ANALYSIS OF SNORNA EXPRESSION AND FUNCTION IN ACUTE MYELOID LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Gerloff D. 06/09/16; 132427; E878
Dr. Dennis Gerloff
Contributions
Contributions
Abstract
Abstract: E878
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) belong to the group of non-protein-coding RNAs with distinct functions in ribosomal RNA (rRNA) modification. snoRNAs are divided into the two major classes: box C/D and box H/ACA snoRNAs. SnoRNAs assembly with small nucleolar ribonucleoproteins (RNPs) and guide those snoRNPs to the rRNA, where they catalyze rRNA modifications. C/D box snoRNAs are involved in 2-O-methylation, whereas H/ACA snoRNAs guide pseudouridylation. Recent studies showed that snoRNAs play a role in cancerogenesis. Hence, we analyzed snoRNAs expression and function in leukemogenesis.
Aims
Investigation of snoRNA expression pattern and function in acute myeloid leukemia.
Methods
To understand the role of snoRNAs in leukemia, we analyzed the snoRNA expression pattern of 63 AML patients at time of diagnosis. Additionally we analyzed snoRNA expression patterns in AML1-ETO or Myc transduced lin- mouse bone marrow cells. Librarys for small RNA (40-200 nt) specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. For functional analysis of single snoRNAs, we performed single Knockouts of 6 snoRNAs (SNORD14D, SNORD34, SNORD35A, SNORD43, SNORD53 and SNORD104) in Kasumi and MV4-11 cells using CRISPR/Cas9 technology. Mutations in the genomic sequence were confirmed by Sanger sequencing. Subsequently, expression of snoRNAs was analyzed by qRT-PCR. To analyze the effect of snoRNA knockout on colony formation ability of mutated cells, we embedded 300 cells/well in methylcellulose. Colonies were counted after 7-10 days.
Results
Next generation sequencing data revealed 229 C/D box snoRNAs and 90 H/ACA box snoRNAs were expressed in one or more patient samples. In further analyses, we compared clinical data and snoRNA expression pattern. SnoRNA expression patterns were associated with specific risk groups. High levels of snoRNA expression associated with intermediate molecular risk (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Further, we could show that snoRNAs are higher expressed in AML patient samples with NPM1 wildtype (124 snoRNAs, p≤0.05). Transduction of the oncogenes AML1-ETO9a or Myc to normal lin- mouse bone marrow cells induced an increased overall snoRNA expression. We found 93 snoRNAs downregulated and 88 upregulated during ATRA induced differentiation of HL60 cells. The functionally analyzed snoRNAs SNORD14D, SNORD34, SNORD35A, SNORD43 and SNORD53 were downregulated. CRISPR/Cas9 induced knockouts for SNORD14D, SNORD34, SNORD35A or SNORD43 significantly inhibited colony formation of Kasumi-1 cells. Also, loss of SNORD14D or SNORD35A decreased colony formation potential of MV4;11 cells.Loss of SNORD34, SNORD35A, SNORD43 and SNORD104 resulted in a significant reduction of 2´-O-methylation on the respective rRNA modification sides (28S U2824, 28S C4506, 18S C1703 and 28S C1327).
Conclusion
SnoRNA expression profiles are altered in specific AML subtypes. Further, we identified a functional relevance of snoRNAs in cell growth of leukemic cells.
Session topic: E-poster
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) belong to the group of non-protein-coding RNAs with distinct functions in ribosomal RNA (rRNA) modification. snoRNAs are divided into the two major classes: box C/D and box H/ACA snoRNAs. SnoRNAs assembly with small nucleolar ribonucleoproteins (RNPs) and guide those snoRNPs to the rRNA, where they catalyze rRNA modifications. C/D box snoRNAs are involved in 2-O-methylation, whereas H/ACA snoRNAs guide pseudouridylation. Recent studies showed that snoRNAs play a role in cancerogenesis. Hence, we analyzed snoRNAs expression and function in leukemogenesis.
Aims
Investigation of snoRNA expression pattern and function in acute myeloid leukemia.
Methods
To understand the role of snoRNAs in leukemia, we analyzed the snoRNA expression pattern of 63 AML patients at time of diagnosis. Additionally we analyzed snoRNA expression patterns in AML1-ETO or Myc transduced lin- mouse bone marrow cells. Librarys for small RNA (40-200 nt) specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. For functional analysis of single snoRNAs, we performed single Knockouts of 6 snoRNAs (SNORD14D, SNORD34, SNORD35A, SNORD43, SNORD53 and SNORD104) in Kasumi and MV4-11 cells using CRISPR/Cas9 technology. Mutations in the genomic sequence were confirmed by Sanger sequencing. Subsequently, expression of snoRNAs was analyzed by qRT-PCR. To analyze the effect of snoRNA knockout on colony formation ability of mutated cells, we embedded 300 cells/well in methylcellulose. Colonies were counted after 7-10 days.
Results
Next generation sequencing data revealed 229 C/D box snoRNAs and 90 H/ACA box snoRNAs were expressed in one or more patient samples. In further analyses, we compared clinical data and snoRNA expression pattern. SnoRNA expression patterns were associated with specific risk groups. High levels of snoRNA expression associated with intermediate molecular risk (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Further, we could show that snoRNAs are higher expressed in AML patient samples with NPM1 wildtype (124 snoRNAs, p≤0.05). Transduction of the oncogenes AML1-ETO9a or Myc to normal lin- mouse bone marrow cells induced an increased overall snoRNA expression. We found 93 snoRNAs downregulated and 88 upregulated during ATRA induced differentiation of HL60 cells. The functionally analyzed snoRNAs SNORD14D, SNORD34, SNORD35A, SNORD43 and SNORD53 were downregulated. CRISPR/Cas9 induced knockouts for SNORD14D, SNORD34, SNORD35A or SNORD43 significantly inhibited colony formation of Kasumi-1 cells. Also, loss of SNORD14D or SNORD35A decreased colony formation potential of MV4;11 cells.Loss of SNORD34, SNORD35A, SNORD43 and SNORD104 resulted in a significant reduction of 2´-O-methylation on the respective rRNA modification sides (28S U2824, 28S C4506, 18S C1703 and 28S C1327).
Conclusion
SnoRNA expression profiles are altered in specific AML subtypes. Further, we identified a functional relevance of snoRNAs in cell growth of leukemic cells.
Session topic: E-poster
Abstract: E878
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) belong to the group of non-protein-coding RNAs with distinct functions in ribosomal RNA (rRNA) modification. snoRNAs are divided into the two major classes: box C/D and box H/ACA snoRNAs. SnoRNAs assembly with small nucleolar ribonucleoproteins (RNPs) and guide those snoRNPs to the rRNA, where they catalyze rRNA modifications. C/D box snoRNAs are involved in 2-O-methylation, whereas H/ACA snoRNAs guide pseudouridylation. Recent studies showed that snoRNAs play a role in cancerogenesis. Hence, we analyzed snoRNAs expression and function in leukemogenesis.
Aims
Investigation of snoRNA expression pattern and function in acute myeloid leukemia.
Methods
To understand the role of snoRNAs in leukemia, we analyzed the snoRNA expression pattern of 63 AML patients at time of diagnosis. Additionally we analyzed snoRNA expression patterns in AML1-ETO or Myc transduced lin- mouse bone marrow cells. Librarys for small RNA (40-200 nt) specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. For functional analysis of single snoRNAs, we performed single Knockouts of 6 snoRNAs (SNORD14D, SNORD34, SNORD35A, SNORD43, SNORD53 and SNORD104) in Kasumi and MV4-11 cells using CRISPR/Cas9 technology. Mutations in the genomic sequence were confirmed by Sanger sequencing. Subsequently, expression of snoRNAs was analyzed by qRT-PCR. To analyze the effect of snoRNA knockout on colony formation ability of mutated cells, we embedded 300 cells/well in methylcellulose. Colonies were counted after 7-10 days.
Results
Next generation sequencing data revealed 229 C/D box snoRNAs and 90 H/ACA box snoRNAs were expressed in one or more patient samples. In further analyses, we compared clinical data and snoRNA expression pattern. SnoRNA expression patterns were associated with specific risk groups. High levels of snoRNA expression associated with intermediate molecular risk (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Further, we could show that snoRNAs are higher expressed in AML patient samples with NPM1 wildtype (124 snoRNAs, p≤0.05). Transduction of the oncogenes AML1-ETO9a or Myc to normal lin- mouse bone marrow cells induced an increased overall snoRNA expression. We found 93 snoRNAs downregulated and 88 upregulated during ATRA induced differentiation of HL60 cells. The functionally analyzed snoRNAs SNORD14D, SNORD34, SNORD35A, SNORD43 and SNORD53 were downregulated. CRISPR/Cas9 induced knockouts for SNORD14D, SNORD34, SNORD35A or SNORD43 significantly inhibited colony formation of Kasumi-1 cells. Also, loss of SNORD14D or SNORD35A decreased colony formation potential of MV4;11 cells.Loss of SNORD34, SNORD35A, SNORD43 and SNORD104 resulted in a significant reduction of 2´-O-methylation on the respective rRNA modification sides (28S U2824, 28S C4506, 18S C1703 and 28S C1327).
Conclusion
SnoRNA expression profiles are altered in specific AML subtypes. Further, we identified a functional relevance of snoRNAs in cell growth of leukemic cells.
Session topic: E-poster
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) belong to the group of non-protein-coding RNAs with distinct functions in ribosomal RNA (rRNA) modification. snoRNAs are divided into the two major classes: box C/D and box H/ACA snoRNAs. SnoRNAs assembly with small nucleolar ribonucleoproteins (RNPs) and guide those snoRNPs to the rRNA, where they catalyze rRNA modifications. C/D box snoRNAs are involved in 2-O-methylation, whereas H/ACA snoRNAs guide pseudouridylation. Recent studies showed that snoRNAs play a role in cancerogenesis. Hence, we analyzed snoRNAs expression and function in leukemogenesis.
Aims
Investigation of snoRNA expression pattern and function in acute myeloid leukemia.
Methods
To understand the role of snoRNAs in leukemia, we analyzed the snoRNA expression pattern of 63 AML patients at time of diagnosis. Additionally we analyzed snoRNA expression patterns in AML1-ETO or Myc transduced lin- mouse bone marrow cells. Librarys for small RNA (40-200 nt) specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. For functional analysis of single snoRNAs, we performed single Knockouts of 6 snoRNAs (SNORD14D, SNORD34, SNORD35A, SNORD43, SNORD53 and SNORD104) in Kasumi and MV4-11 cells using CRISPR/Cas9 technology. Mutations in the genomic sequence were confirmed by Sanger sequencing. Subsequently, expression of snoRNAs was analyzed by qRT-PCR. To analyze the effect of snoRNA knockout on colony formation ability of mutated cells, we embedded 300 cells/well in methylcellulose. Colonies were counted after 7-10 days.
Results
Next generation sequencing data revealed 229 C/D box snoRNAs and 90 H/ACA box snoRNAs were expressed in one or more patient samples. In further analyses, we compared clinical data and snoRNA expression pattern. SnoRNA expression patterns were associated with specific risk groups. High levels of snoRNA expression associated with intermediate molecular risk (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Further, we could show that snoRNAs are higher expressed in AML patient samples with NPM1 wildtype (124 snoRNAs, p≤0.05). Transduction of the oncogenes AML1-ETO9a or Myc to normal lin- mouse bone marrow cells induced an increased overall snoRNA expression. We found 93 snoRNAs downregulated and 88 upregulated during ATRA induced differentiation of HL60 cells. The functionally analyzed snoRNAs SNORD14D, SNORD34, SNORD35A, SNORD43 and SNORD53 were downregulated. CRISPR/Cas9 induced knockouts for SNORD14D, SNORD34, SNORD35A or SNORD43 significantly inhibited colony formation of Kasumi-1 cells. Also, loss of SNORD14D or SNORD35A decreased colony formation potential of MV4;11 cells.Loss of SNORD34, SNORD35A, SNORD43 and SNORD104 resulted in a significant reduction of 2´-O-methylation on the respective rRNA modification sides (28S U2824, 28S C4506, 18S C1703 and 28S C1327).
Conclusion
SnoRNA expression profiles are altered in specific AML subtypes. Further, we identified a functional relevance of snoRNAs in cell growth of leukemic cells.
Session topic: E-poster
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