Study On The Mechanism Of YTHDF2 Regulating The Cellular Proliferation Through M6A Methylation Pathway In Multiple Myeloma

Background and ObjectiveMultiple Myeloma(MM)is a plasma cell malignant tumor characterized by monoclonal immunoglobulin and abnormal proliferation of bone marrow plasma cells.MM is often accompanied by clinical symptoms such as osteolytic damage,hypercalcemia,anemia,and kidney damage.The treatment of MM mainly includes radiotherapy and chemotherapy,and hematopoietic stem cell transplantation.In addition to traditional chemotherapeutics,targeted drugs such as proteasome inhibitors(bortezomib,carfilzomib)and immunomodulators are currently the first-line drugs used in the treatment of MM,as well as some glucocorticoids such as dexamethasone and prednisone Wait.Although most MM patients have achieved a certain degree of remission,the survival of patients is significantly affected due to recurrence,metastasis,and drug resistance.Therefore,in-depth study of the pathological and molecular mechanisms of MM and discovery of novel therapeutic targets for MM brook no delay.In this project,we first constructed a MM cell line with stable knockdown of YTH domain family 2(YTHDF2)expression based on the YTHDF2 gene screened from the gene database and conducted phenotypic studies on cell proliferation and cell cycle as well as animal experiments to verify YTHDF2 function.Next,we screened for the downstream genes that YTHDF2 may regulate through the high-throughput sequencing of N6-methyladenosine(m6A)methylation,and then explored the potential mechanism that regulated the growth and proliferation of MM cells through m6A methylation modification.The research is intended to provide novel molecular targets and treatment strategies for MM.Methods1.Lentiviral transfection system was performed to construct YTHDF2 knockdown(KD)and overexpression(OE)MM cell lines,and Western blot experiment and fluorescence microscope observation were used to verify the transfection effect.In addition,Dot Blot experiment was conducted to verify the effect of KD/OE of YTHDF2 on m6A levels of MM cells.Afterward,MTT proliferation test detected the proliferation of YTHDF2-KD cells and YTHDF2-OE cells,as well as flow cytometry measuring the effect of KD and OE of YTHDF2 on the mitotic cycle of cells.By injecting wild-type(ARP1-WT)and ARP1-YTHDF2-KD MM cells to construct an immunodeficient mouse xenograft tumor model,we planned to study the effect of targeting YTHDF2 on the growth of MM cells in vivo.2.Through the comparison of the m6A-seq library(IP)and RNA-seq library(input)two high-throughput sequencing libraries,combined with the MM patient gene library,we found the special MM-related targets downstream of YTHDF2 as Signal transducer and activator of transcription 5a(STAT5A).RNA immunoprecipitation(RIP)-qPCR experiments,Real-time(RT)-qPCR and WB experiments were utilized to verify the effect of YTHDF2 on STAT5A mRNA and protein expression.The mRNA stability experiment was used to confirm the degradation effect of YTHDF2 on STAT5A mRNA.3.STAT5A-OE cells were constructed to verify the effect of STAT5A on the proliferation rate of MM cells.STAT5A-Chromatin immunoprecipitation(ChIP)sequencing and RNA sequencing were used to find Mitogen-activated protein kinase kinase 2(MAP2K2)as a downstream gene of STAT5A regulated by transcription factors.4.ChIP-qPCR was applied to verify that STAT5A directly binds to MAP2K2 and regulates its transcription.The protein levels of MAP2K2,Extracellular regulated MAP kinase(ERK),and phosphorylated Extracellular regulated MAP kinase(p-ERK)in YTHDF2-KD and STAT5A-OE cells were verified by WB.The connection between MAP2K2 and ERK was validated by Cooperation immunoprecipitation(CoIP)and RNA interference experiments.Interleukin-2(IL-2)was used to verify that unphosphorylated STAT5A(u-STAT5A)played an indispensable role in inhibiting the expression of MAP2K2.Results1.Based on the high-throughput and the MM patient gene database,we selected the m6A methylation reading protein YTHDF2(YTH domain family 2),which was closely related to the disease process of MM patients.Secondly,gene chip technology and clinical data of MM patients confirmed that the YTHDF2 gene was significantly related to the survival rate of MM patients.The survival rate of MM patients with high YTHDF2 expression was lower than that of MM patients with low YTHDF2 expression.Next,cell proliferation and cell cycle phenotype research of YTHDF2 gene KD/OE stable transduction MM cell lines showed that blocking YTHDF2 can significantly inhibit the proliferation rate of MM cells,but it is not caused by regulating the cell cycle distribution.2.The results of high-throughput sequencing of m6A methylation showed that knocking down YTHDF2 can make changes in the expression levels of multiple genes.The STAT5A gene was screened in combination with the gene database of MM patients.The results of studies on the correlation between YTHDF2 and STAT5A expression showed that after knocking down YTHDF2,the mRNA level of STAT5A was up-regulated,as was the protein level.Therefore,it is preliminarily speculated that the proliferation effect of YTHDF2 on MM cells may be achieved by regulating STAT5A expression.3.Compared with WT cells,the proliferation rate of STAT5A-OE MM cells was significantly decreased,suggesting that STAT5A may exist as a tumor suppressor in MM cells.The above results suggest that STAT5A exerts diametrically opposite effects on the proliferation of MM cells before and after translation modification and phosphorylation.4.ChIP sequencing revealed that STAT5A may inhibit their expression by regulating the transcription of genes related to the MAPK signaling pathway,thereby regulating the proliferation of MM cells.Combined with the results of RNA sequencing,it was indicated that MAP2K2 plays an important role in STAT5A’s inhibition of MM cell proliferation.The results of the study found that STAT5A inhibited the expression of MAP2K2,thereby repressing the phosphorylation of ERK,which was manifested as an inhibitory effect on the proliferation of MM cells.At the same time,it was found that unphosphorylated STAT5A(u-STAT5A)inhibits the expression of MAP2K2,and when STAT5A is phosphorylated,its inhibitory effect will be lifted.ConclusionThis paper explored the role of YTHDF2 in MM cells and found the axial regulation between YTHDF2,STAT5A,MAP2K and ERK:YTHDF2 recognized m6A methylation on STAT5A mRNA and promoted its degradation.u-STAT5A can inhibit the transcription of MAP2K2,reduce its expression,and decrease the phosphorylation level of ERK,thereby inhibiting the proliferation of MM cells to a certain extent.This study intends to provide a new molecular target for the treatment of MM.