LncRNA MALAT1 Promotes Ox-LDL-Induced Autophagy In HUVECs By Inhibiting The PI3K/AKT Pathway

Objective 1.To explore the effect of oxidized low density lipoprotein(ox-LDL)on autophagy of human umbilical vein endothelial cells(HUVEC).2.To investigate the expression level of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1(MALAT1)and the activation of PI3K/AKT signal pathway in HUVECs induced by ox-LDL.3.To identify the effects of MALAT1 on autophagy and PI3K/AKT pathway in ox-LDL-induced HUVECs.Methods 1.HUVECs were collected after incubation with 100 ug/ml ox-LDL for 12 h,and Western blot analysis were used to detect the expression level of autophagy related proteins LC3-II of HUVECs,immunofluorescence staining were used to detect the accumulation of LC3 puncta in cells using a confocal laser scanning microscopy,the form and quantity change of autophagic vesicles were examined under transmission electron microscopy.Futher,we detected the expression level of LC3-II,the number of LC3 puncta and autophagic vesicles of ox-LDL-induced HUVECs in the presence of lysosomal inhibitor Bafilomycin A1.2.HUVECs were cultured with 100 ug/ml ox-LDL for 12 h,the level of MALAT1 were detected by q RT-PCR analysis,the protein expression levels of p-PI3 K,PI3K,AKT,p-AKT,RHEB,p-p70S6 K and p70S6 K in cells were detected by Western blot analysis.3.Tansfected the MALAT1 expression vector and its negative control into HUVECs using lipofectamine 2000,then treated HUVECs with 100 ug/ml ox-LDL for 12 h,the effect of MALAT1 overexpression on autophagy of ox-LDL-induced HUVECs were detected by Western blot,immunofluorescence staining and transmission electron microscope,the effect of MALAT1 overexpression on p-PI3 K,PI3K,AKT,p-AKT,RHEB,p-p70S6 K,and p70S6 K protein levels of ox-LDL-induced HUVECs were detected by Western blot.si MALAT1 and the negative control(si Control)were transfected into HUVECs to knockdown the expression of MALAT1,after 24 h,HUVECs were treated with ox-LDL for 12 h,Western blot,immunofluorescence staining and transmission electron microscope were used to detected the effect of MALAT1 downregulation on autophagy and PI3K/AKT signal pathway in ox-LDL induced in HUVECs.Results 1.After treatment with ox-LDL for 12 h,the results of western blot showed LC3-II protein levels were upregulated,immunofluorescence staining showed an increase of the number of LC3 puncta,transmission electron microscopy showed an increase of the number of autophagic vesicles.Moreover,ox-LDL led to further accumulation of LC3-II levels,LC3 puncta and autophagic vesicles in the presence of bafilomycin A1.2.Ox-LDL upregulated the expression of MALAT1 in HUVECs according to q RT-PCR;Ox-LDL treatment downregulated the p-PI3 K,p-Akt,RHEB,and p-p70S6 K levels without affecting the total expression levels of PI3 K,AKT,p70S6 K according to western blot.3.p MALAT1 transfection showed an increase in LC3-II protein levels by Western blot,and the MALAT1 overexpression group had a higher number of LC3 puncta according to the fluorescence microscopy images and a higher number of autophagic vesicles as observed under a transmission electron microscope.Western blot analysis showed that p MALAT1 transfection inhibited PI3 K,Akt and p70S6 K phosphorylation,decreased RHEB in ox-LDL-induced HUVECs.MALAT1 downregulation decreased the LC3-II protein level and the number of LC3 puncta and autophagic vesicles,increased the p-PI3 K,p-Akt,RHEB and p-p70S6 K protein levels in HUVECs.Conclusion Ox-LDL could induce autophagy,upregulate the expression of MALAT1 and inhibit the PI3K/Akt pathway in HUVECs.MALAT1 promotes ox-LDL-induced autophagy in HUVECs partly through the PI3K/AKT signaling pathway.