The Mechanism Of Histone Demethylase RBP2 And RNA Binding Protein Lin28B Mediating Blast Crisis Of Chronic Myeloid Leukemia

BackgroundChronic Myeloid Leukemia(CML)is a myeloproliferative disorder characterized by BCR-ABL fusion gene which caused by chromosome translocation t(9;22).BCR-ABL gene can encode a fusion protein has abnormal tyrosine kinase activity and activate its related downstream signaling pathways.The progression of CML can be divided into chronic phase(CP),accelerated phase(AP)and crisis phase(BP).Tyrosine kinase inhibitors(TKIs)is the first-line treatment of CML,which can inhibit the tyrosine kinase activity of BCR-ABL.Treated with TKIs,most of CML CP patients can survive for a long time.However,once CML CP patients progress to the crisis phase,TKIs have little effect on these patients,the fatality rate of these patients are very high and the median survival is only 6 months.The mechanism of CML is complex and has not been fully elucidated.Therefore,new mechanism of CML blast crisis and key molecules need to be found for effective prevention and control of CML.CML blast crisis is a multi-step and changeable process and usually accompanied by high expression and excessive activation of BCR-ABL.BCR-ABL activation getting out of control is considered as the driving force to promote CML blast crisis.Therefore,there are many BCR-ABL dependent pathways considered to mediate CML blast crisis.In addition,studies have found that excepting genetic strikes such as activation or amplification of oncogenes and functional loss or mutation of tumor suppressor genes,abnormal regulation of key targets in the signaling pathway and epigenetic imbalances are also involved in CML blast crisis.Thus,there are many BCR-ABL independent pathways to mediate CML blast crisis.Retinoblastoma binding protein 2(RBP2)belongs to the KDM5 family,targets the di-methylation and tri-methylation(Me2/Me3)of histone H3 fourth lysine(H3-K4)to promote demethylation of histone specifically.Due to the JmjC domain of RBP2 histone demethylase motif,RBP2 can regulate genes transcription to mediate a variety of cellular biological processes.In recent years,RBP2 has been reported to be involved in the onset and progression of multiple cancers,such as gastric cancer,lung cancer,breast cancer,malignant glioma,kidney cancer,ovarian cancer and liver cancer.In our team's previous study,we found that RBP2 is significantly down-regulated in the CML blast phase and regulates miR-21 to mediate CML blast crisis through BCR-ABL independent pathway.However,whether RBP2 can mediate the progression of CML through BCR-ABL dependent pathway remains unclear and needs further study.Lin28B is an RNA binding protein belonging to the Lin28 family,which can recognize RNA 3'UTR motif to regulate the expression of target genes,and mediates a variety of biological functions.Lin28B is overexpressed in variety of cancers,including lymphoma,neuroblastoma,colon cancer,liver cancer and so on.Studies found Lin28B is regulated by transcription factor NF-?B.Meanwhile Lin28B plays a key role in the maintenance of leukemia stem cells through its regulation on miRNA let-7 to promote proliferation and cell-cycle,Thus,Lin28B is a potential target for effective treatment of AML.Recent studies also show that Lin28A,which share the same family with Lin28B can act as a DNA binding protein.However,there is no relevant study about Lin28B in CML.Whether Lin28B plays a role in CML blast crisis still needs to be explored.Abnormal expressions of histone demethylase RBP2 and RNA binding protein Lin28B in CML blast phase were found in this study.We found that histone demethylase RBP2 through BCR-ABL dependent pathway and the regulation of miR-181d by RNA binding protein Lin28B through BCR-ABL independent pathways promoted CML blast crisis based on our investigations at cellular and molecular level,in animal bodies and human bone marrow tissue.This study is to provide a new sight for the roles and mechanisms of RBP2 and Lin28B in CML blast crisis.Purpose:1.The effect and mechanism of RBP2 mediated by BCR-ABL dependent pathway in CML blast crisis.2.The effect and mechanism of Lin28B mediated by BCR-ABL independent pathway in CML blast crisis.Methods and Results:1.Histone demethylase RBP2 regulates PTEN promoting CML blast crisis via BCR-ABL dependent pathway.(1)Overexpression of RBP2 inhibits the proliferation of leukemia cellsRBP2 overexpression plasmids were transfected into K562 and MEG01 cells,and EdU and soft agar clonal formation experiments were applied to detect the effect of RBP2 on proliferation of leukemia cells.Results suggested that the proliferation of RBP2 overexpressed cells were significantly inhibited.(2)To explore the relationship between RBP2 and BCR-ABLWestern blot assay was applied to detect the expression of RBP2 in BCR-ABL positive cell lines(K562 and MEG01)and BCR-ABL negative cell lines(HL60 and U937).It was found that RBP2 expression in BCR-ABL positive cell lines was significantly lower than that in BCR-ABL negative cell lines,suggesting the correlation between RBP2 and BCR-ABL.RBP2 overexpression plasmids were transfected into K562,MEG01 cells and primary cells of a CML patient to apply Western blot and qRT-PCR assays.It was found that phosphorylated BCR-ABL(p-BCR-ABL,the active form of BCR-ABL)was significantly reduced with RBP2 overexpression.While the total protein and mRNA expression of BCR-ABL were not significantly changed.The results above suggested that RBP2 regulated the phosphorylation level of BCR-ABL at the post-transcriptional level.(3)The mechanism of RBP2 regulating the phosphorylation level of BCR-ABL(p-BCR-ABL)With transfecting RBP2 overexpressed plasmids into K562 and MEG01 cells,the phosphorylation of BCR-ABL(p-BCR-ABL)related enzymes were detected by qRT-PCR,including PTEN,SHP1,PP2A,SET and PTP1B.We found that only the expression of PTEN was regulated by RBP2.42 Bone marrow samples of CML chronic phase and blast phase patients were collected from Qilu hospital.qRT-PCR assays were applied and found that a positive correlation between the expression of RBP2 and PTEN.Transfecting RBP2 overexpressed plasmids into K562 and MEG01 cell lines,the protein expression of PTEN increased with the overexpression of RBP2 by Western blot assays.RBP2 overexpressed plasmids were transfected into bone marrow cells of a CML patient,and we found that PTEN expression was significantly decreased by applying qRT-PCR assays.Bone marrow cells were isolated from RBP2 heterozygous mice and wild-type mice,and qRT-PCR was used to verify that the expression of PTEN was reduced along with RBP2 knockdown in vivo.All above indicate that RBP2 can regulate the expression of PTEN in primary cells,leukemia cell lines and mice.(4)The mechanism of RBP2 regulating the expression of PTENEmpty plasmid,wild-type RBP2 overexpression plasmid and RBP2 enzyme overexpression plasmid with enzyme activity mutant(RBP2 H483A)were transfected into K562 and MEG01 cells to apply Western blot and qRT-PCR assays.Results showed that cells transfected with wild-type RBP2 overexpression plasmid could upregulate the mRNA and protein expression of PTEN,while cells transfected with mutant RBP2 overexpression plasmid(RBP2 H483A)had no effect on the expression of PTEN.Thus,RBP2 regulated PTEN expression depending on its enzyme activity.ChIP experiment was performed in K562 cells and it was found that RBP2 protein can bind to the promoter region of PTEN.Then the ChIP assay was repeated in K562 cells transfected with RBP2 overexpressed plasmid and the results showed that,with the overexpression of RBP2,this protein was significantly enriched in the promoter region of PTEN.H3K4me2 and H3K4me3,however,were sharply diminished in the same region.These findings suggested that RBP2 depends on its demethylase activity to bind to the promoter region of PTEN.RBP2 wild-type overexpression plasmid or RBP2 overexpression plasmid without enzyme active(RBP2 H483A)and the wild-type or mutant dual luciferase reporter plasmid of PTEN promoter were co-transfected into HEK293 cells.The results of the dual luciferase experiment indicated that the PTEN promoter activity was significantly increased with RBP2 overexpression plasmid transfected.While the PTEN promoter activity had no significantly change with RBP2 mutant plasmid(RBP2 H483A)transfected,also the activity of PTEN promoter with binding site mutant had no significantly change.These results indicated that RBP2 regulates the activity of PTEN promoter depending on its enzyme activity.(5)The mechanism of PTEN regulating the phosphorylation of BCR-ABL(p-BCR-ABL)PTEN siRNA was transfected into K562 cells and PTEN overexpression plasmids were transfected into MEG01 cells to apply Western blot and qRT-PCR experiments.Western blot suggested that the phosphorylation of BCR-ABL,STAT5 and ERK significantly increased with the decrease of PTEN expression.With the increase of PTEN expression,the phosphorylation of BCR-ABL,STAT5 and ERK significantly decreased.There was no significant difference in the mRNA expression of BCR-ABL whether the expression of PTEN was down-regulated or up-regulated.We applied Co-immunoprecipitation(Co-IP)assays in K562 cells and HEK293 cells with BCR-ABL and PTEN overexpression plasmids co-transfected.Results proved that PTEN and BCR-ABL could bind to each other endogenously and exogenously.These results indicated that PTEN mediated the dephosphorylation of BCR-ABL by binding to BCR-ABL.(6)BCR-ABL regulates the expression of RBP2K562 cells were treated with the tyrosine kinase inhibitor imatinib(IM)atdifferent time and concentrations.Western blot and qRT-PCR were applied,and it was found that mRNA and protein expression of RBP2 gradually increased with imatinib incubation time and concentration increasing.It is suggested that inhibition of BCR-ABL enzyme activity could induce the expression of RBP2.Transfecting BCR-ABL overexpression plasmid or BCR-ABL siRNA into K562 cells,then we detected the protein and mRNA expression of RBP2 and BCR-ABL by applying Western blot and qRT-PCR experiments.We found that mRNA and protein expression of RBP2 were negatively regulated by BCR-ABL.These results shownthat BCR-ABL could negatively regulate the expression of RBP2 at the transcriptional level.(7)RBP2 inhibits the proliferation of leukemia cells by BCR-ABL dependent pathwayK562 and MEG01 cells co-transfected with RBP2 and BCR-ABL overexpression plasmids,or only transfected RBP2 overexpression plasmid or empty plasmid.Then EdU and soft agar clonal formation experiments were applied to verify the proliferation of leukemia cells.The results suggested that RBP2 overexpression inhibited the proliferation and clonal formation of leukemia cells,while BCR-ABL overexpression partially restored the ability of RBP2 overexpression.Above all,RBP2 overexpression inhibited the proliferation of leukemia cells through BCR-ABL pathway.2.The mechanism of RNA binding protein Lin28B regulates miR-181d promoting CML blast crisis via BCR-ABL independent pathway.(1)The expression of RNA binding protein Lin28B increased with CML progressionThe expression of 29 RNA binding proteins were compared between CML chronic phase and blast phase patients in GEO database GSE4170.It was found that Lin28B was the most significantly up-regulated RNA binding protein with CML progression.We collected bone marrow samples of CML CP and BP patients from Qilu hospital to apply qRT-PCR and immunofluorescence(IF).We found that the mRNA and protein expression of Lin28B were significantly increased in the blast phase compared with chronic phase,suggesting that Lin28B might be involved in the progression of CML.(2)To detect the effect of Lin28B on the proliferation of leukemia cells Lentivirus was used to infect K562 and HL60 cells to build stable Lin28B knockdown cell lines.Then EdU assays were applied.It was confirmed that the proliferation of leukemia cells decreased significantly with Lin28B knockdown.(3)Lin28B is related to leukemia cell differentiationDMSO and PMA were applied to induce K562 and MEG01 cells differentiating into macrophage-like and granulocyte-like.The expression change of Lin28B in differentiated cells was detected by Western blot and qRT-PCR.The mRNA and protein expression of Lin28B decreased significantly with cell differentiation.These results indicated that the expression of Lin28B was associated with the differentiation of leukemia cells.(4)The mechanism of Lin28B regulating miR-181dK562 and MEG01 cell lines were infected with lentivirus to build stable Lin28B knockdown cells to apply qRT-PCR assays.It was confirm that the expression of miR-181d was downregulated with Lin28B knockdown.This suggested that miR-181d might be a potential target of Lin28B.Lin28B antibody was used for ChIP assay in K562 and HL60 cells and found that Lin28B could bind to the promoter region of miR-181d.Then K562 and HL60 cell lines were infected with lentivirus to knock down Lin28B to apply dual luciferase assay.It is suggested that the activity of miR-181d promoter was significantly decreased with Lin28B knockdown.The above results indicated that Lin28B activated transcription of miR-181d by binding to its promoter region.(5)the mechanism of miR-18 1d regulating target PDCD4K562 and HL60 cells were transfected with miR-181 d mimics,miR-181 d inhibitor or the corresponding controls to apply Western blot.With the increase of miR-181d expression,the protein expression of PDCD4 decreased significantly.This suggested that PDCD4 might be a potential target of miR-181d.Then,the sequences of dual luciferase reporter plasmids of the PDCD4 3'UTR and PDCD4 3'UTR with binding site mutated were built.miR-181 d mimics,miR-181d inhibitor or the corresponding control were transfected into K562 and HL60 cells,co-transfected with luciferase reporter plasmids to apply dual luciferase reporter assay.The results suggested that overexpression of miR-181d inhibited the activity of PDCD4 3'UTR,and the decreased expression of miR-181d could up-regulate the activity of PDCD4 3'UTR.But change of miR-181d expression had no effect on the activity of PDCD4 3'UTR with binding site mutated.These assays indicated that miR-181d regulated PDCD4 expression by binding to its 3'UTR region.These results suggested that miR-181d regulated the expression of PDCD4 by binding to PDCD4 3'UTR.(6)Lin28B knockdown inhibited the growth of leukemia cells in NOD-SCID miceNOD-SCID mice were given a sublethal dose of 2Gy X-ray radiation.After 24 hours,1 ×106 stable knockdo1n Lin28B cells and control cells were respectively injected subcutaneously into the both sides of the mice armpit.The tumor volumes were measured every 3 days,and the mice were sacrificed 18 days to measure the tumor weights.The tumor weight and volume of Lin28B knockdown group were significantly smaller than the control group.This indicated that Lin28B could inhibit the proliferation of leukemia cells in vivo.Conclusions:1.Histone demethylase RBP2 activates PTEN transcription by binding to its promoter region;PTEN mediates the dephosphorylation of BCR-ABL by binding to BCR-ABL;Thus,the "RBP2/PTEN/BCR-ABL" axis mediates CML blast crisis via BCR-ABL dependent pathway.2.RNA binding protein Lin28B activates miR-181d transcription by binding to its promoter region;miR-181d inhibits the expression of PDCD4 by binding to its 3'UTR region;Thus,"Lin28B/mir-181d/PDCD4" axis mediates CML blast crisis via BCR-ABL independent pathway.