The Study Of Hypoxia Promoting DKK1 Expression In Multiple Myeloma

Background:Myeloma bone disease is the most frequent feature of myeloma,occurring in approximately two-thirds of patients at diagnosis and in nearly90% of patients with multiple myeloma(MM).The bone marrow microenvironment of MM is hypoxic,and hypoxia-inducible factor 1α(HIF-1α)enhances angiogenesis,promotes abnormal glucose metabolism,and induces the expression of anti-apoptotic proteins.DKK1 is closely related to bone metastasis and bone destruction in various malignant tumors including myeloma.At present,the mechanism of hypoxia regulating DKK1 expression has not been reported.Objective :The purpose of this study is to determine the relationship between the high expression of DKK1 in MM patients and hypoxia microenvironment in the bone marrow.(1)Hypoxia caused by the rapid proliferation of MM cells is also an important factor induced DKK1 expression.(2)Hypoxia leads to the activation of MMSET and CREB in MM cells,which may play a key role in histone modification and regulate DKK1 expression(3)Simultaneity of blocking hypoxia and CREB may have a more significant effect on the reversal of bone destruction than targeting DKK1 alone.Methed:1.RNA-seq sequencing study: After 24 h hypoxic induction of m M.1S cells and LP-1 cells,the genes significantly differentially expressed were analyzed by transcriptome m RNA-seq sequencing and expressed by heat map.Cells were cultured under hypoxia(1%O2)for 6,12 and 24 h,respectively.The changes of DKK1 level were detected by q PCR and WB.The level of DKK1 secreted in the culture medium under normal and hypoxia conditions was determined by ELISA assay.2.Cytological phenomenon study: Western blot was used to detect the changes of hypoxia-related signaling pathway protein marker in m M.1S and LP-1 cells cultured for 24 h under hypoxia conditions,such as the changes in the phosphorylation of Akt and p38 kinase.Cells were treated with SB203580,a p38 inhibitor,for 24 h,or sh RNA was constructed to knock down p38α and DKK1 m RNA expression levels in cells.Cell immunofluorescence assay was used to detect the nuclear translocation of transcription factor CREB protein,and WB was used to detect the phosphorylation level of the protein.The overexpression of CREB and MMSET proteins in MM cells was mediated by lentivirus,and the expression of DKK1 was detected by q PCR and WB.Luciferase analysis showed that p GL3-DKK1-Luc reported on the activity of the p GL3-DKK1-Luc in the presence of increased CREB overexpression.The expression of histone methylation enzyme MMSET was detected by WB after 24 hours of hypoxia.After treating MM cells with hypoxia-inducing factor HIF-1α inhibitor(LW6),the reversal of MMSET under hypoxia-inducing condition was detected.3.Clinical data collection and index detection: 50 cases of bone marrow of clinically confirmed myeloma patients and 16 cases of bone marrow t(4;14)specimens from patients with mutation and 5 healthy people.Collected from Department of Hematology,Yongchuan Hospital Affiliated to Chongqing Medical University,subjects and their families were informed of the purpose and procedure of this study.Patient age,blood M-protein level,patient condition,and medication status were retrospectively analyzed.Subject to approval by the ethics committee.Immunohistochemistry(IHC)was used to detect the expression levels of DKK1 and MMSET in bone marrow tissue sections.The P value was determined by one-way analysis of variance in an independent experiment.The level of DKK1 in bone marrow serum was determined by ELISA,and the P value was determined by Pearson coefficient.4.Cytological mechanism study: Co-immunoprecipitation assay(Co-IP)confirmed that there was a direct interaction between CREB and MMSET.The co-precipitation assay(Co-IP)confirmed the direct correlation between different truncated MMSET(Δ PWWP1,Δ PHD,Δ SET)and CREB at the protein level.Co-IP detected the correlation changes between CREB and MMSET after CREB phosphorylation.Luciferase assay of DKK1-luc reporter in MM cells electroporated with plasmids encoding CREB and MMSET full length(FL),depletion of SET domain(ΔSET),or tyrosine to alanine mutation at 1179 amino acid site governing MMSET methyltransferase activity(Y1179A).CHX was used to detect the stability of CREB protein when infected with lentivirus-carrying vectors expressing full-length MMSET(FL),SET domain depletion truncation,or a Y1179 A mutant for loss of themethyl-transferase function in the SET domain for 72 h.The enrichment of CREB,MMSET and H3K36Me2 in DKK1 promoter after 24 h hypoxia was verified by Ch IP-q PCR.The HIF-1α inhibitor LW6 or CREB inhibitor KG-501 were used for the change of the enrichment.5.Targeting CREB attenuates DKK1 expression and bone disruption in cells and Xenograft experiment in nude mice: After 24 hours of hypoxia culture,The MSC cells was co-cultured with the supernatant,and the osteogenic ability of MSC cells was detected by Alizarin red-S staining intensity and alkaline phosphatase(ALP)activity.NSG/SCID mice bearing bortezomib-resistant MM.1S cells in femur bone marrow were treated with either the hypoxia-activated pro-drug TH-302 or the CREB inhibitor KG-501 or their com-bination.Micro-CT scanning was used to detect the number and thickness of trabecular bones.Results:1.After 24 h of hypoxia induction,the differentially expressed genes were analyzed by transcriptome m RNA-seq sequencing.1536 differentially expressed genes were identified in m M.1S cells and 987 genes were identified in LP-1 cells.47 of the 58 genes overlapping between the two cell lines were significantly increased.Among them,are closely associated with myeloma progress and bone lesions of DKK1,A T G P combination box and the family member 2(ABCG2),the structure of the nuclear receptor SET domain protein 2(NSD2)and BCL2 apoptosis regulating factor(BCL2)expression increased significantly,activation of transcription factor 3(ATF3),MAX polymerization protein(MXI1)1,2 and tin calcium protein 2(STC2)is hypoxia inducing factor(HIF)-1 alpha signaling pathways known targets.KEGG analysis showed that the p38-MAKP signaling pathway was significantly activated,and enzyme-linked immunosorbent assay(ELISA)also confirmed that hypoxia promoted the secretion of DKK1 into the medium.2.Cells cultured in hypoxia for 24 h,P38 kinase was significantly activated.P38 blocker can reduce hypoxia-induced DKK1 expression.Hypoxia increases CREB phosphorylation at Ser133 and increases intrico-nuclear transfer of CREB protein.Cells were treated with CREB inhibitor KG501 to inhibit hypoxia-induced DKK1 expression.CREB overexpression stimulates transcriptional activity of the DKK1 luciferase reporter gene.Hypoxia induces MMSET expression,and this effect can be reversed with the hypoxia-inducible factor inhibitor LW6.3.Immunohistochemical analysis of bone marrow biopsy samples from clinical patients showed a positive correlation between MMSET and DKK1 levels in the same specimen.MMSET was overexpressed in t(4:14)positive patients.There was a strong positive correlation between DKK1 and MMSET expression in myeloma cells in patients with t(4:14)positive compared with those with other chromatin abnormalities.4.Co-IP showed that CREB and MMSET proteins directly interacted with each other in MM cells.Co-IP also showed that full-length MMSET protein and MMSET protein without PHD domain deletion could interact with CREB protein.Cell immunofluorescence assay showed that there was intracellular co-localization between the two proteins.After down-expression MMSET,the instability of CREB was significantly reduced.With CREB present,while the ectopic expression of full-length MMSET protein resulted in a greater than eightfold activation of the DKK1-luciferase reporter in MM.1S and LP-1 cells,when the SET domain was deleted or loss of function mutated,the activation of the DKK1-luciferase reporter was very limited.H3K36me2 levels was increased in myeloma cells under hypoxia by WB assay.Chromatin immunoprecipitation(Ch IP)analysis showed that hypoxia increased the enrichment of CREB,MMSET,and H3K36Me2 at the DKK1 promoter.HIF-1α inhibitor LW6,the p38 inhibitor SB203580,or the CREB inhibitor KG-501 decreased the enrichment at the DKK1 promoter.5.When CREB was knocked down,the expression of DKK1 was significantly decreased detected by WB,while the stability of MMSET protein remained unchanged by CHX.After the MM cells were infected with sh CREB lentivirus and cultured in hypoxia,the supernatant of the cells was collected and co-cultured with mesenchymal stem cells.Alizarin red-S staining and alkaline phosphatase staining showed enhanced osteogenic activity.NSG/SCID mice were injected with BTZ-resistant MM.1S cells into the femoral cavity,respectively or in combination with TH-302(20 mg/kg)and KG-501(10 mg/kg)drugs.Micro CT showed that the significant recovery of trabecula disruption in the combination group,with significantly higher numbers of trabeculae and trabecula thickness.Conclusion:In summary,this study revealed that the cooperation of the p38-CREB cascade with MMSET promotes DKK1 expression in response to hypoxia in myeloma cells.Regulating DKK1 expression by targeting hypoxia and CREB together may have therapeutic significance in the management of myeloma patients with chemoresistance and lytic bone disease.