摘要
目的探究金丝桃苷(Hyp)通过miR-139-5p/DDIT4信号通路调节脑胶质瘤细胞的增殖、凋亡、迁移和侵袭。方法体外培养人恶性胶质瘤细胞U87 MG,(1)分别给予0、25、50、100μmol·L-1Hyp处理[按Hyp不同剂量分为对照组、低剂量Hyp(L-Hyp)组、中剂量(M-Hyp)组、高剂量(H-Hyp)组。(2)利用Lipofectamine-2000转染试剂分别将抑制物-NC、miR-139-5p抑制物转染至U87 MG细胞中(分为抑制物-NC组、miR-139-5p抑制物组),另设未转染的对照组;U87 MG细胞转染后,再分别给予0、H-Hyp(100μmol·L-1)处理,分为对照组、H-Hyp组、H-Hyp+抑制物-NC组和H-Hyp+miR-139-5p抑制物组。CCK-8检测各组U87 MG细胞增殖;Annexin V-FITC/PI法检测各组U87 MG细胞凋亡率;Transwell实验检测各组细胞迁移数和侵袭数;qRT-PCR检测各组miR-139-5p、DDIT4表达。双荧光素酶报告基因(DLR)法检测验证miR-139-5p与DDIT4的靶向关系。(3)建立裸鼠异种移植模型,分为对照组和Hyp(30 mg·kg^(-1))组,测量肿瘤体积和重量,用qRT-PCR检测肿瘤组织中miR-139-5p、DDIT4表达。结果(1)与对照组比较,L-Hyp组、M-Hyp组、H-Hyp组U87 MG细胞A450值、迁移数和侵袭数减少,凋亡率增加,miR-139-5p表达增加,DDIT4表达减少,以H-Hyp组变化显著(均P<0.05)。(2)与对照组和抑制物-NC组比较,miR-139-5p抑制物组U87 MG细胞miR-139-5p表达减少,DDIT4表达增加,A450值、迁移数和侵袭数增加,凋亡率减少(均P<0.05);DLR法验证显示,miR-139-5p与DDIT4存在靶向关系。(3)与对照组比较,H-Hyp组U87 MG细胞miR-139-5p表达增加,DDIT4表达减少,A450值、迁移数和侵袭数减少,凋亡率增加(均P<0.05);与H-Hyp组、H-Hyp+抑制物-NC组比较,H-Hyp+miR-139-5p抑制物组U87 MG细胞miR-139-5p表达减少,DDIT4表达增加,A450值、迁移数和侵袭数增加,凋亡率下降(均P<0.05)。(4)与对照组比较,Hyp组肿瘤体积和重量减少、miR-139-5p表达增加、DDIT4表达下降(均P<0.05)。结论Hyp能通过上调miR-139-5p,靶向下调DDIT4表达,进而抑制U87 MG细胞增殖、迁移和侵袭,并促进凋亡。
Aim To explore the regulation of hyperoside(Hyp)on the proliferation,apoptosis,migration and invasion of glioma cells through the miR-139-5p/DDIT4 signaling axis.Methods Human malignant glioma cells U87 MG were cultured in vitro.①0,25,50,and 100μmol·L-1 Hyp were administered,respectively[Ctrl group,low-dose Hyp(L-Hyp)group,medium-dose(M-Hyp)group,and high-dose(H-Hyp)group];②inhibitor-NC and miR-139-5p inhibitor were transfected into U87 MG cells(inhibitor-NC group,miR-139-5p inhibitor group)using Lipofectamine-2000,and an untransfected Ctrl group was also set;③U87 MG cells were treated with 0 and H-Hyp(100μmol·L^(-1))after transfection,respectively,and they were separated into Ctrl group,H-Hyp group,H-Hyp+inhibitor-NC group,and H-Hyp+miR-139-5p inhibitor group.The proliferation of U87 MG cells in each group was detected by CCK-8;Annexin V-FITC/PI method was used to detect the apoptosis in each group;Transwell assay was used to detect the migration and invasion in each group;qRT-PCR was used to detect the expressions of miR-139-5p and DDIT4 in U87 MG cells in each group,and the targeting relationship between miR-1395p and DDIT4 was verified by dual luciferase reporter gene assay(DLR).Xenotransplantation models of nude mice were established and divided into control group and Hyp group(30 mg·kg^(-1)).The tumor volume and weight were measured,and the expressions of miR-139-5p and DDIT4 in tumor tissues were detected by qRT-PCR.Results①Compared with Ctrl group,the A450 value,the number of migration and the number of invasion of U87 MG cells in the L-Hyp group,M-Hyp group,H-Hyp group decreased,the apoptosis rate increased,and the expression of miR-139-5p increased,the expression of DDIT4 decreased,and the changes were more in the H-Hyp group(P<0.05).②Compared with the Ctrl group and the inhibitor-NC group,the expression of miR-139-5p in U87 MG cells in the miR-139-5p inhibitor group decreased,the expression of DDIT4 increased,the A450 value,the numbers of migration and the numbers of invasion increased,and the apoptosis rate decreased(P<0.05);DLR results showed that miR-139-5p had a targeting relationship with DDIT4.③Compared with the Ctrl group,the expression of miR-1395p in U87 MG cells in the H-Hyp group increased,the expression of DDIT4,the A450 value,the numbers of migration and the numbers of invasion decreased,and the apoptosis rate increased(P<0.05).Compared with H-Hyp group and H-Hyp+inhibitor-NC group,the expression of miR-139-5p in U87 MG cells in the H-Hyp+miR-139-5p inhibitor group decreased,the expression of DDIT4 increased,the A450 value,the numbers of migration and the numbers of invasion increased,and the apoptosis rate decreased(P<0.05).④Compared with the control group,tumor volume and tumor weight decreased,miR-139-5p expression increased,and DDIT4 expression decreased in Hyp group(P<0.05).Conclusion Hyp can inhibit the proliferation,migration and invasion of U87 MG cells,and promote their apoptosis by up-regulating miR 139-5p expression and targeting down DDIT4 expression.
作者
宋志远
任洪波
牛国栋
韩晓正
胡志恒
曲大成
SONG Zhi-yuan;REN Hong-bo;NIU Guo-dong;HAN Xiao zheng;HU Zhi-heng;QU Da-cheng(Department of Neurosurgery,Handan Central Hospital,Handan 056000,China)
出处
《中国临床神经科学》
2025年第2期136-145,共10页
Chinese Journal of Clinical Neurosciences
基金
邯郸市科学技术研究与发展计划项目任务合同(编号:1723208069-1)。
