Contributions
Abstract: EP951
Type: E-Poster Presentation
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
Our previous results indicated that the demethylase of RNA N-6 methylation (m6A) modification, ALKBH5, played an oncogenic role in multiple myeloma via modulating HIPPO pathway(the results have not yet been published). While latest researches have also showed m6A modification on non-coding RNAs. Besides, the interaction between m6A RNA and histone modifications have arouse the researchers’ interest. Here we find that ALKBH5 demethylases the long non-coding RNA (lncRNA)-small nucleolar RNA host gene 15 (SNHG15) and positively regulates its expression. Further experiments also disclose that ALKBH5 may alter the H3K36me3 level and promotes the pathogenesis of multiple myeloma through regulating lncRNA SNHG15 m6A and RNA expression.
Aims
We aim to further display the potential epigenetic mechanisms between ALKBH5, ncRNA and histone modification involved in the progression of MM
Methods
The expression of SNHG15 and SETD2 were detected by RT-qPCR and western blot. ALKBH5-siRNA lentivirus and NC were transfected into MM cells to knockdown ALKBH5 gene. SNHG15-siRNA lentivirus, SNHG15-cNDA lentivirus and NC were transfected into MM cells to knockdown and knockin SNHG15 gene. MeRIP-seq was previously accomplished (the results have not yet been published). CCK8 assays were performed to determine the proliferation of myeloma cells. Flow cytometry assays were performed to determine the apoptosis of myeloma cells. Transwell assays were performed to determine the migration of myeloma cells.
Results
1. MeRIP-qPCR reveals that the m6A level of lncRNA SNHG15 is increased after ALKBH5 knockdown. RT-qPCR finds that the expression of lncRNA SNHG15 is increased and decreased after ALKBH5 knockin and knockdown. 2. Through overexpressing lncRNA SNHG15 in MM PMI8226 ALKBH5- cells, we established the in vitro compensated model (marked control, ALKBH5- and ALKBH5-SNHG15+ group). CCK8 assays show that lncRNA SNHG15 rescues the inhibited cell proliferation caused by ALKBH5 knockdown. Transwell assays display that lncRNA SNHG15 compensates the suppressed cell migration caused by ALKBH5 knockdown. And flow cytometry assays show that the rate of cell apoptosis in ALKBH5-SNHG15+ group RPMI8226 cells is lower in ALKBH5- group RPMI8226 cells. 3. RT-qPCR shows that lncRNA SNHG15 is upregulated in primary MM cells (cells from the bone marrow of MM patients sorted by CD138 antibody beads) and MM cell lines (RPMI8226, NCL- H929,U266, MM1R, MM1S and ARH-77 cells). After downregulation of lncRNA SNHG15 by SNHG15-siRNA lentivirus in RPMI8226 cells, the proliferation of SNHG15- group PMI8226 cells is decreased. 4. Western blot analysis reveals the expression changes of histone H3K36me3 methyltransferase SETD2 among control, ALKBH5-, ALKBH5-SNHG15+ group MM RPMI8226 cells.
Conclusion
The RNA m6A demethylase ALKBH5 acts on lncRNA SNHG15 in multiple myeloma and positively regulates its expression. The overexpression of SNHG15 in ALKBH5- cells rescued the inhibited cell proliferation, the suppressed cell migration, and the accelerated cell apoptosis caused by ALKBH5 knockdown. LncRNA SNHG15 was highly expressed in CD138 + plasma cells from MM Patients and MM cell lines. LncRNA SNHG15 positively regulates MM cells proliferation. ALKBH5 regulates histone modification through lncRNA SNHG15.
Keyword(s): Multiple myeloma
Abstract: EP951
Type: E-Poster Presentation
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
Our previous results indicated that the demethylase of RNA N-6 methylation (m6A) modification, ALKBH5, played an oncogenic role in multiple myeloma via modulating HIPPO pathway(the results have not yet been published). While latest researches have also showed m6A modification on non-coding RNAs. Besides, the interaction between m6A RNA and histone modifications have arouse the researchers’ interest. Here we find that ALKBH5 demethylases the long non-coding RNA (lncRNA)-small nucleolar RNA host gene 15 (SNHG15) and positively regulates its expression. Further experiments also disclose that ALKBH5 may alter the H3K36me3 level and promotes the pathogenesis of multiple myeloma through regulating lncRNA SNHG15 m6A and RNA expression.
Aims
We aim to further display the potential epigenetic mechanisms between ALKBH5, ncRNA and histone modification involved in the progression of MM
Methods
The expression of SNHG15 and SETD2 were detected by RT-qPCR and western blot. ALKBH5-siRNA lentivirus and NC were transfected into MM cells to knockdown ALKBH5 gene. SNHG15-siRNA lentivirus, SNHG15-cNDA lentivirus and NC were transfected into MM cells to knockdown and knockin SNHG15 gene. MeRIP-seq was previously accomplished (the results have not yet been published). CCK8 assays were performed to determine the proliferation of myeloma cells. Flow cytometry assays were performed to determine the apoptosis of myeloma cells. Transwell assays were performed to determine the migration of myeloma cells.
Results
1. MeRIP-qPCR reveals that the m6A level of lncRNA SNHG15 is increased after ALKBH5 knockdown. RT-qPCR finds that the expression of lncRNA SNHG15 is increased and decreased after ALKBH5 knockin and knockdown. 2. Through overexpressing lncRNA SNHG15 in MM PMI8226 ALKBH5- cells, we established the in vitro compensated model (marked control, ALKBH5- and ALKBH5-SNHG15+ group). CCK8 assays show that lncRNA SNHG15 rescues the inhibited cell proliferation caused by ALKBH5 knockdown. Transwell assays display that lncRNA SNHG15 compensates the suppressed cell migration caused by ALKBH5 knockdown. And flow cytometry assays show that the rate of cell apoptosis in ALKBH5-SNHG15+ group RPMI8226 cells is lower in ALKBH5- group RPMI8226 cells. 3. RT-qPCR shows that lncRNA SNHG15 is upregulated in primary MM cells (cells from the bone marrow of MM patients sorted by CD138 antibody beads) and MM cell lines (RPMI8226, NCL- H929,U266, MM1R, MM1S and ARH-77 cells). After downregulation of lncRNA SNHG15 by SNHG15-siRNA lentivirus in RPMI8226 cells, the proliferation of SNHG15- group PMI8226 cells is decreased. 4. Western blot analysis reveals the expression changes of histone H3K36me3 methyltransferase SETD2 among control, ALKBH5-, ALKBH5-SNHG15+ group MM RPMI8226 cells.
Conclusion
The RNA m6A demethylase ALKBH5 acts on lncRNA SNHG15 in multiple myeloma and positively regulates its expression. The overexpression of SNHG15 in ALKBH5- cells rescued the inhibited cell proliferation, the suppressed cell migration, and the accelerated cell apoptosis caused by ALKBH5 knockdown. LncRNA SNHG15 was highly expressed in CD138 + plasma cells from MM Patients and MM cell lines. LncRNA SNHG15 positively regulates MM cells proliferation. ALKBH5 regulates histone modification through lncRNA SNHG15.
Keyword(s): Multiple myeloma