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Identification of a novel lncRNA induced by the nephrotoxin ochratoxin A and expressed in human renal tumor tissue

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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.

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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).

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Authors and Affiliations

  1. Julius Bernstein Institute of Physiology, Martin Luther University Halle-Wittenberg, Magdeburgerstrasse 6, 06112, Halle (Saale), Germany

    Mirjana Polovic, Sandro Dittmar, Isabell Hennemeier, Patrick Azinovic, Alexander Nolze, Marcel Köhn, Gerald Schwerdt & Michael Gekle

  2. Institute of Food Chemistry, Westphalian Wilhelm University Muenster, Münster, Germany

    Hans-Ulrich Humpf

  3. Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle, Germany

    Barbara Seliger

  4. Clinic of Urology, University Hospital, Halle, Germany

    Paolo Fornara & Gerit Theil

  5. Institute of Molecular Medicine, Martin Luther University Halle-Wittenberg, Halle, Germany

    Marcel Köhn

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  1. Mirjana Polovic
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Correspondence to Michael Gekle.

Electronic supplementary material

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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)

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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

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