651–669
Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166
Rion N, Ruegg MA (2017) LncRNA-encoded peptides: more than translational noise? Cell Res 27:604–605
Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, Guernec G, Martin D, Merkel A, Knowles DG, Lagarde J, Veeravalli L, Ruan X, Ruan Y, Lassmann T, Carninci P, Brown JB, Lipovich L, Gonzalez JM, Thomas M, Davis CA, Shiekhattar R, Gingeras TR, Hubbard TJ, Notredame C, Harrow J, Guigo R (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775–1789
Wright MW (2014) A short guide to long non-coding RNA gene nomenclature. Hum Genom 8:7–11
Bar C, Chatterjee S, Thum T (2016) Long noncoding RNAs in cardiovascular pathology, diagnosis, and therapy. Circulation 134:1484–1499
Hon CC, Ramilowski JA, Harshbarger J, Bertin N, Rackham OJ, Gough J, Denisenko E, Schmeier S, Poulsen TM, Severin J, Lizio M, Kawaji H, Kasukawa T, Itoh M, Burroughs AM, Noma S, Djebali S, Alam T, Medvedeva YA, Testa AC, Lipovich L, Yip CW, Abugessaisa I, Mendez M, Hasegawa A, Tang D, Lassmann T, Heutink P, Babina M, Wells CA, Kojima S, Nakamura Y, Suzuki H, Daub CO, de Hoon MJ, Arner E, Hayashizaki Y, Carninci P, Forrest AR (2017) An atlas of human long non-coding RNAs with accurate 5′ ends. Nature 543:199–204
Volders PJ, Verheggen K, Menschaert G, Vandepoele K, Martens L, Vandesompele J, Mestdagh P (2015) An update on LNCipedia: a database for annotated human lncRNA sequences. Nucleic Acids Res 43:D174–D180
Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP (2010) A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465:1033–1038
Cheetham SW, Gruhl F, Mattick JS, Dinger ME (2013) Long noncoding RNAs and the genetics of cancer. Br J Cancer 108:2419–2425
Engreitz JM, Sirokman K, McDonel P, Shishkin AA, Surka C, Russell P, Grossman SR, Chow AY, Guttman M, Lander ES (2014) RNA–RNA interactions enable specific targeting of noncoding RNAs to nascent pre-mRNAs and chromatin sites. Cell 159:188–199
Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT, Freier SM, Bennett CF, Sharma A, Bubulya PA, Blencowe BJ, Prasanth SG, Prasanth KV (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39:925–938
Kawakami T, Zhang C, Taniguchi T, Kim CJ, Okada Y, Sugihara H, Hattori T, Reeve AE, Ogawa O, Okamoto K (2004) Characterization of loss-of-inactive X in Klinefelter syndrome and female-derived cancer cells. Oncogene 23:6163–6169
Qiao HP, Gao WS, Huo JX, Yang ZS (2013) Long non-coding RNA GAS5 functions as a tumor suppressor in renal cell carcinoma. Asian Pac J Cancer Prev 14:1077–1082
Kino T, Hurt DE, Ichijo T, Nader N, Chrousos GP (2010) Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor. Sci Signal 3:ra8
Mayama T, Marr AK, Kino T (2016) Differential expression of glucocorticoid receptor noncoding RNA repressor Gas5 in autoimmune and inflammatory diseases. Horm Metab Res 48:550–557
Hennemeier I, Humpf HU, Gekle M, Schwerdt G (2014) Role of microRNA-29b in the ochratoxin A-induced enhanced collagen formation in human kidney cells. Toxicology 324:116–122
Zhu L, Zhang B, Dai Y, Li H, Xu W (2017) A review: epigenetic mechanism in ochratoxin a toxicity studies. Toxins (Basel) 9:E113
Hennemeier I, Humpf HU, Gekle M, Schwerdt G (2012) The food contaminant and nephrotoxin ochratoxin A enhances Wnt1 inducible signaling protein 1 and tumor necrosis factor expression in human primary proximal tubule cells. Mol Nutr Food Res 56:1375–1384
Rottkord U, Röhl C, Ferse I, Schulz M, Rückschloss U, Gekle M, Schwerdt G, Humpf HU (2017) Structure–activity relationship of ochratoxin A and synthesized derivatives: importance of amino acid and halogen moiety for cytotoxicity. Arch Toxicol 91:1461–1471
Schwerdt G, Königs M, Holzinger H, Humpf HU, Gekle M (2009) Effects of the mycotoxin FB1 on cell death in human kidney cells and human lung fibroblasts in primary culture. J Appl Toxicol 29:174–182
Lane RD, Federman D, Flora JL, Beck BL (1986) Computer-assisted determination of protein concentrations from dye-binding and bicinchoninic acid protein assays performed in microtiter plates. J Immunol Methods 92:261–270
Schwerdt G, Frisch A, Mildenberger S, Hilgenfeld T, Grossmann C, Gekle M (2012) Influence of aldosterone and salt or ouabain in A10 rat aorta smooth muscle cells. J Vasc Res 49:231–241
Bergmeyer HU, Bernt E (1974) Laktat-dehydrogenase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 3. Verlag Chemie, Weinheim, Germany, pp 607–612
Finotello F, Di Camillo B (2015) Measuring differential gene expression with RNA-seq: challenges and strategies for data analysis. Br Funct Genom 14:130–142
Reimand J, Arak T, Adler P, Kolberg L, Reisberg S, Peterson H, Vilo J (2016) g:Profiler—a web server for functional interpretation of gene lists (2016 update). Nucleic Acids Res 44:W83–W89
Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z (2009) GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinform 10:48
Zambelli F, Pesole G, Pavesi G (2009) Pscan: finding over-represented transcription factor binding site motifs in sequences from co-regulated or co-expressed genes. Nucleic Acids Res 37:W247–W252
Harris H (2013) History: non-coding RNA foreseen 48 years ago. Nature 497:188
Malek E, Jagannathan S, Driscoll JJ (2014) Correlation of long non-coding RNA expression with metastasis, drug resistance and clinical outcome in cancer. Oncotarget 5:8027–8038
Hsieh JJ, Purdue MP, Signoretti S, Swanton C, Albiges L, Schmidinger M, Heng DY, Larkin J, Ficarra V (2017) Renal cell carcinoma. Nat Rev Dis Primers 3:17009
Fava LL, Bock FJ, Geley S, Villunger A (2012) Caspase-2 at a glance. J Cell Sci 125:5911–5915
Kumar S (2009) Caspase 2 in apoptosis, the DNA damage response and tumour suppression: enigma no more? Nat Rev Cancer 9:897–903
Jaluria P, Konstantopoulos K, Betenbaugh M, Shiloach J (2008) Egr1 and Gas6 facilitate the adaptation of HEK-293 cells to serum-free media by conferring enhanced viability and higher growth rates. Biotechnol Bioeng 99:1443–1452
Hasanbasic I, Cuerquis J, Varnum B, Blostein MD (2004) Intracellular signaling pathways involved in Gas6-Axl-mediated survival of endothelial cells. Am J Physiol Heart Circ Physiol 287:H1207–H1213
Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft DFG (GRK 1591 to Michael Gekle, Gerald Schwerdt and Barbara Seliger; SCHW 1515/2-1 and HU 730/12-1) and by the Deutsche Krebshilfe (Barbara Seliger).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
18_2017_2731_MOESM1_ESM.pdf
Supplementary file 1: Original amplification and melting curves of representative qPCR measurements using primers against intron 4 (detect WISP1-AS1), exon 5 (detect WISP1-AS1 and WISP1 mRNA) or exon1 and exon 2 (detect exclusively WISP1 mRNA), performed with samples from cells incubated for 48h with either control media or media containing 100 nM OTA (green = controls, red = OTA-treated). Inserts show the respective melting curves. Additionally, we compared the PCR results from samples when reverse transcriptase was omitted during the RT step and from samples when the random primers where omitted during the RT step with regular transcribed samples. Supplementary file 2: Sequence of WISP1-AS1. Blue letters represent the 3’ UTR part, black represents the exon 5 part and red the intron 4 part of the host gene WISP1. Supplementary file 3: A) In silico ribosome profiling. Upper part of the panel (in red) shows translational events. Besides translation of exon 4 and exon 5 that belongs to WISP1 mRNA, there is no substantial translation in intron 4 nor in 3` UTR, from where WISP1-AS1 is transcribed. These data support non-coding feature of WISP1-AS1. http://gwips.ucc.ie/cgi-bin/hgTracks?db=hg38&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr8%3A133225257-133227653&hgsid=53858_QXJ74F9Fr6YEETdobe4ec7GFB3q4. B) In silico RNA-seq (encode matrix) on NHEK cells. From this data, there is a transcriptional activity on minus strand (antisense), in poly(A) fraction. https://genome.ucsc.edu/cgi-bin/hgTracks?db=hg19&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr8%3A134236181-134243318&hgsid=598156345_o0TLNDLt0t05GWvHKmJICQGEdmfd. Supplementary file 4: A) Quantitative RT-PCR of WISP1-AS1 using random hexamer and oligo(dT)18 primers for reverse transcription. N = 3. HEK-293T cells were exposed for 48 hours to 100 nM OTA. B) Hybridization specificity of biotinylated probes against WISP1-AS1 (positive control probe) and complementary biotinylated probes (negative control probe). Samples are in vitro transcribed 364 nt sense or antisense RNA fragments of WISP1-AS1. Each biotinylated probe hybridizes only to its complementary fragment, confirming specificity regarding orientation. C) Northern Blot with the probe directed against WISP1-AS1. Numbers on the left = number of bases. D) Northern Blot with the probe directed against the complementary sequence of WISP1-AS1. E) Induction of WISP1-AS1 by OTA (100 nmol/l over 48h, n=3) in cell lines derived from renal clear cell carcinomas. *=p<0.05 versus respective control (PDF 1051 kb)
18_2017_2731_MOESM2_ESM.pdf
Supplementary file 5: WISP1-AS1-dependent downregulated mRNAs in HEK-293T cells after 48 hours exposure to 100 nM OTA (PDF 947 kb)
18_2017_2731_MOESM3_ESM.pdf
Supplementary file 6: WISP1-AS1-dependent upregulated mRNAs in HEK-293T cells after 48 hours exposure to 100 nM OTA (PDF 50 kb)
18_2017_2731_MOESM4_ESM.xlsx
Supplementary file 7: Enrichment analysis for WISP1-AS1-dependent upregulated mRNAs (FPM > 10 for controls; FC>1.5) (XLSX 83 kb)
18_2017_2731_MOESM5_ESM.xlsx
Supplementary file 8: Enrichment analysis for WISP1-AS1-dependent downregulated mRNAs (FPM > 10 for OTA-treated samples; FC<0.66) (XLSX 101 kb)
18_2017_2731_MOESM6_ESM.pdf
Supplementary file 9: WISP1-AS1-dependent upregulation of GAS6 mRNA in HEK-293T cells after 48 hours exposure to 100 nM OTA. N=4. *=p<0.05 versus control (PDF 35 kb)
Rights and permissions
About this article
Cite this article
Polovic, M., Dittmar, S., Hennemeier, I. et al. Identification of a novel lncRNA induced by the nephrotoxin ochratoxin A and expressed in human renal tumor tissue. Cell. Mol. Life Sci. 75, 2241–2256 (2018). https://doi.org/10.1007/s00018-017-2731-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00018-017-2731-6