YTHDF2 Promotes The Growth Of Chronic Myelogenous Leukemia As An M6A Reader Protein

Objective:This thesis aims to investigate the role of N6-methyladenosine(m6A)modification in the growth and drug response of chronic myeloid leukemia(CML)cells and its underlying mechanisms.Methods:(1)The m6A modification in CD34+cells from CML patients and those from healthy donors was quantified;Q-RT-PCR was used to assess the expression of m6A modification related genes.(2)The effects of YTHDF2 silencing or overexpressed on the m6A modification,the growth,colony-formation cell(CFC)production and IM response were analyzed.The effect of YTHDF2 silencing on CFC production of CML CD34+cells was also studied.(3)RNA-seq was utilized to analyse the gene expression influenced by YTHDF2 knockdown in K562 and the downstream target gene TINAGL1 was further validated by Q-RT-PCR.Knock down of TINAGL1 with spermine-introduced pullulan(PS)delivery was able to "rescue" the inhibitory effect on CFC and elevated imatinib sensitivity caused by YTHDF2 silencing.Methylated RNA Immunoprecipitation(MeRIP)and RNA immunoprecipitation(RIP)were performed to study the regulatory mechanism of YTHDF2 to TINAGL1.We measured m6A modification level,growth and CFC ability and TINAGL1 expression in K562 cell with ZC3H13 silencing.Results:(1)m6A modification was downregulated in CML CD34+cells.YTHDF2 and ZC3H13 were higly expressed in CML CD34+cells.(2)YTHDF2 regulated the m6A modification level in K562,and the expression of YTHDF2 was negatively correlated with m6A modification level.YTHDF2 promoted the cell growth and CFC ability in K562,and silence of YTHDF2 reduced the CFC production of CML CD34+cells.Silencing YTHDF2 increased the sensitivity of K562 cells to IM and overexpressed YTHDF2 increased the tolerance to IM.(3)Silencing YTHDH2 elevated the expression of TINAGL1,a putative tumor suppressor,through recognizing and binding to the m6A sites.Knock down of TINAGL1 together with YTHDF2 was able to "rescue" the inhibitory effect of YTHDF2 silencing on cell growth and elevated imatinib sensitivity.Silence of ZC3H13,a component of methyltransferase complex,inhibited the growth of K562 cells while enhanced the expression of TINAGL1.Conclusion:The m6A modification is significantly reduced in CML CD34+cells than their normal control cells,with the elevated expression of both YTHDF2(encoding a m6A "reader")and ZC3H13(encoding a component of methytransferase complex).YTHDF2 promotes the growth of CML cells and confers the resistance of these cells upon imatinib treatment,possible cooperating with ZC3H13 to regulate TINAGL1 in an m6A dependent manner.