Long Noncoding RNA XXYLT1-AS2 Regulates Proliferation And Adhesion By Targeting The RNA Binding Protein FUS In HUVEC

Objective This study investigated that underlying mechanism of lncRNA XXYLT1-AS2 on endothelial dysfunction in atherosclerosis,leading to provided a novel lncRNA to serve as a novel therapeutic strategies for atherosclerosis.Methods(1)The bioinformatics method was used to analyze the GEO database to obtain differentially expressed genes,and a rat model of carotid balloon injury was constructed.RT-PCR and RT-qPCR were used to identify the species conservation of lncRNA-XXYLT1-AS2 and detect the expression in cell lines and carotid arteries in rat balloon injury models.(2)To synthesize the target piRNA to overexpress RNA and inhibit the expression of RNA,the ability of proliferation and migration was evaluated by EdU assay,CCK-8 assay,and scratch test in HUVEC respectively.(3)The effects of XXYLT1-AS2 on endothelial cell adhesion and related adhesion and inflammatory factor expression with the treatment of inflammatory stimulation of TNF-? were detected by Western blot,monocyte adhesion experiment and RT-qPCR.(4)The cellular localization of XXYLT1-AS2 was explored by FISH,and we utilized CatRAPID,an online tool to identify supposed protein binding to lncRNA,to evaluate the interaction of XXYLT1-AS2 and FUS which predicted by the StarBase Browser.Moreover,RIP,IF,RNA pulldown,and FISH were used to analyze the interaction of XXYLT1-AS2 with FUS.(5)si-FUS was synthesized,and the effects of FUS on the biological functions of HUVEC proliferation,migration,and adhesion inflammation were detected by CCK8,EdU,cell scratch test,Western blot,monocyte adhesion test,and RT-qPCR.(6)Co-transfection of lncRNA and target protein treated with ox-LDL in HUVEC.Western blot,CCK8,EdU,and cell scratch tests were performed to test whether XXYLT1-AS2 could target the downstream protein FUS to regulate CCND1,which functioned in HUVEC biological function.(7)Normal arteries in healthy individuals and diseased arteries in patients with atherosclerosis were collected.IHC and FISH experiments were performed to test the expression of lncRNA and FUS in each group.Results(1)XXYLT1-AS2 was identified by the analysis of GEO datasets,which was highly conserved in rats rather than the mouse and XXYLT1-AS2 was abundantly expressed in HUVEC and THP-1.Moreover,we observed that XXYLT1-AS2 clearly decreased in the carotid artery of the rat model of balloon injury at different time points(P<0.05).(2)SiRNAs and plasmid were constructed to test the efficiency of knockdown and overexpression in HUVEC,the results of CCK8,EdU and cell scratch experiments showed that cells transfected with lncRNA-XXYLT1-AS2 had a negative effect on proliferation and migration compared with the NC group(P<0.05).(3)The monocyte adhesion assay and RT-qPCR were examined to observe the effect of XXYLT1-AS2 on cell adhesion,adhesion and inflammatory factors expression.The results showed that compared with NC,the lncRNA overexpression group significantly inhibited cell adhesion and expression of adhesion inflammatory factors(P<0.05).(4)XXYLT1-AS2 was detected to locate in nucleus rather than cytoplasm by FISH,and CatRAPID,an online tool,was utilized to identify supposed protein binding to lncRNA.The interaction of XXYLT1-AS2 and FUS was evaluated by RIP,IF,RNA pulldown,and FISH.(5)Reduced FUS expression enhanced cell proliferation and migration by EDU,CCK8,and cell scratch test assay.Meanwhile,the knockdown of FUS led to facilitate adhesion of THP-1 and resulted in increased VCAM-1 and MCP-1 expression.(6)HUVECs were stimulated by ox-LDL,CCK8,EdU,and scratch detection results showed that compared with the lncRNA overexpression group,cell proliferation and migration efficiency were significantly increased in the group which co-transfected XXYLT1-AS2 and si-FUS(P<0.05).(7)Compared with the NC group,the expressions of lncRNA-XXYLT1-AS2 and FUS were decreased in aorta of AS group(P<0.05).Conclusions XXYLT1-AS2 was significantly downregulated in atherosclerotic plaques which exerted a protective role in atherosclerosis via regulation of endothelial dysfunction by directly targeting FUS/CCND1 pathway.Moreover,XXYLT1-AS2 and FUS also alleviated monocyte recruitment and inflammation by mediating activation AKT and NF-?B signaling.In summary,all these evidences provided a novel evidence that lncRNA modulates endothelial dysfunction to serve as a novel therapeutic strategies for atherosclerosis.