Long Non-coding RNA XIST Promotes Ox-LDL Induced Pyroptosis Of Endothelial Cells By Inhibiting MiR-22

Backgrounds and aims: Cardiovascular disease(CVD)is the main cause of death around the world,with 70% of CVD mortalities due to sequelae of atherosclerosis.Previous studies have shown that the injury or dysfunction of vascular endothelial cells is associated with the occurrence and development of cardiovascular diseases,including atherosclerosis.Pyroptosis,a type of regulated cell death with inflammatory response,is a new way of cell death and widely involved in the occurrence and development of atherosclerosis.Recent studies have shown that long noncodingRNA regulates gene expression at many levels and is closely related to the pathological process of atherosclerosis.LncRNA X inactivate-specific transcript(XIST)is upregulated in ox-LDL-treated human umbilical vein endothelial cells(HUVECs),and knockout of XIST can improve cell viability and inhibit cell apoptosis.The purpose of this experiment is to explore whether LncRNA XIST could interact with miR-22 to promote endothelial cell pyrotosis induced by oxidized low density lipoprotein.Methods:(1)HUVECs were treated with different concentrations of ox-LDL,and cells were collected at different time points for detection.Cell viability detected by cell morphology and CCK8 assay was used to determine the optimal concentration and time of ox-LDL treatment.(2)To determine the effect of ox-LDL on cell pyroptosis,cells were divided into two groups: Control group(without ox-LDL treatment)and 100μg/m L oxLDL-treated 24 h group.Western blot(WB)was used to detect the expression changes of pyroptosis-related proteins level(NLRP3,Caspase-1 and IL-1β).(3)In order to determine the transfection efficiency of siXIST,cells were divided into three groups: Control group(without any siRNA);si-NC group(transfection with si-NC)and si-XIST group(transfection with si-XIST).The expression levels of LncRNA XIST was detected by RT-PCR.(4)To determine the effects of si-XIST on HUVECs pyroptosis,cells were divided into two groups: ox-LDL + si-NC group and ox-LDL + si-XIST group.CCK8 assay were used to detect the cell activity,and Western blot were used to detect the expression changes of pyroptosisrelated proteins(NLRP3,Caspase-1 and IL-1β).(5)Bioinformatics database was used to predict the target miRNA of LncRNA XIST and double luciferase reporter assay was used to analyze whether LncRNA XIST combined with miR-22.The HUVECs,which were respectively transfected with miR-22 mimic or its negative control group,miR-22 inhibitor or its negative control group,si-XIST or its negative control group,were treated with 100 μg/m L ox-LDL for 24 hours,and the expression levels of miR-22 and XIST were detected by RT-PCR.(6)The cells were transfected with miR-22 mimic or its negative control,miR-22 inhibitor or its negative control,and then adding 100 μg/m L ox-LDL treatment for 24 h.The expression levels of pyroptosis-related proteins(NLRP3,IL-1β,Caspase-1)were detected by WB.(7)In order to determine the effects of down-regulation of miR-22 on the expression changes of pyroptosis proteins in HUVECs influenced by LncRNA XIST upon ox-LDL stimuli,the cells were divided into 4 groups: ox-LDL+ anti-NC inhibitor,ox-LDL+ miR-22 inhibitor group,ox-LDL + miR-22 inhibitor+ si-NC group and ox-LDL + miR-22 inhibitor+ si-XIST group.The expression changes of pyroptosis-related proteins level(NLRP3,Caspase-1 and IL-1β)were detected by WB.Results:(1)HUVECs were stimulated with ox-LDL at different concentrations(0,50,100,150,200 μg/m L)for 12 h,24h and 48 h,respectively.It was found that the cell viability was obviously inhibited after ox-LDL treatment.Considering the results of concentration gradient experiment and time gradient experiment,100μg/m L and 24 h were selected as the optimal concentration and time for establishing ox-LDLinduced cell injury model.(2)After ox-LDL treatment of HUVECs,the expression levels of NLRP3,Caspase-1 and IL-1β were increased.(3)Compared with the control group,the expression levels of LncRNA XIST were successfully inhibited after transfection with si-XIST(P<0.05).(4)Compared with si-NC+ ox-LDL group,the cell viability increased and the expression levels of NLRP3,Caspase-1 and IL-1β decreased significantly after inhibiting the expression of LncRNA XIST.(5)The bioinformatics prediction results showed that LncRNA XIST contained potential binding sites of miR-22.Luciferase report assay showed that miR-22 directly bound to LncRNA XIST.RT-PCR experiments showed that miR-22 mimic and miR-22 inhibitor were successfully transfected into HUVECs.Moreover,lncRNA XIST can inhibite the expression of miR-22.(6)The overexpression of miR-22 can inhibit the ox-LDL-induced HUVECs pyroptosis.On the contrary,the inhibition of miR-22 expression can promote the ox-LDL-induced HUVECs pyroptosis.(7)The promotive effects of miR-22 inhibitor on ox-LDL-induced expression changes of pyroptosis-related proteins in HUVECs were reversed by down-regulation of XIST.Conclusion: Long non-codingRNA XIST promotes ox-LDLinduced endothelial cell pyrotosis by inhibiting miR-22.