同型半胱氨酸和lncRNA ANRIL对动脉粥样硬化炎症反应的影响

Effects of Hcy and lncRNA ANRIL on atherosclerotic inflammatory response

目的研究血清同型半胱氨酸(Hcy)水平与长链非编码RNA(lncRNA)细胞周期依赖激酶抑制剂2B基因的反义非编码基因(ANRIL)的相关性及对动脉粥样硬化炎症,即人脐静脉内皮细胞(HUVEC)表达白介素-10(IL-10)和单核细胞趋化蛋白-1(MCP-1)的影响。方法体外培养HUVEC,给予不同浓度梯度(空白对照组、0.5、1.0、2.0、5.0 mmol/L)Hcy处理细胞。用实时荧光定量聚合酶链式反应(qRT-PCR)检测lncRNA ANRIL表达水平。酶联免疫吸附(ELISA)法检测MCP-1及IL-10含量。用LipoFiter转染试剂分别将shANRIL、shNC转染入不同细胞内。再选取前述实验中最佳Hcy浓度(5.0 mmol/L)干预24 h进行实验。蛋白免疫印迹(Western blot)检测MCP-1及IL-10的蛋白表达水平。结果用不同浓度Hcy干预HUVEC 24 h后,Hcy明显损伤内皮细胞,且Hcy浓度越高,细胞损伤越严重。与空白对照组相比,Hcy干预组lncRNA ANRIL、MCP-1增加,IL-10降低(P<0.05);随着Hcy干预浓度增加,IL-10降低,lncRNA ANRIL、MCP-1升高(均P<0.05)。与空白对照组相比,Hcy组、shNC+Hcy组、shANRIL+Hcy组IL-10蛋白表达量较低、MCP-1蛋白表达量较高(均P<0.05)。与shANRIL+Hcy组相比,Hcy组、shNC+Hcy组IL-10蛋白表达量较低、MCP-1蛋白表达量较高(均P<0.05)。shNC+Hcy组及Hcy组间IL-10蛋白、MCP-1蛋白表达量差异无统计学意义(均P>0.05)。结论Hcy通过促进lncRNA ANRIL表达,使MCP-1表达上调、IL-10表达下调,从而促进细胞炎症反应,参与动脉粥样硬化。

Objective To study the relationship between the level of serum homocysteine(Hcy)and the antisense non coding gene(ANRIL)of long chain non coding RNA(lncRNA)cell cycle dependent kinase inhibitor 2B gene,and the effect on Atherosclerosis inflammation,that is,the expression of interleukin-10(IL-10)and Monocyte chemoattractant protein-1(MCP-1)in Human umbilical vein endothelial cell(HUVEC).Methods HUVEC was cultured in vitro and cells were treated with different concentration gradients(blank control group,0.5,1.0,2.0,5.0 mmol/L)of Hcy.The expression level of lncRNA ANRIL was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of MCP-1 and IL-10.LipoFilter transfection reagents were used to transfect shANRIL and shNC into different cells,respectively.In the above experiment,the optimal Hcy concentration(5.0 mmol/L)was selected for intervention for 24 hours.Western blot was used to detect the protein expression levels of MCP-1 and IL-10.Results After 24 hours of intervention with different concentrations of Hcy in HUVEC,Hcy significantly damaged endothelial cells,and the higher the Hcy concentration,the more severe the cell damage.Compared with the blank control group,the Hcy intervention group showed an increase in lncRNA ANRIL and MCP-1,while IL-10 decreased(all P<0.05);As the concentration of Hcy intervention increases,IL-10 decreases,while lncRNA ANRIL and MCP-1 increased(all P<0.05).Compared with the blank control group,the Hcy group,the shNC+Hcy group,and the shANRIL+Hcy group had lower levels of IL-10 protein expression and higher levels of MCP-1 protein expression(all P<0.05).Compared with the shANRIL+Hcy group,the Hcy group and the shNC+Hcy group had lower levels of IL-10 protein expression and higher levels of MCP-1 protein expression(all P<0.05).There was no statistically significant difference in the expression levels of IL-10 protein and MCP-1 protein between the shNC+Hcy group and the Hcy group(all P>0.05).Conclusions Hcy upregulates MCP-1 expression and downregulates IL-10 expression by promoting lncRNA ANRIL expression.Thus,it can promote cellular inflammatory reaction and participate in Atherosclerosis.