The Roles Of IDH2 And ITGA6 And Their Underlying Mechanisms In Multiple Myeloma

Part 1 IDH2 contributes to tumorigenesis by regulating RNA m6A methylation in multiple myelomaObjective:This study aims to explore the role of IDH2-mediated m6A methylation in multiple myeloma and the molecular mechanisms it exerts this function.Methods:The GSEA(Gene Set Enrichment Analysis)analysis was performed with differentially expressed genes between healthy subjects and myeloma patients.The differential genes were screened in GEO four databases.Survival analysis was performed to evaluate the value of the genes.In vitro,the cell proliferation,cell cycle distribution and apoptosis were detected.The role of IDH2 in vivo was evaluated in orthotopic MM xenografts.Subsequently,the effect of IDH2 on the global m6A methylation level,5-mC methylation level and histone methylation level,the activity of RNA demethylases and DNA demethylases were detected.CCK-8 assay was performed to detect the cell proliferation ability in down-regulated the DNA demethylases or FTO cells.The genes related to FTO were analyzed by KOBAS(KEGG Ortho logy Based Annotation System).The methylation level and expression of WNT7B were detected respectively in cells knockdown FTO.The dual luciferase reporter assay was performed to verify the combination of FTO and WNT7B.The expression of WNT7B was determined by qPCR and the stability of WNT7B was measured by α-Amanita experiment.Dual luciferase reporter assay was performed with WNT7B 3’untranslated region wild-type or mutant sites.The effect of WNT7B on WNT signaling pathway was detected.Results:GSEA indicated the differential genes between healthy donors and myeloma patients were mainly enriched in tricarboxylic acid cycle pathway.The result showed that three genes were concordantly increased in the four databases(>1.5 fold).Among them,IDH2 expression was significantly increased in the progression of plasma cell dyscrasias.Survival analysis showed IDH2 was related to prognostic of myeloma patients.Depletion of IDH2 reduced cell proliferation,arrested cell cycle in G1 phase,and significantly increased apoptosis.In vivo,downregulated IDH2 MM cells in mice led to a significant reduction in tumor volume.Down-regulating of IDH2,the global m6A RNA level was reduced in both cell lines,but the DNA methylation level changed inconsistently in the two cell lines,while the histone methylation level did not change significantly.The activity of RNA demethylases and DNA demethylases decreased.DNA demethylases increased the cell proliferation capacity.After down regulating FTO,the proliferation ability of cells decreased.The genes related to FTO were enriched in WNT signaling pathway.Among them,WNT7B was significantly positively correlated with FTO.The m6A level of WNT7B was significantly upregulated,while the expression of WNT7B was downregulated in cells suppressed of FTO.qPCR demonstrated that the expression and half-live of WNT7B were increased in YTHDF2-deficient cells.The dual luciferase experiment proved that YTHDF2 could bind to WNT7B.Suppression of WNT7B,the activity of the WNT pathway was reduced.Conclusion:IDH2 regulated global m6A RNA modification in myeloma cells via targeting RNA demethylase FTO.Down-regulation of IDH2 increased m6A RNA methylation levels,inhibited cell proliferation,arrested cell cycle,and increased apoptosis in myeloma.The imbalance of m6A methylation enhanced the expression of WNT7B,activated the WNT signaling pathway,and promoted the development of myeloma.Part 2 ITGA6-AS1 mediates cell invasion by regulating ITGA6 in multiple myelomaObjective:The purpose of this study is to find the genes and the underlying molecular mechanisms during the progression of multiple myeloma(MM)into plasma cell leukemia(PCL),and to reveal the regulatory mechanism of long non-coding RNA.Methods:The GSEA analysis was performed with differentially expressed genes between MM and PCL patients in two datasets(GSE2113 and GSE39925).The univariate and multivariate survival analysis were carried out to validate whether the changed genes were related to the prognosis of MM.AUC(Area Under the Curve)analysis was performed to assess ISS and ISS+ ITGA6 models.The cell proliferation and invasion ability were detected in MM cells overexpressed of ITGA6.CD138+cells were extracted from 30 MM patients,and the expression levels of ITGA6 and ITGA6-AS1 were detected by qPCR.The location of ITGA6-AS1 was analyzed by cell nucleus/cytoplasm fraction isolation experiment.Northern blot was performed to detect the ITGA6-AS1 expression in MM cells.The coding potential was predicted by bio informatics analysis.The expression levels of ITGA6 pre-mRNA and ITGA6 mRNA were detected by qPCR,and the protein level of ITGA6 was detected by western blot in myeloma cells suppressed ITGA6-AS1.The effect of ITGA6-AS1 on the stability of ITGA6 pre-mRNA was detected by nuclease protection experiment and RNA stability experiment.The effect of ITGA6AS1 on the invasive ability was detected by transwell assay.To evaluate that ITGA6-AS1 contributed to the invasion of myeloma cells by regulating ITGA6,ITGA6-AS1 was upregulated while ITGA 6 was down-regulated to compare whether the invasive ability could be recovered.Results:GSEA indicated that gene signatures for the positive regulation of cell metastasis were significantly enriched in PCL samples.Both datasets identified the eight genes which were differential expressed in PCL versus MM.ITGA6 expression was significantly reduced by the progression of PCL.Survival analysis suggests that ITGA6 could be used as an independent prognostic factor for myeloma,which helps to improve the efficacy of the ISS prognosis model.The overexpression of ITGA6 greatly reduced the invasion of myeloma cells,but had no effect on cell proliferation.ITGA6-AS1 expression was positively correlated with the expression of ITGA6 in CD138+cells.ITGA6-AS1 was located in both the cytoplasm and the nucleus by qPCR.Northern blot assay demonstrated the presence of ITGA6-AS1 in myeloma cells.Furthermore,the result of bio informatics analysis displayed that ITGA6-AS1 had no coding potentiality.The upregulation of ITGA6-AS1 led to significant increases in ITGA6 pre-mRNA and ITGA6 at both the mRNA and protein levels.ITGA6-AS1 binded to ITGA6 pre-mRNA and formed an RNA duplex,thereby enhancing the stability of ITGA6 pre-mRNA.The overexpression of ITGA6-AS1 dramatically downregulated the invasion of myeloma cells compared to that of control cells.Moreover,the downregulation of ITGA6 in ITGA6-AS1 overexpressed cells significantly abrogated the impeded invasion of myeloma cells.Conclusion:ITGA6 acts as an independent prognostic factor of myeloma and regulates the invasion ability of myeloma cells.ITGA6-AS1 affected invasion by regulating ITGA6 expression in myeloma cells.ITGA 6-AS1 and ITGA6 could be used as markers to predict the progression of myeloma to plasma cell leukemia.