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miR-101 is dysregulated in malignant melanoma and targets MITF
New Biotechnology · Volume 27S · April 2010
POSTERS ABSTRACTS 1
liferation, migration and apoptosis. Nevertheless, the knowledge of molecular mechanism of miR-21 function in promoting cancer development is limited. Although multiple miRNA targets have been predicted by bioinformatics approaches, only few miRNA targets have been experimentally verified so far. In the present study, we performed two-dimensional differential in-gel electrophoresis (2-DIGE) of LNCaP prostate cancer cells treated with miR-21 or negative control and found 24 differentially expressed proteins. Validation of candidate targets showed the regulation of a tumor suppressor by miR-21. We detected the regulation of the identified protein in various cell lines using miR-21 precursor as well as antimiR-21 oligonucleotides by Western Blot analyses. Overexpression of miR-21 in LNCaP resulted in enhanced cell vitality. Knockdown of the identified tumor suppressor by siRNA mimics the proliferative effect of miR-21 to a comparable extent. These results suggest that the tumor suppressor identified by 2-DIGE is an important mediator of the biological effects of miR-21 in prostate cancer cells. doi:10.1016/j.nbt.2010.01.042
[P1.36] Genome-wide expression profile associated with renal aging S.J. Noppert 1,3,∗ , M. Rudnicki 1,3 , J. Muehlberger 2,3 , G. Mayer 1,3
Enrich 1,3 , P.
Perco 2,3 , I.
1
Medical University Innsbruck, Austria Emergentec Biodevelopment GmbH, Austria 3 Medical University of Vienna, Austria 2
Kidney transplantation can successfully restore quality of life to patients with end-stage renal disease, however, the number of patients requiring a kidney transplant exceeds the available organs. Currently kidneys from older donors may be excluded from transplantation, even though chronological age does not always reflect organ viability. Genomic profiling of donor kidneys may provide insight into the biological age of a donor organ and potentially expand the number of kidneys available for transplantation. 77 zero-hour renal transplant biopsies were obtained from collection centres in Austria and Germany. RNA was extracted from these samples and hybridized to Agilent Whole Human Genome (4x44K) microarrays. Biopsies were classified into five age groups roughly corresponding to decades and significant differences in gene expression determined by ANOVA and SAM statistical analysis as well as correlation between age and expression of normalized data. We identified 349 genes with altered expression associated with age, representing biological processes including immunity, apoptosis, cell structure and motility and stress response. Three predominant patterns of gene expression were evident: (1) increasing expression across the age groups, (2) genes decreasing with age and (3) increasing with age but reduced expression in donors over 70 years. Group 3 was dominated by metallothionein family members. Identification of age-regulated genes may enable assessment of biological age of a donor kidney independent of chronological age, hence providing a measure of functional capacity. The expression
profile of a panel of genes identified in this study may provide a useful measure of the fitness of a donor organ for transplant. Of particular interest is the metallothionein genes that increase expression with age, however, show reduced expression in kidneys of donors aged over 70 years, who may be considered successful agers. This study has identified candidate genes whose expression can assess the biological age of a donor kidney. doi:10.1016/j.nbt.2010.01.043
[P1.37] miR-101 is dysregulated in malignant melanoma and targets MITF C. Luo ∗ , P. Merz, D. Schadendorf, S.B. Eichmüller German Cancer Research Center, Germany
Malignant melanoma is the most aggressive form of skin cancer in human. Present treatment modalities are unsatisfactory and the five year survival rates are below 15% for patients with distant metastasis. MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression. They participate in the regulation of almost every cellular process investigated in cancer. In this study, we performed miRNA expression profiling for normal human epithelial melanocytes (NHEMs) and melanoma cell lines of patients with different survival times from the first diagnosis of stage IV melanoma, using Illumina beadbased chips. A number of miRNAs were found to be differentially expressed between NHEM and melanoma, as well as between short survival and long survival patients. Expression of candidate miRNAs was validated by Taqman miRNA assay. Among them, miR-101 was up-regulated in melanoma cell lines compared to NHEMs. Expression of miR-101 was constantly low in NHEMs but variable in melanoma cell lines. Interestingly, miR-101 was also upregulated in melanoma cell lines derived from the long survival patients compared to the short survival patients. We confirmed that MITF (microphthalmia-associated transcription factor), the key regulator of melanoma, is a direct target of hsa-miR-101 by luciferase reporter assay. Sixty-five to eighty-eight percent reduction of luciferase activity was observed after co-transfection of miR-101 overexpression plasmid and MITF-3 UTR reporter plasmid into HEK293T cells or melanoma cells. MITF is known as a key regulator both in NHEM and melanoma, and its protein expression has to be carefully controlled. Our results suggest that miR-101 may play an important role in the control of MITF and thus might have a strong impact on melanoma development and aggressiveness. doi:10.1016/j.nbt.2010.01.044
www.elsevier.com/locate/nbt S35
POSTERS ABSTRACTS 1
liferation, migration and apoptosis. Nevertheless, the knowledge of molecular mechanism of miR-21 function in promoting cancer development is limited. Although multiple miRNA targets have been predicted by bioinformatics approaches, only few miRNA targets have been experimentally verified so far. In the present study, we performed two-dimensional differential in-gel electrophoresis (2-DIGE) of LNCaP prostate cancer cells treated with miR-21 or negative control and found 24 differentially expressed proteins. Validation of candidate targets showed the regulation of a tumor suppressor by miR-21. We detected the regulation of the identified protein in various cell lines using miR-21 precursor as well as antimiR-21 oligonucleotides by Western Blot analyses. Overexpression of miR-21 in LNCaP resulted in enhanced cell vitality. Knockdown of the identified tumor suppressor by siRNA mimics the proliferative effect of miR-21 to a comparable extent. These results suggest that the tumor suppressor identified by 2-DIGE is an important mediator of the biological effects of miR-21 in prostate cancer cells. doi:10.1016/j.nbt.2010.01.042
[P1.36] Genome-wide expression profile associated with renal aging S.J. Noppert 1,3,∗ , M. Rudnicki 1,3 , J. Muehlberger 2,3 , G. Mayer 1,3
Enrich 1,3 , P.
Perco 2,3 , I.
1
Medical University Innsbruck, Austria Emergentec Biodevelopment GmbH, Austria 3 Medical University of Vienna, Austria 2
Kidney transplantation can successfully restore quality of life to patients with end-stage renal disease, however, the number of patients requiring a kidney transplant exceeds the available organs. Currently kidneys from older donors may be excluded from transplantation, even though chronological age does not always reflect organ viability. Genomic profiling of donor kidneys may provide insight into the biological age of a donor organ and potentially expand the number of kidneys available for transplantation. 77 zero-hour renal transplant biopsies were obtained from collection centres in Austria and Germany. RNA was extracted from these samples and hybridized to Agilent Whole Human Genome (4x44K) microarrays. Biopsies were classified into five age groups roughly corresponding to decades and significant differences in gene expression determined by ANOVA and SAM statistical analysis as well as correlation between age and expression of normalized data. We identified 349 genes with altered expression associated with age, representing biological processes including immunity, apoptosis, cell structure and motility and stress response. Three predominant patterns of gene expression were evident: (1) increasing expression across the age groups, (2) genes decreasing with age and (3) increasing with age but reduced expression in donors over 70 years. Group 3 was dominated by metallothionein family members. Identification of age-regulated genes may enable assessment of biological age of a donor kidney independent of chronological age, hence providing a measure of functional capacity. The expression
profile of a panel of genes identified in this study may provide a useful measure of the fitness of a donor organ for transplant. Of particular interest is the metallothionein genes that increase expression with age, however, show reduced expression in kidneys of donors aged over 70 years, who may be considered successful agers. This study has identified candidate genes whose expression can assess the biological age of a donor kidney. doi:10.1016/j.nbt.2010.01.043
[P1.37] miR-101 is dysregulated in malignant melanoma and targets MITF C. Luo ∗ , P. Merz, D. Schadendorf, S.B. Eichmüller German Cancer Research Center, Germany
Malignant melanoma is the most aggressive form of skin cancer in human. Present treatment modalities are unsatisfactory and the five year survival rates are below 15% for patients with distant metastasis. MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression. They participate in the regulation of almost every cellular process investigated in cancer. In this study, we performed miRNA expression profiling for normal human epithelial melanocytes (NHEMs) and melanoma cell lines of patients with different survival times from the first diagnosis of stage IV melanoma, using Illumina beadbased chips. A number of miRNAs were found to be differentially expressed between NHEM and melanoma, as well as between short survival and long survival patients. Expression of candidate miRNAs was validated by Taqman miRNA assay. Among them, miR-101 was up-regulated in melanoma cell lines compared to NHEMs. Expression of miR-101 was constantly low in NHEMs but variable in melanoma cell lines. Interestingly, miR-101 was also upregulated in melanoma cell lines derived from the long survival patients compared to the short survival patients. We confirmed that MITF (microphthalmia-associated transcription factor), the key regulator of melanoma, is a direct target of hsa-miR-101 by luciferase reporter assay. Sixty-five to eighty-eight percent reduction of luciferase activity was observed after co-transfection of miR-101 overexpression plasmid and MITF-3 UTR reporter plasmid into HEK293T cells or melanoma cells. MITF is known as a key regulator both in NHEM and melanoma, and its protein expression has to be carefully controlled. Our results suggest that miR-101 may play an important role in the control of MITF and thus might have a strong impact on melanoma development and aggressiveness. doi:10.1016/j.nbt.2010.01.044
www.elsevier.com/locate/nbt S35