敲低lncRNA LINC01296通过Wnt/β-catenin通路抑制骨肉瘤细胞增殖能力及干性特征  

Knockdown of lncRNA LINC01296 Inhibited Proliferation and Stemcell Characteristics of Osteosarcoma Cells through Wnt/β-catenin Pathway

作　　者：刘广飞[1] 郭志远 王艳花 卢守亮 郭磊[3] 程才[1] LIU Guangfei;GUO Zhiyuan;WANG Yanhua;LU Shouliang;GUO Lei;CHENG Cai(Department of Orthopedics,the Central Hospital of Cangzhou,Cangzhou,Hebei,061000,China;ECG Room,the Central Hospital of Cangzhou,Cangzhou,Hebei,061000,China;Department of Rehabilitation,Hejian Branch of Cangzhou Central Hospital,Cangzhou,Hebei,062450,China)

机构地区：[1]沧州市中心医院骨一科,河北省沧州市061000 [2]沧州市中心医院心电图室,河北省沧州市061000 [3]沧州市中心医院河间分院康复科,河北省沧州市062450

出　　处：《医学分子生物学杂志》2025年第2期166-172,共7页Journal of Medical Molecular Biology

基　　金：河北省卫生健康委科研基金(No.20220381)。

摘　　要：目的探究敲低长链非编码RNA(lncRNA)LINC01296对人骨肉瘤(osteosarcoma,OS)细胞增殖能力和干性特征的影响,初步探究相关机制。方法培养人正常成骨细胞系hFOB1.19与人OS细胞系MG63、U2OS、Saos2,实时荧光定量PCR(qRT-PCR)检测lncRNA LINC01296的表达情况。将MG63细胞分为对照组、siRNA阴性对照组(阴性对照NC siRNA转染至细胞)、LINC01296 siRNA干扰组(LINC01296 siRNA转染至细胞)、LINC01296 siRNA+LiCl组(LINC01296 siRNA转染至细胞并用10 mmol/L Wnt/β-catenin通路激活剂LiCl培养24 h),检测各组中lncRNA LINC01296表达情况,CCK-8法检测各组细胞存活率,EdU染色观察各组细胞增殖情况,肿瘤细胞球体形成实验检测各组细胞的球体数目,蛋白质印迹法检测各组细胞中干性相关蛋白(Nanog、OCT-4、SOX2)及Wnt/β-catenin通路相关蛋白(β-catenin、c-Myc、Cyclin D1)的表达水平。结果人OS细胞系MG63、U2OS、Saos2中lncRNA LINC01296相对表达量显著高于hFOB1.19细胞(P<0.05)。与对照组比较,LINC01296 siRNA组MG63细胞存活率显著降低(P<0.05),EdU阳性细胞比例显著降低(P<0.05),肿瘤细胞球体数目显著减少(P<0.05),细胞中Nanog、OCT-4、SOX2及β-catenin、c-Myc、Cyclin D1的蛋白相对表达量均显著下调(P<0.05);与LINC01296 siRNA组比较,LINC01296 siRNA+LiCl组MG63细胞存活率显著升高(P<0.05),EdU阳性细胞比例也显著升高(P<0.05),肿瘤细胞球体数目显著增加(P<0.05),同时细胞中Nanog、OCT-4、SOX2及β-catenin、c-Myc、Cyclin D1的蛋白相对表达量均显著上调(P<0.05)。结论在OS细胞中靶向敲低lncRNA LINC01296表达能够抑制细胞增殖能力,降低细胞干性特征,该机制可能与抑制Wnt/β-catenin通路的激活有关。Objective To investigate the effect of knocking down long non-coding RNA(lncRNA)LINC01296 on the proliferation capacity and stem-cell characteristics of human osteosarcoma(OS)cells,and to explore the related mechanisms.Methods Human normal osteoblast cell line hFOB1.19 and human OS cell lines MG63,U2OS and Saos2 were cultured,and the expression level of lncRNA LINC01296 was detected by qRT-PCR.MG63 cells were divided into 4 groups:control group,siRNA negative control group(negative control siRNA was transfected into cells),LINC01296 siRNA interference group(LINC01296 siRNA was transfected into cells),LINC01296 siRNA+LiCl group(LINC01296 siRNA was transfected into cells and cultured with 10 mmol/L Wnt/β-catenin pathway activator LiCl for 24 h).The expression level of lncRNA LINC01296 in each group was detected,the survival rate of cells in each group was detected by CCK-8 method,the proliferation of cells was observed by EdU staining,and the spheroid number in each group was detected by tumor cell spheroid formation assay,the expression levels of stem-cell related proteins(Nanog,OCT-4,SOX2)and Wnt/β-catenin pathway-related proteins(β-catenin,c-Myc,Cyclin D1)in cells of each group were detected by Western blotting.Results The relative expression level of lncRNA LINC01296 in the human OS cell lines MG63,U2OS and Saos2 was significantly higher than that in the hFOB1.19 cells(P<0.05).The survival rate,the proportion of EdU positive cells,and the number of tumor cell spheres of MG63 cells in the LINC01296 siRNA group was significantly decreased when compared with those in the control group(all P<0.05),in addition,the relative expression levels of Nanog,OCT-4,SOX2,β-catenin,c-Myc and Cyclin D1 in the LINC01296 siRNA group were significantly down-regulated(P<0.05).The survival rate,the proportion of EdU positive cells,and the number of tumor cell spheres of MG63 cells in the LINC01296 siRNA+LiCl group was significantly increased when compared with those in the LINC01296 siRNA group(all P<0.05),at the same time,the re

关 键 词：骨肉瘤 长链非编码RNA LINC01296 增殖 肿瘤干性 WNT/Β-CATENIN通路 

分 类 号：R738.1[医药卫生—肿瘤]