The Mechanism And Significance Of Dexamethasone-mediated DOT1L Downregulation In The Killing Of Lymphoma And Leukemia Cells

Backgrounds:Dexamethasone(Dex,a synthetic GC),functioning through binding to GR,as one of the most widely prescribed drugs in the world today,has been widely used in the therapeutics of hematologic malignancies including leukemias and lymphoma.However,its precise antitumor mechanism has not been fully elucidated.DOT1L(disruptor of telomere silencing 1 like)is the the only non-SET domain comprising methytransferase which could catalyze the methylation of histone H3 at lysine 79 site to date.Previous studies have reported that DOT1 L involved in telomere silencing,gene expression regulation,cell aging and DNA damage response.Recently,it has been demonstrated that DOT1 L could promote the onset and progression of various tumorous cells,such as lung cancer,breast cancer,ovarian cancer,renal clear cell carcinoma,neuroblastoma and so on.In addition,DOT1 L has been proved to have strong correlation with MLL(MLL-rearranged leukemias).The mechanism by DOT1 L promotes the initiation of MLL is that DOT1 L leads to aberrant H3K79 methylation patterns,contributing to the overexpression of MLL target genes such as MEIS1 and Hoxa9.Therefore,DOT1 L is assumed as a novel therapeutic target against MLL-rearranged leukemias and its specific inhibitors are investigatd in clinical research.However it has not yet been reported that whether DOT1 L involves in lymphoma.Besides,it is still unknown whether DOT1 L could be regulated by Dex.Objective:To investigate the effects of dexamethasone on B lymphoma and MLL-rearranged leukemia cells proliferation and DOT1 L expression,and to explore the potential molecular mechanism.Methods:(1)Extracting the histone of B lymphoma cell line(Raji,Namalwa,Daudi,Jeko-1)?acute monocytic leukemia cell line(THP-1)and MLL-rearranged leukemia cell line(MV4-11),to measure the basic protein level of DOT1L/H3K79 by Western blot;analyzing the differential expression of DOT1 L in hematological tumors and normal tissues by using TCGA database.(2)B lymphoma cell line(Raji),acute monocytic leukemia cell line(THP-1)and MLL-rearranged leukemia cell line(MV4-11)were treated with GR agonist Dex,then both mRNA and histone were extracted to assess the expression of DOT1 L and target gene MEIS1 by Real-time PCR and Western blot at transcriptional and translational level;identification of growth inhibition with CCK-8 assay.(3)Aboved cell lines were treated with GR antagonist RU486(Mifepristone)then incubated with Dex or not,to detect the expression of DOT1 L and target gene MEIS1 by Real-time PCR and Western blot at transcriptional and translational level;cell proliferation was measured by CCK-8 assay.(4)The specific si RNA targeting DOT1 L were synthesized and transfected into Raji cell,to measure cell proliferation level by CCK-8.(5)We predict whether there is a GR binding site in DOT1 L promoter region by analyzing a nuclear website,subsequently,construct recombinant plasmids of DOT1 L promoter region termed as pGL3-DOT1 L.Eventually,dual luciferase reporter assay was used to detect the influence of GR agonist on the transcriptional activity of DOT1 L gene promoter.(6)Pre-treated with Actinomycin D(Act D),then incubated with Dex to measure the expression of DOT1 L at post-transcription level.Results:(1)The expression of DOT1 L in hematological tumors were strikingly higher than normal tissues;and compared to MV4-11 cell in which DOT1 L was overexpressed,DOT1 L has a relatively high level in lymphoma cells,which is contrary to THP-1 cell.(2)Dex could suppress the proliferation of Raji and MV4-11 cell,downregulate the expression of DOT1 L and MEIS1 at both mRNA and protein level,while sparing THP-1 cell line which is insensitive to dex.(3)Dex inhibits cell proliferation,DOT1 L and MEIS1 expression by activating GR.(4)It is intriguing that we decipher knocking down DOT1 L could significantly suppress the proliferation of lymphoma cell(Raji).(5)Dual luciferase reporter assay turns out that Dex does not repress DOT1 L expression via influencing DOT1 L promoter activity,which indicating t he existence of other mechanisms.(6)Dex via suppresses the stability of DOT1 L m RNA to downregulate DOT1 L expression at post-transcription level in Raji.Conclusion:(1)DOT1L is highly expressed in B lymphoma cells.(2)For the first time,we found that the antitumor effect of Dex,at least partially,induced by downregulation of DOT1 L.(3)In the B lymphoma cell(Raji),Dex inhibits DOT1 L expression through attenuating DOT1 L m RNA stability.(4)Regarding to si DOT1 L causes growth inhibition of the B lymphoma cell(Raji),we inspiringly and firstly found that DOT1 L is suggested to B lymphoma.