摘要
目的探究微RNA-27a(microRNA-27a,miR-27a)调控磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)通路对脂多糖(lipopolysaccharide,LPS)诱导的人肺腺癌细胞株A549凋亡的影响,并初步探讨其机制。方法Starbase分析miR-27a与磷脂酰肌醇-3激酶催化亚基δ(phosphatidylinositol-3 kinase catalytic subunit delta,PIK3CD)存在互补的结合位点,并以双荧光素酶验证。将A549细胞分为正常组、LPS组、LPS+miR-27a模拟物阴性对照组、LPS+miR-27a模拟物组、LPS+miR-27a模拟物+PI3K激活剂组。LPS+miR-27a模拟物阴性对照组、LPS+miR-27a模拟物组、LPS+miR-27a模拟物+PI3K激活剂组分别转染miR-27a模拟物阴性对照、LPS+miR-27a模拟物、LPS+miR-27a模拟物,培养细胞6 h,更换为完全培养液培养细胞24 h,然后除正常组外,其余各组细胞添加10 mg/L LPS刺激24 h,且LPS+miR-27a模拟物+PI3K激活剂组加入PI3K激活剂740 Y-P,正常组细胞完全培养液正常培养相同时间。实时荧光定量聚合酶链反应检测细胞中miR-27a表达水平;细胞计数试剂盒8检测细胞增殖情况;Hoechst33342染色与流式细胞术检测细胞凋亡情况;透射电镜观察A549细胞自噬;蛋白免疫印迹检测细胞中PIK3CD、磷酸化AKT(phosphorylated-AKT,p-AKT)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、活化的半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)、微管相关蛋白1轻链3Ⅱ(microtubule-associated protein 1light chain 3 II,LC3Ⅱ)蛋白表达。结果miR-27a与PIK3CD存在结合位点,并经双荧光素酶验证。与正常组相比,LPS组、LPS+miR-27a模拟物阴性对照组A549细胞中miR-27a表达水平、增殖率、Bcl-2蛋白表达水平降低(P<0.05),凋亡率、细胞中PIK3CD、p-AKT、Bax、cleaved caspase-3、LC3Ⅱ蛋白表达水平升高(P<0.05);分别与LPS组、LPS+miR-27a模拟物阴性对照组相比,LPS+miR-27a模拟物组A549细胞中miR-27a表达水平、增殖率、细胞中Bcl-2蛋白表达水平升高(P<0.05),凋亡率、细胞中PIK3CD、p-AKT、Bax、cleaved caspase-3、LC3Ⅱ蛋白表达水平降低(P<0.05);与LPS+miR-27a模拟物组相比,LPS+miR-27a模拟物+PI3K激活剂组细胞中miR-27a表达水平、增殖率降低(P<0.05),凋亡率、细胞中PIK3CD、p-AKT、cleaved caspase-3、LC3Ⅱ蛋白表达水平升高(P<0.05)。正常组细胞数量较多,细胞间排列紧密,可见核大小均一,细胞器结构正常;LPS组和LPS+miR-27a模拟物阴性对照组可见细胞变圆,细胞核固缩,形成团块现象,显现多个大小不一的圆形自噬泡;LPS+miR-27a模拟物组核固缩细胞数降低,LPS+miR-27a模拟物+PI3K激活剂组核固缩细胞数较LPS+miR-27a模拟物组升高,均可见少量圆形自噬泡,但数目不一。结论高表达miR-27a可以抑制PI3K/AKT通路,从而减轻LPS诱导的人肺腺癌细胞A549凋亡,而这一途径可能与自噬减少相关。
Objective To investigate the effect of microRNA-27a(miR-27a)on the apoptosis of human lung adenocarcinoma cells A549 induced by lipopolysaccharide(LPS)by regulating the phosphatidylinositol-3-kinase(PI3K)/protein kinase B(AKT)pathway,and its mechanism is discussed preliminarily.Methods The complementary binding sites of miR-27a and phosphatidylinositol-3 kinase catalytic subunit delta(PIK3CD)were analyzed by Starbase and verified by double luciferase.The A549 cells were divided into normal group,LPS group,LPS+miR-27a mimic negative control group,LPS+miR-27a mimic group,LPS+miR-27a mimic+PI3K activator group.In the LPS+miR-27a mimic negative control group,LPS+miR-27a mimic group and LPS+miR-27a mimic+PI3K activator group,the cells were transfected with miR-27a mimic negative control,miR-27a mimic and miR-27a mimic,respectively,and were cultured for6 h.After that,the cells were cultured in complete medium for 24 h,and then,except for the normal group,the cells in the other groups were stimulated with 10 mg/L LPS for 24 h,and the PI3K activator 740 Y-P was added to the LPS+miR-27a mimic+PI3K activator group,and cells in normal group were cultured in complete medium for the same time.Real-time quantitative polymerase chain reaction was used to detect the expression level of miR-27a in cells;cell counting kit 8 was used to detect cell proliferation;Hoechst33342 staining and flow cytometry was used to detect apoptosis;autophagy of A549 cells was observed by transmission electron microscope;Western blot was used to detect the expression of PIK3CD,phosphorylated-AKT(p-AKT),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved caspase-3 and microtubule-associated protein 1 light chain 3 II(LC3II)protein.Results There was a binding site between miR-27a and PIK3CD,which was verified by double luciferase.Compared with those in normal group,the expression level of miR-27a,proliferation rate and protein expression level of Bcl-2 in LPS group and LPS+miR-27a mimic negative control group were lower(P<0.05),the apoptosis rate,protein expression levels of PIK3CD,p-AKT,Bax,cleaved caspase-3,LC3Ⅱwere higher(P<0.05);compared with those in LPS group and LPS+miR-27a mimic negative control group,the expression level of miR-27a,proliferation rate and protein expression level of Bcl-2 in LPS+miR-27a mimic group were higher(P<0.05),the apoptosis rate,protein expression levels of PIK3CD,p-AKT,Bax,cleaved caspase-3,LC3Ⅱwere lower(P<0.05);compared with those in LPS+miR-27a mimic group,the expression level of miR-27a and proliferation rate in LPS+miR-27a mimic+PI3K activator group were lower(P<0.05),the apoptosis rate,protein expression levels of PIK3CD,p-AKT,cleaved caspase-3,LC3Ⅱwere higher(P<0.05).The number of cells in the normal group was more,the cells were closely arranged,the nucleus size was uniform,and the organelle structure was normal;in LPS group and LPS+miR-27a mimic negative control group,cells became round,nuclei pyknosis,formed clumps,and showed multiple round autophagic vesicles of different sizes;the number of nuclear pyknotic cells in LPS+miR-27a mimic group decreased,and the number of nuclear pyknotic cells in LPS+miR-27a mimic+PI3K activator group increased compared with LPS+miR-27a mimic group,a small number of circular autophagic vesicles were observed,but the number was different.Conclusion Overexpression of miR-27a can inhibit PI3K/Akt pathway and reduce LPS induced apoptosis of human lung adenocarcinoma cells A549,which may be related to the reduction of autophagy.
作者
朱锦琪
陈剑波
杨红忠
高欣
ZHU Jinqi;CHEN Jianbo;YANG Hongzhong;GAO Xin(Department of Respiratory and Critical Care Medicine,Changsha Central Hospital Affiliated to Nanhua University,Changsha,Hunan 410004,P.R.China;Department of Lung,Changsha Central Hospital Affiliated to Nanhua University,Changsha,Hunan 410004,P.R.China)
出处
《中国呼吸与危重监护杂志》
CAS
CSCD
北大核心
2022年第2期118-125,共8页
Chinese Journal of Respiratory and Critical Care Medicine
基金
湖南省卫生健康委科研立项课题(20200645)。
