长链非编码RNA淋巴样增强因子1反义RNA 1调控miR-339-5p对骨肉瘤细胞增殖和凋亡作用的研究

Effect of LEFl-AS1 on osteosarcoma cell proliferation and apoptosis regulated by miR-339-5p

背景:长链非编码RNA(lncRNA)淋巴样增强因子1反义RNA 1(LEF1-AS1)在骨肉瘤中的生物学意义及机制尚不清楚。目的:研究LEF1-AS1对微小RNA(miR)-339-5p的靶向调控及对骨肉瘤细胞增殖和凋亡的影响。方法:实时荧光定量PCR(qRT-PCR)检测LEF1-AS1、miR-339-5p在人骨肉瘤组织和瘤旁组织中的表达。在细胞U2OS中转染si-LEF1-AS1,细胞计数试剂盒8(CCK-8)和克隆形成测定细胞增殖与克隆能力,流式细胞术检测细胞凋亡,免疫印迹实验检测P21和含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)蛋白表达。生物信息学预测和双荧光素酶报告实验分析LEF1-AS1和miR-339-5p的靶向关系。在细胞U2OS中共转染si-LEF1-AS1和anti-miR-339-5p,或共转染siLEF1-AS1和miR-339-5p,观察其在细胞增殖和凋亡中的作用。结果:与瘤旁组织比较,骨肉瘤组织中的LEF1-AS1表达量明显升高,miR-339-5p表达量显著减少(P<0.05)。抑制LEF1-AS1明显降低细胞U2OS的存活率和克隆形成数,显著提高凋亡率、P21和Caspase-3蛋白水平(P<0.05)。LEF1-AS1靶向、调控miR-339-5p的表达。si-LEF1-AS1和anti-miR-339-5p共转染明显减少细胞U2OS中miR-339-5p表达量、P21、Caspase-3蛋白表达量、细胞凋亡率,显著增加细胞U2OS存活率和克隆形成数(P<0.05)。si-LEF1-AS1和miR-339-5p共转染明显提高miR-339-5p表达量、P21、Caspase-3蛋白表达量和细胞凋亡率,显著降低细胞U2OS存活率和克隆形成数(P<0.05)。结论:LEF1-AS1靶向调控miR-339-5p的表达,抑制LEF1-AS1可抑制骨肉瘤细胞的增殖并诱导细胞凋亡。

Background:The biological significance and mechanism of long-chain non-coding RNA(lncRNA)lymphoid enhancer-binding factor 1 antisense RNA 1(LEF1-AS1)in osteosarcoma is still unclear.Objective:To study the targeted regulation of LEF1-AS1 on microRNA(miR)-339-5p and its effect on osteosarcoma cell proliferation and apoptosis.Methods:Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of LEF1-AS1 and miR-339-5p in human osteosarcoma tissue and adjacent tissues.si-LEF1-AS1 was transfected in cell U20S.Cell counting kit-8(CCK-8)and colony formation were employed to determine cell proliferation and cloning ability;flow cytometry measured the apoptosis of cell;western blot was applied to assay expression of P21 and cysteinyl aspartate specific proteinase 3(Caspase-3)protein.Bioinformatics prediction and dual luciferase reporting experiments analyzed the targeting relationship between LEF1-AS1 and miR-339-5p.Co-transfected si-LEF1-AS1 and anti-miR-339-5p,or co-transfected si-LEF1-AS1 and miR-339-5p in U20S were observed for their roles in cell proliferation and apoptosis.Results:Compared with adjacent tissue,the expression level of LEF1-AS1 in osteosarcoma tissue significantly increased,while the expression level of miR-339-5p greatly decreased(P<0.05).Inhibition of LEF1-AS1 dramatically reduced the survival rate of U20S and the number of colony formation,and evidently improved the apoptosis rate,P21 and Caspase-3 protein levels(P<0.05).LEF1-AS1 targeted and regulated the expression of miR-339-5p.miR-339-5p expression,P21,Caspase-3 protein expression,and apoptosis rate reduced obviously after co-transfection of si-LEF1-AS1 and anti-miR-339-5p in cell U20S,and cell U20S survival and cloning formation number increased apparently(P<0.05).Co-transfection of si-LEF1-AS1 and miR-339-5p notably improved miR-339-5p expression,P21,Caspase-3 protein expression,and apoptosis rate,and remarkably declined cell survival and the number of colony formation of U20S cells(P<0.05).Conclusions:Inhibition of LEF1-AS1 can inhibit the proliferation of osteosarcoma cells and induce apoptosis through targeted regulation of miR-339-5p expression.