The Study Of ALKBH5 Promoting Pathogenesis Of Multiple Myeloma By Regulating The Expression Of LncRNA SNHG15

Objective:1.To verify the expression level of ALKBH5 in CD 138+cells from multiple myeloma(MM)patients and MM cell lines;2.To investigate whether ALKBH5 regulates the expression level of lncRNA SNHG15;3.To study whether ALKBH5 modulates the biological functions of multiple myeloma through regulating the expression of lncRNA SNHG15.Methods:1.Clinical experimentsThe primary MM cells were collected from the bone marrow of 10 MM patients,and the magnetic beads tagged with CD138 were used to sort CD138+MM cells.Meanwhile,the Normal control cell were collected from the bone marrow of 5 healthy donors,and the magnetic beads tagged with CD 13 8 were used to sort CD 138+primary cells.The mRNA expression levels of lncRNA SNHG15 and ALKBH5,as well as the protein expression levels of ALKBH5 in these samples were detected by RT-qPCR and Western Blot assay.The correlation between ALKBH5 mRNA and lncRNA SNHG15 was compared by linear regression analysis.2.In vitro experiments(1)The mRNA and protein expression levels of ALKBH5 in multiple myeloma cell lines(NCI-H929、RPMI-8226、ARH-77、U266)were detected by RT-qPCR and Western Blot assay.(2)LV-ALKBH5-RNAi,LV-ALKBH5-OE and their control lentivirus were designed and synthesized in vitro,and then they were transfected into MM RPMI-8226 cell line("8226" for short)to down-regulate or overexpress the expression level of ALKBH5,marked as ALKBH5-group and control group,ALKBH5+and control group.(3)The lncRNAs potentially modulated by ALKBH5 were screened by MeRIP-seq,and they were furtherly validated in 8226 cells by RT-qPCR.(4)The expression differences of lncRNA SNHG15 between ALKBH5-versus control group and between ALKBH5+versus control group were compared by RT-qPCR assay,to make sure that ALKBH5 positively regulated the expression of lncRNA SNHG15.(5)The mRNA expression levels of lncRNA SNHG15 in multiple myeloma cell lines(NCI-H929、RPMI-8226、ARH-77、U266)were detected by RT-qPCR.(6)LV-lncRNA SNHG15-OE and control lentivirus were designed and synthesized in vitro.They were transfected into ALKBH5-and control 8226 cells,constructing ALKBH5-SNHG15+group,ALKBH5-SNHG15NC group and ALKBH5NC SNHG15NC group,to compensate for the reduction of lncRNA SNHG15 caused by the down-regulation of ALKBH5 and to explore the impacts of ALKBH5 on the biological functions of multiple myeloma through regulating the expression level of lncRNA SNHG15:1)The expression changes of lncRNA SNHG15 among ALKBH5-SNHG15+group,ALKBH5-SNHG15NC group and ALKBH5NC SNHG15NC group were verified by RT-qPCR experiments;2)CCK8 assay was used to detect the differences of cell proliferation among three groups;3)AnnexinV PE/7AAD double staining flow cytometry method was used to detect the differences of apoptosis among three groups;4)Transwell assay was used to detect the differences of migration among three groups.Results:1.The results of RT-qPCR and Western Blot assays showed that the expression levels of ALKBH5 mRNA and protein in CD138+ cells from bone marrow of multiple myeloma patients were significantly higher than those in Normal control cells.And the expression of ALKBH5 in multiple myeloma cell lines RPMI8226,NCI-H929,U266 and ARH-77 was significantly higher than that in Normal control group;2.LV-ALKBH5-RNAi lentivirus significantly down-regulated the mRNA and protein expressions of ALKBH5 in RPMI-8226 cells.LV-ALKBH5-OE lentivirus significantly up-regulated the expression of ALKBH5 in RPMI-8226 cells;3.MeRIP-seq was performed on ALKBH5-group,control group and Normal control group,10 downstream lncRNAs were screened out.Among them,the expression of lncRNA SNHG15 in myeloma 8226 cells was significantly higher than that in Normal control cells,so this lncRNA was selected for follow-up studies;4.The results of RT-qPCR showed that the relative expression level of lncRNA SNHG15 in CD138+cells from bone marrow of multiple myeloma patients and MM cell lines was significantly higher than that in the Normal controls,and the relative expression level of lncRNA SNHG15 in MM patients was positively correlated with the expression of ALKBH5 mRNA;5.RT-qPCR assays validated that the expression of lncRNA SNHG15 was decreased in ALKBH5-group than in Control group,while the expression of lncRNA SNHG15 was increased in ALKBH5+group than in Control group;6.RT-qPCR assays further confirmed that the expression of lncRNA SNHG15 was decreased in ALKBH5-SNHG15NC group than in ALKBH5NC SNHG15NC group,while the expression of lncRNA SNHG15 was increased in ALKBH5-SNHG15+group than in ALKBH5-SNHG15NC group;7.CCK8 results showed that the cell viability of ALKBH5-SNHG15NC group cells was significantly lower than that in ALKBH5NC SNHG15NC group,while the cell viability of ALKBH5-SNHG15+group was higher than that in ALKBH5-SNHG15NC group;8.AnnexinV PE/7AAD double staining flow cytometry showed that the apoptosis rate of ALKBH5-SNHG15NC group cells was significantly higher than that in the ALKBH5NC SNHG15NC group,while the apoptosis rate of ALKBH5-SNHG15+group was lower than that in ALKBH5-SNHG15NC group;9.Transwell experiment showed that the migration ability of ALKBH5-SNHG15NC group cells was significantly lower than that in ALKBH5NC SNHG15NC group,while the migration ability of ALKBH5-SNHG15+group was higher than that in ALKBH5SNHG15NC group.Conclusion:1.ALKBH5 was highly expressed in primary multiple myeloma cells and MM cell lines;2.ALKBH5 positively regulated the expression level of lncRNA SNHG15;3.LncRNA SNHG15 was highly expressed in primary multiple myeloma cells and MM cell lines,and it was positively correlated with the mRNA expression of ALKBH5;4.ALKBH5 promoted the pathogenesis of multiple myeloma by regulating the expression of lncRNA SNHG15.