CAJ | 학술논문

目的探讨长链非编码RNA HOX转录反义RNA (HOX transcript antisense RNA,HOTAIR)对人脑胶质瘤细胞代谢和生长的影响.方法采用荧光标记的小干扰HOTAIR (si-HOTAIR)转染胶质瘤U87和U251细胞株,通过实时定量荧光聚合酶链式反应(RT-PCR)检测HOTAIR在胶质瘤细胞中的表达水平;利用CCK8实验和克隆形成实验检测细胞的生长变化;采用海马生物公司糖酵解应激实验和线粒体应激实验试剂盒检测细胞有氧糖酵解和线粒体功能.通过Western blot检测有氧糖酵解相关酶[包括葡萄糖-6-磷酸异构酶(GPI)、丙酮酸激酶M2(PKM2)及乳酸脱氢酶A(LDHA)]的表达水平.结果荧光显微镜下观察U87和U251细胞中si-HOTAIR转染效率均＞90％.与阴性对照(NC)组比较,si-HOTAIR组细胞中HOTAIR的表达均明显下调(P＜0.01);si-HOTAIR组U87和U251胶质瘤细胞的增殖能力和克隆形成能力明显低于NC组(P＜0.01);下调HOTAIR后,胶质瘤细胞有氧糖酵解能力减弱,表现为基础糖酵解水平降低(P＜0.05)、最大糖酵解能力减弱(P＜0.01)及糖酵解剩储备量下降(P＜0.01);si-HOTAIR组GPI、PKM2和LDHA表达水平较NC组降低(P＜0.01).下调HOTAIR后,胶质瘤细胞线粒体功能受损,与NC组比较,表现为基础呼吸水平下降(其中U87细胞P＜0.01)、线粒体偶联效率降低及剩余呼吸能力减弱(U87和U251组均P＜0.05).结论下调HOTAIR表达可以使人胶质瘤细胞糖酵解能力和线粒体功能受损,同时影响胶质瘤细胞的生长能力.
목적 탐토장련비편마RNA HOX전록반의RNA (HOX transcript antisense RNA,HOTAIR)대인뇌효질류세포대사화생장적영향.방법 채용형광표기적소간우HOTAIR (si-HOTAIR)전염효질류U87화U251세포주,통과실시정량형광취합매련식반응(RT-PCR)검측HOTAIR재효질류세포중적표체수평;이용CCK8실험화극륭형성실험검측세포적생장변화;채용해마생물공사당효해응격실험화선립체응격실험시제합검측세포유양당효해화선립체공능.통과Western blot검측유양당효해상관매[포괄포도당-6-린산이구매(GPI)、병동산격매M2(PKM2)급유산탈경매A(LDHA)]적표체수평.결과 형광현미경하관찰U87화U251세포중si-HOTAIR전염효솔균＞90％.여음성대조(NC)조비교,si-HOTAIR조세포중HOTAIR적표체균명현하조(P＜0.01);si-HOTAIR조U87화U251효질류세포적증식능력화극륭형성능력명현저우NC조(P＜0.01);하조HOTAIR후,효질류세포유양당효해능력감약,표현위기출당효해수평강저(P＜0.05)、최대당효해능력감약(P＜0.01)급당효해잉저비량하강(P＜0.01);si-HOTAIR조GPI、PKM2화LDHA표체수평교NC조강저(P＜0.01).하조HOTAIR후,효질류세포선립체공능수손,여NC조비교,표현위기출호흡수평하강(기중U87세포P＜0.01)、선립체우련효솔강저급잉여호흡능력감약(U87화U251조균P＜0.05).결론 하조HOTAIR표체가이사인효질류세포당효해능력화선립체공능수손,동시영향효질류세포적생장능력.
Objective To investigate the influence of long noncoding RNA HOX transcript antisense RNA (HOTAIR) on human glioma cell metabolism and growth.Methods The fluorescently labeled small interfering (si-HOTAIR) was used to transfect glioma U87 and U251 cell lines.The real-time polymerase chain reaction (RT-PCR) was used to detect the expression level of HOTAIR in glioma cells.CCK8 test and colony formation assay were used to detect the growth changes of the glioma cells.Seahorse BioScience XF glycilysis stress test kit and XF mitochondrial stress test kit were used to detect the cell aerobic glycolysis and mitochondrial function.Western blot was used to detect glycolysis related enzymes,including the expression levels of glucose-6-phosphate isomerase (GPI),Pyruvate kinase M2 (PKM2),and lactate dehydrogenase A (LDHA).Results The transfection efficiency of si-HOTAIR in U87and U251cells observed under a fluorescence microscope was more than 90％.The HOTAIR expression in the cells of the si-HOTAIR group was downregulated significantly compared with the negative control group (P ＜ 0.01).The proliferative ability and clonal formation ability of the U87and U251 glioma cells of the si-HOTAIR group were significantly lower that than those of the negative group (P ＜ 0.01).After downregulation of HOTAIR,the aerobic glycolysis capacity of glioma cells were diminished,showing reduced based glycolysis level (P ＜ 0.05),diminished maximum glycolytic capacity (P ＜ 0.01),and decreased remaining amount of glycolysis (P ＜ 0.01).The expression levels of GPI,PKM2 and LDHA of the si-HOTAIR group were lower than those of the negative control group (P ＜ 0.01).After downregulation of HOTAIR,the mitochondrial function in glioma cells was impaired,showing decreased basal respiration level (U87 cells P ＜ 0.01),decreased mitochondrial coupling efficiency,and diminished residual respiration ability (P ＜ 0.05).Conclusions Downregulation of HOTAIR expression may impair the glycolytic capacity and mitochondrial function of glioma cells,simultaneously influence the growing ability of glioma cells.