Role Of Long Non-codingrna-NONHSAT247851.1 In PM_2.5-induced Inflammation In Human Umbilical Vein Endotheilial Cells ?HUVECs?

Objective:To study the inflammatory response of human umbilical vein endothelial cells?HUEVCs?induced by haze PM_2.5 collected in Guangzhou and reveal the regulation mechanism of long non-coding RNA?lncRNA?in the inflammatory effect of cardiovascular system,and further understand the molecular toxicological mechanisms of PM_2.5 exposure-induced inflammation.Method:HUVECs were cultured in vitro and exposed to PM_2.5 suspension to establish an inflammatory model.CCK8 method was used to detect the toxicity of PM_2.5.5 of different concentrations on HUVECs,Elisa and qRT-PCR were used to detect the expression level of inflammatory factors in HUVECs.A high throughput sequencing was used to obtain inflammation-related lncRNA,and qRT-PCR was used to verify the authenticity of the sequencing results.Combined with sequencing results,lncRNA?NONHSAT24751.1?was selected to continue the subsequent experiments.The cytoplasmic nuclear subcellular localization experiment was performed on.siRNA interference was used to construct a loss-of-function system,NONHSAT24751.1 was silenced and then exposed to PM_2.5 to verify its role in the process of PM_2.5 induced endothelial injury.Untreated HUVECs were collected for ChIRP-MS experiments to explore proteins that specifically bind to NONHSAT24751.1.Small RNA interference experiments combined with PM_2.5 exposure for 48 hours,then extract proteins for Western blot.Bioinformatics analysis revealed that NF?B may be one of the transcripts of NONHSAT24751.1,so specific blockers BAY11-7082?10?M?were used to treat cells and then exposed to PM_2.5,which verified whether NF?B was involved in the expression of PM_2.5 induced NONHSAT24751.1.Results:?1?PM_2.5 induces an inflammatory response in HUVECs.The results of CCK8 showed that compared with the control group,with the increase of PM2.5 exposure dose,HUVECs cell viability decreased gradually.However,the transcriptional and protein levels of inflammatory proteins IL1?and IL6 increased with increasing doses,showing a positive dose-effect correlation.?2?Screening of inflammatory lncRNAsBiological analysis was performed on abnormal lncRNAs obtained by high-throughput sequencing,and 6 lncRNAs related to inflammation were screened out.The qRT-PCR verified that the expression levels of NONHSAT247851.1 increased with the dose of PM_2.5,showing a certain dose-effect relationship.?3?Functional verification of NONHSAT247851.1siRNA was used to reduce the expression of NONHSAT24751.1 in HUVECs,and then stimulated with PM_2.5.qRT-PCR and elisa showed that the transcription and protein levels of IL1?in the transfected group decreased compared with the non-transfected group.?4?Study on the proinflammatory mechanism of NONHSAT247851.1The cytoplasmic nuclear subcellular localization experiment found that NONHSAT247851.1 is located in the nucleus.The ChIRP-MS experiment found that raf-1 can specifically bind to NONHSAT247851.1.Western blot analysis showed that the phosphorylation degree of raf-1 and p65 protein in NC+PM_2.5 group was much higher than that in siRNA+PM_2.5 group.qRT-PCR results showed that NONHSAT247851.1transcription was decreased after BAY11-7082?10?M?pretreatment,and the protein level of IL1?was also significantly decreased by ELISA.Conclusion:?1?NONHSAT24751.1 plays a positive role in the inflammatory response of HUVECs induced by PM_2.5.?2?NONHSAT24751.1 binds to raf-1 protein to form RNA-protein complex.NONHSAT24751.1 promotes the phosphorylation of raf-1 and p65proteins when PM_2.5.5 exposure to HUVECs.?3?NF?B can promote the expression of NONHSAT24751.1 when PM_2.5stimulates HUVECs.