Study On The Role And Mechanism Of FTO In Foam Cell Formation

BackgroundStroke has become the leading cause of death and disability among Chinese residents.Ischemic stroke is the most common type of stroke,and atherosclerosis(AS)is closely related to ischemic stroke.The formation of foam cell caused by lipid disorder is an important pathological process of AS.Therefore,how to reduce the formation of foam cells is the key to the prevention and treatment of atherosclerosis.m~6A is the most common chemical modifications in eukaryotic m RNA.Increasing studies have revealed that the m~6A modification may affect the occurrence and development of AS.However,it is not clear how the m~6A modification-related proteins regulates foam cell formation.MethodsIn this study,we determined the expression changes of m~6A modification-related proteins in the process of foam cell formation and revealed the underlying mechanism by which m~6A modification-related proteins regulate foam cell formation.The foam cells were constructed by stimulating RAW264.7 cells with ox-LDL,and the phagocytosis of lipoproteins by macrophages was observed by oil red staining,oil red semi-quantitative and cholesterol ester content determination.Meanwhile,the changes in the overall level of m~6A were detected by Dot Blot.RT-PCR and Western Blot were used to detect the changes in m~6A modification-related proteins,including METTL3,METTL14,WTAP,FTO,ALKBH5.Then,FTO overexpression/knockdown RAW264.7 cells were constructed and treated with ox-LDL to transform into foam cells.The expression of FTO and LDLR were detected by q PCR and Western Blot.At last,LDLR was verified as a target gene of FTO by Me RIP.Results(1)The foam cell model was established by treating RAW264.7cells with 80μg/m L ox-LDL,and the results showed that with the prolongation of the treatment time(within 48h)of ox-LDL,the foam cell formation increased.(2)Compared with the control group,the group treated with80μg/m L ox-LDL for 48h group significantly increased the m RNA expression of METTL3,FTO and Al KBH5,significantly decreased the FTO protein expression level,and significantly enhanced the overall m~6A modification level(P<0.01).(3)Compared with RAW264.7 cells,IGF2BP2 m RNA expression was significantly down-regulated(P<0.05)and protein expression was significantly up-regulated(P<0.01)in RAW264.7 cells treated with80μg/m L ox-LDL for 48h.(4)After sh/ov FTO-RAW264.7 cells were treated with 80μg/m L ox-LDL for 48h,the data proved that foam cell formation was increased in sh FTO-RAW264.7 group compared with sh NC-RAW264.7 group and foam cell formation was decreased in the ov FTO-RAW26.47 group compared with the ov NC-RAW264.7 group.(5)Compared with the control group,the m RNA expression levels of FDPS,DHCR24,FABP4,FDFT1,INSIG1,PLIN2,FABP5,HEBX,LDLR and the expression level of LDLR protein in RAW264.7 cells treated with 80μg/m L ox-LDL for 48h were all decreased(P<0.01).(6)The expression level of LDLR m RNA and protein in sh/ov FTO-RAW264.7 cells was detected.The expression of LDLR m RNA and protein in sh FTO-RAW26.47 group was lower than that in sh NC-RAW264.7 group,while the expression of LDLR m RNA and protein in ov FTO-RAW264.7 group was higher than that in ov NC-RAW264.7group(P<0.05).(7)Compared with the ov NC-RAW264.7 group,the level of m~6A modification of LDLR m RNA was significantly reduced in the ov FTO-RAW26.47 group(P<0.01).ConclusionIn the process of foam cell formation,the down-regulated FTO increased the methylation level of LDLR m RNA,further inhibiting the expression of LDLR.