The Mechanism Of METTL3 Mediated M6A Methylation Upregulation Of EGR1 Promoting T2DM Vascular Lesions And The Intervention Effect Of Hdaxc

Objective 1.In this study,we investigated the mechanism of METTL3-mediated m6 A methylation-regulated EGR1 on endothelial to mesenchymal transition(EndMT)in type 2 diabetic vascular lesions through cellular experiments.And,to investigate the intervention effect of Huangdi Anxiao Capsules on type 2 diabetic vascular lesions rats through animal experiments.Methods 1.METTL3 mediated m6 A modification regulates EGR1 m RNA to promote EndMT and participates in T2 DM vascular lesions Cell model of type 2 diabetic vascular lesions was established by H+T-induced human umbilical vein cell endothelial cells(HUVECs),the overall methylation level was verified by colorimetric assay,WB detected the expression of major methylesterase and demethylase proteins,and Screening for MEETL3,a methylation enzyme differentially expressed in vascular lesion cell models.The expression of METTL3 was down-regulated by si RNA technology,and the transfection level of METTL3 was detected by WB and RT-q PCR.The protein expression of endothelial calmodulin(VE-Cadherin)and α-smooth muscle actin(α-SMA)were detected by WB and IF to evaluate the occurrence of EndMT.And the effects of METTL3 on cell proliferation and migration were detected by CCK8 and Transwell assays.Preliminary screening of METTL3-mediated m6 A modification of downstream target gene EGR1 using Me RIP-seq and Me RIP-q PCR;WB and RT-q PCR were used to detect the effect on METTL3 and EGR1 under METTL3 inhibition state.By replying to experiments,we further verified that METTL3 regulates EGR1 expression,evaluated the occurrence of EndMT,and detected cell proliferation and migration.2.HDAXC regulates METTL3 and EGR1 to inhibit EndMT and improve T2 DM vascular lesions High glucose and high fat combined with streptozotocin(STZ)were injected intraperitoneally into SD rats to replicate the type 2 diabetic vascular lesions rat model.Under the intervention state of the Huangdi Anxiao capsules,HE staining was performed to observe the endothelial cell injury in rat thoracic aorta;the expression of METTL3 and EGR1 in rat thoracic aorta was detected by WB and RT-q PCR;the protein expression of endothelial calmodulin(VE-Cadherin)and α-smooth muscle actin(α-SMA),markers of EndMT,was measured by WB.Results 1.METTL3 mediated m6 A modification regulates EGR1 m RNA to promote EndMT and participates in T2 DM vascular lesions METTL3 was highly expressed in H+T-induced HUVECs and the overall methylation level was highly expressed.WB experiments showed that compared with the H+T group,the expression of VE-Cadherin was significantly increased and the expression of α-SMA was decreased in the si-METTL3 group.In addition,IF experiments also confirm the above results.It indicates that METTL3 promotes the occurrence of EndMT in H+T-induced HUVECs.Compared with the H+T group,the si-METTL3 group significantly inhibited the proliferation and migration ability of H+T-induced HUVECs.METTL3 targeted to EGR1 m RNA,Me RIP-q PCR experiments showed that inhibition of METTL3 expression significantly decreased the m6 A level of EGR1;the m6 A level of EGR1 showed a significant increase after METTL3 overexpression.WB,RT-q PCR experiments revealed that by reducing METTL3 expression,EGR1 expression was subsequently changed.It is suggested that METTL3 promotes the expression of EGR1 m RNA through m6 A methylation modification.VE-Cadherin expression was significantly increased and α-SMA expression was significantly reduced,partially reversing the effect of METTL3 deficiency on EndMT.The rescue experiment showed that the cell proliferation and migration ability were reduced in the si-METTL3+EGR1 group,partially reversing the effect of METTL3 deficiency on proliferation and migration.It can be seen that METTL3-mediated m6 A methylation promotes EGR1 expression,thereby promoting EndMT progression in type 2 diabetic vascular lesions.2.HDAXC regulates METTL3 and EGR1 to inhibit EndMT and improve T2 DM vascular lesions After HDAXC intervention,HE staining revealed that endothelial cell injury in rat thoracic aorta was significantly reduced,the expression of METTL3 and EGR1 was significantly decreased;the protein expression level of VE-Cadherin was significantly increased and that of α-SMA was significantly decreased.Therefore,HDAXC inhibited EndMT and improve T2 DM vascular lesions by reducing the expression of METTL3 and EGR1.Conclusions 1.In H+T-induced HUVECs,the overall level of m6 A modification was increased and METTL3 expression was increased.METTL3 is involved in the development of type 2 diabetic vascular lesions by targeting EGR1 m RNA dependent on m6 A modification to promote EndMT progression.2.The mechanism of HDAXC treatment of T2 DM vascular lesions may be related to METTL3,EGR1 expression and EndMT development.