摘要
目的 探讨MIR22HG对肺炎支原体诱导的RAW246.7细胞凋亡的影响及分子机制.方法 培养小鼠巨噬细胞RAW246.7,将其分为空白对照组(RAW246.7+10µL磷酸盐缓冲液)、模型细胞(RAW246.7+10 μL肺炎支原体菌株)、模型细胞+si-NC组(RAW246.7+10μL肺炎支原体菌株+转染MIR22HG干扰载体阴性对照)和模型细胞+si-MIR22HG组(RAW246.7+10 μL肺炎支原体菌株+转染MIR22HG干扰载体);采用实时荧光定量PCR(RT-qPCR)检测MIR22HG表达水平,细胞计数试剂盒8(CCK-8)和EdU染色检测细胞增殖,流式细胞术检测细胞凋亡,酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平,蛋白印迹法测定caspase-9、Bax、p-p65、caspase-3及p-IκBα蛋白表达.结果 肺炎支原体诱导的RAW246.7细胞中MIR22HG水平增加,细胞活性和EdU阳性细胞比率下降、凋亡增加,TNF-α、IL-6水平升高,Bax、caspase-9、caspase-3表达水平升高,Bcl-2表达水平降低,p-p65、p-IκBα水平增加,差异有统计学意义(P＜0.05);敲减MIR22HG后,细胞活性、EdU阳性细胞比率和Bcl-2表达增加,凋亡率、IL-6、TNF-α水平、caspase-9、Baxp-p65、caspase-3、p-IKBα表达水平降低,差异有统计学意义(P＜0.05).结论 敲减MIR22HG可能通过下调NF-κB信号通路抑制肺炎支原体诱导的RAW246.7细胞炎症反应和凋亡.
Abstract
Objective To explore the effect and molecular mechanism of MIR22HG on RAW246.7 cell apoptosis induced by Mycoplasma pneumoniae(MP).Methods Macrophages RAW246.7 cells were cultured and divide them into the control group(10 μL phosphate buffer),the model group(10 μL MP strain),the model+si-NC group(transfected with MIR22HG interference vector negative control+10 µL MP strain)and the model+si-MIR22HG group(transfected with MIR22HG interference vector+10 pL MP strain).The expression of MIR22HG was detected by RT-qPCR;cell proliferation was detected by cell counting kit 8(CCK-8)and EdU staining.Flow cytometry was used to detect cell apoptosis,and the levels of TNF-α and IL-6 were detected by enzyme-linked immunosorbent assay(ELISA).Western blot was used to detect Bax,caspase-9,caspase-3,p-p65,and p-IκBα protein expression.Results In RAW246.7 cells induced by MP,the expression level of MIR22HG increased,while the cell activity and EdU positive cell ratio decreased,and the apoptosis rate increased;the levels of TNF-α,IL-6,and the expression levels of Bax,caspase-9,and caspase-3 increased.Bcl-2 expression decreased,while p-p65,p-IκBα expressions increased(P＜0.05).After knocking down MI22HG,cell activity and EdU positive cell ratio increased,while cell apoptosis rate,TNF-α,IL-6 levels,and Bax,caspase-9,caspase-3 expression levels all decreased;Bcl-2 expression increased,while the expression levels of p-p65 and p-IκBα decreased(P＜0.05).Conclusions Knockdown of MIR22HG inhibits the apoptosis and inflammation of RAW246.7 cells induced by MP,which may be related to the NF-κB signaling pathway.
基金项目
山东省医药卫生科技发展计划项目(202205020092)
NETL
NSTL
万方数据