ALKBH5 Promoted The Pathogenesis Of Multiple Myeloma Via Modulating Hippo Pathway

Object: To explore the expression level of RNA demethylase ALKBH5 in mutiple myeloma(MM)patients and cell lines,analyze the possible target genes of ALKBH5 through Me RIP sequencing,study the effect of ALKBH5 on MM cell apoptosis,proliferation and number of stem cells,and explore the molecular mechanism of ALKBH5 in the progression of MM.To investigate the effect of ALKBH5 on the biological function of multiple myeloma cells in vivo and in vitro.Methods: The expression levels of ALKBH5 in bone marrow-derived CD138+ cells of MM patients and peripheral blood mononuclear cells(PBMCs)of normol volunteers were detected by q RT-PCR.LV-ALKBH5-RNAi virus,negative control virus,LV-ALKBH5-OE virus and control virus were synthesized and transfected into RPMI8226 cells.The expression levels of ALKBH5 were down-regulated and overexpressed respectively.RNA methylation assay kit was used to detect the changes in RNA methylation levels.Target genes regulated by ALKBH5 in RPMI8226 cell line were analyzed by Me RIP-seq.CCK8 method was used to detect the effect of down-regulated ALKBH5 on myeloma cell proliferation.Annexin V PE/7AAD double-staining flow cytometry was used to detect the effect of down-regulated ALKBH5 on the apoptosis of myeloma cells.The number of tumor-initiating cells in RPMI8226 cell line after ALKBH5 down-regulation was detected by CD138/CD34double-staining flow cytometry.Hoechst/PI staining was used to detect the effect of down-regulation of ALKBH5 on the number of side population cells in myeloma cells by flow cytometry.q RT-PCR was used to examine the expression of NANOG、COT4、SOX2.Western blotting was used to detect the effect of ALKBH5 on apoptosis related protein BCL-2 /BAX in myeloma cells and the effect on Hippo pathway proteins SAV1,MST1/2,YAP and P-YAP.Finally,Subcutaneous xenograft model of human multiple myeloma in NOD/SCID was established to observe the effect of down-regulation of ALKBH5 on the growth of myeloma cells in mice.Results:(1)ALKBH5 was highly expressed in multiple myeloma patients and four myeloma cell lines,with the highest level in RPMI8226 cell.(2)LV-ALKBH5-RNAi virus can effectively down-regulate the expression level of ALKBH5 in RPMI8226 cell and increase the level of RNA methylation.(3)Me RIP sequencing results determined the target gene SAV1,whose expression level was down-regulated and methylation level was increased.(4)CCK8 assay showed that the proliferation of RPMI8226 was significantly inhibited after ALKBH5 was down-regulated.(5)After the down-regulation of ALKBH5,the apoptosis of RPMI8226 cells increased,accompanied by the down-regulation of BCL-2 /BAX.(6)Down-regulation of ALKBH5 can reduce the proportion of stem cells and expression of genes associated with stemness in RPMI8226 cells.(7)Down-regulating the expression of ALKBH5 can increase the RNA methylation level of RPMI8226 cells,significantly reduce the expression level of SAV1,inhibit the Hippo pathway,increase the expression level of YAP and P73 target gene puma,and promote cell apoptosis.(8)Down-regulation of ALKBH5 could signif icantly inhibit the growth of subcutaneous xenograft in nude mice.Conclusion: Compared with normal PBMC,ALKBH5 expression level in bone marrow-derived CD138+ cells of MM patients and myeloma cell lines are raised.Inhibiting ALKBH5 expression can increase cell RNA methylation and apoptosis,repress myeloma cell proliferation,along with the Hippo signaling pathways suppressed,YAP expression increased,eventually inhibit the growth of myeloma cells in vivo.