Lnc000440 Regulates The Molecular Mechanism Of Lipids Uptake In Monocytes/Macrophages Through ERK1/ATF2 Pathway

Objective:To study the regulatory effect of long non-coding RNA lnc000440 on ox-LDL induced THP-1 monocytes/macrophage lipid uptake and its molecular mechanism.Methods: As a new long-chain non-coding RNA,we first theoretically predict whether long-chain non-coding RNAlnc000440 has the potential to encode proteins by using phylogenetic codon replacement frequency(Phylo CSF).Then GFP sequence was fused to the carboxyl end of the open reading frame(ORF)of long non-coding RNAlnc000440,and the initiation codon of ORF ATG was mutated to ATT as a negative control to further verify whether long non-coding RNAlnc000440 has the ability to encode proteins in the experiment.Then we detected the subdistribution status of lnc000440 in THP-1 cells by isolating the cytoplasmic and nuclear RNA of normal THP-1 cells,using β-actin as the cytoplasmic internal control and U6 as the nuclear internal control,and detecting the content of lnc000440 in the cytoplasm and nucleus by q PCR.Then we use q PCR to detect the expression of long non-coding RNAlnc000440 on treated THP-1 monocytes/macrophages with oxidized low-density lipoproteins(ox-LDL)(0ug/ml,50ug/ml,100ug/ml,150ug/ml,200ug/ml)different concentration gradients to investigate whether lnc000440 is involved in the regulation of cellular atherosclerosis model induced by ox-LDL.We intervene THP-1 cells or THP-1 derived macrophages induced by PMA by constructing long-chain non-coding RNAlnc000440 overexpression lentiviral vector.And we detect the effect of long non-coding RNAlnc000440 on the lipid uptake of THP-1 cells or PMA-induced THP-1derived macrophages through cell oil red O staining,Dil-ox-LDL uptake experiment,Western Blot,q PCR methods to detect the expression levels of SR-A,CD36.In order to further explore the molecular mechanism,we used the Western Blot method to detect the expression levels of T-ERK1/2,p-ERK1/2,T-ATF2,and p-ATF2,and simultaneously detect the expression levels of T-ERK1/2,p-ERK1/2,T-ATF2,and p-ATF2 in the cytoplasm and nucleus to further study whether long-chain non-coding RNAlnc000440 regulates the lipid uptake ability of THP-1 cells by regulating the change of the nucleoplasmic localization of p-ERK1/2-p-ATF2.Finally,all datas use Graph Pad Prism7.00,Adobe Illustrator CS6,Image J software for statistics and data analysis.Results: Our research indicates that lnc000440 is a new long non-coding RNA mainly distributed in the cytoplasm and regulates cellular function by regulating related genes in the cytoplasm.The expression level of lnc000440 decreases in a dose-dependent manner with the concentration of ox-LDL increases in THP-1monocytes/macrophages.Overexpression of lnc000440 can inhibit expression level of CD36 furthermore inhibit the ox-LDL uptake of THP-1 monocytes/macrophages.What’s more,p-ERK1 mainly distributes in cytoplasm and p-ATF2 mainly locates in nucleus in THP-1cells.In ox-LDL-induced THP-1 monocytes/macrophages,the increase of p-ERK1 expression level in the cytoplasm promotes the p-ATF2 transportation to the nucleus,so p-ATF2 expression in the nucleus is also significantly increased.Under ox-LDL induced,over-expression lnc000440 can significantly inhibit the expression of THP-1 monocyclyte/macrophage p-ERK1 and p-ATF2.Conclusions:The long-chain non-coding RNA lnc000440 can delay the foaming of THP-1 monocytes/macrophages by inhibiting lipid uptake.The mechanism is achieved by inhibiting ERK1 phosphorylation,reducing p-ATF2 into the nucleus and reducing the expression of CD36.